熊本大学学術リポジトリ

Kumamoto University Repository System

Title Studies on basic principles for developing

peripherally acting antitussives : Pharmacological

study …

Author(s) Zhou, Jianrong

Citation

Issue date 2006-03-24

Type Thesis or Dissertation

URL http://hdl.handle.net/2298/2649

Right

PhD Thesis

Studies on basic principles for developing peripherally acting antitussives

: Pharmacological study of paratracheal ganglia neurons

Kumamoto University

Graduate School of Pharmaceutical Sciences

Department of Environmental and Molecular Health Sciences

Jianrong Zhou

2006

平成十七年度平成十七年度平成十七年度平成十七年度

博士論文博士論文博士論文博士論文

末梢性鎮咳薬開発末梢性鎮咳薬開発末梢性鎮咳薬開発末梢性鎮咳薬開発のためののためののためののための基本原理基本原理基本原理基本原理にににに関関関関するするするする研究研究研究研究：：：： 傍気管神経節傍気管神経節傍気管神経節傍気管神経節ニューロンニューロンニューロンニューロンのののの薬理学的研究薬理学的研究薬理学的研究薬理学的研究

熊本大学熊本大学熊本大学熊本大学 大学院大学院大学院大学院 薬学教育部薬学教育部薬学教育部薬学教育部

生命薬科学専攻生命薬科学専攻生命薬科学専攻生命薬科学専攻 生命生命生命生命・・・・環境科学講座環境科学講座環境科学講座環境科学講座

環境分子保健学分野環境分子保健学分野環境分子保健学分野環境分子保健学分野

周周周周 建建建建 融融融融

Studies on basic principles for developing peripherally acting antitussives : Pharmacological study of paratracheal ganglion neurons

Graduate School of Pharmaceutical Sciences

Department of Environmental and Molecular Health Sciences

Jianrong Zhou

The number of patients complaining the unprofitable chronic coughs lasting for more than 8 weeks is recently increasing in clinic. Cause of chronic chough is not simple and currently available antitussive therapy is often ineffective. Therefore, great current need is to develop the effective nonspecific antitussive therapy. To construct a novel strategy for developing new class of antitussives for chronic coughs, it is important to understand physiology and pharmacology of the nervous systems, because cough reflex is generated via neuronal reflex arc. In this study, the paratracheal ganglia (PTG) of the parasympathetic nervous system were focused on because it works as a part of cough reflex arc and predominantly controls the airway function.

Since bradykinin (BK), a potent inflammatory peptide, has been implicated as a stimulant of intractable coughs, I first studied effect of BK on cholinergic responses and synaptic currents in PTG neurons (Part I, Fundamental study). At second, I studied effect of suplatast on BK-induced and related responses in PTG neurons, as well as in sensory neurons (Part II, Applied study), because our own experimental research and other clinical trials revealed that suplatast has antitussives effects on chronic coughs such as cough variant asthma and on codeine-resistant coughs in animal models.

The experiments were carried out by whole-cell and outside-out mode of patch clamp technique in the acutely dissociated rat PTG neurons attached with or without synaptic boutons. Trigeminal ganglia neurons or rats were also used. Data obtained from this study are summarized as follows:

I Fundamental study: Effect of BK on cholinergic responses and synaptic currents in PTG neurons

1. Bradykinin B2 receptor-mediated response was additive on M1 muscarinic ACh receptor-mediated responses in PTG neurons. This result must conclusively affect pathological condition via increment in length constant of membrane.

2. BK at low concentrations potentiated nicotinic current (INic) probably via following pathway: bradykinin B2 receptor → PTX-sensitive G protein → PLC. The results also suggested that bradykinin B2 receptor couples with two

distinct G proteins, PTX-sensitive and PTX-insensitive. 3. BK potentiated both amplitude and frequency of EPSCs via bradykinin B2

receptor. Thus, it was suggested that BK stimulates bradykinin B2 receptors at both presynaptic and postsynaptic site, and facilitate synaptic transmission in PTG neurons via three different mechanisms, membrane depolarization and INic potentiation at postsynaptic site and increase in ACh release at presynaptic site.

II Applied study: Effect of suplatast on BK-induced and related responses in PTG neurons, as well as in sensory neurons

1. Suplatast did not affect directly on sensory neurons. However, I found that histamine potentiated capsaicin-induced currents in rat sensory neurons. Since it has been known that suplatast suppresses histamine release from mast cells, it is possible that suplatast inhibits the activation of nociceptive fibers in pathological condition via prevention of histamine-induced potentiation of TRPV1 receptor-mediated currents.

2. Suplatast inhibited INic in noncompetitive- and voltage-dependent manners. Suplatast also reduced the open time of nicotinic receptor/channels and caused flickering in channel open, suggesting an open channel block.

3. In addition, suplatast inhibited the EPSC amplitude and its frequency in PTG neurons. EPSC frequency was much sensitive to suplatast than EPSC amplitude. IC50 for EPSC frequency was similar to the effective concentration to inhibit histamine release from mast cells and was lower than that for inhibition of cytokine production. Suplatast also inhibited the EPSCs potentiated by BK, but had not effect on the potentiation process itself by BK.

In this study, I revealed novel effects of BK that probably contribute to airway inflammation and aggravation of airway function. Suplatast did not affect on BK-induced responses in both sensory and PTG neurons. However, suplatast inhibited the function of PTG neurons at presynaptic and postsynaptic sites. Suplatast became the second example of antitussive drug, which inhibits the function of PTG neurons. Since suplatast, but not previous medicines, inhibits both codeine-sensitive and insensitive cough in vivo, suplatast might become a good seed for development of searching the novel antitussives. Here, I propose a working hypothesis that the chemicals inhibiting the functions of both nociceptive sensory fibers and PTG neurons may become a candidate of useful antitussives for chronic coughs. Finally, PTG neurons, in particular, those attached with synaptic boutons, are a useful preparation for studying effects of peripherally-acting antitussives and might become a new target of novel antitussives effective for chronic coughs.

Abbreviations

AA arachidonic acid

ACh acetylcholine

ACE angiotensin-converting enzyme

AHR airway hyperresponsiveness

4-AP 4-aminopyridine

ASIC acid sensing ion channel

ATP adenosine trisphosphate

BK bradykinin

BKCa large conductance Ca2+ activated K+ channel

CGRP calcitonin gene related peptide

CNS central nervous system

CVA cough variant asthma

CysLT cysteinyl leukotrienes

DMEM Dulbecco’s modified eagle medium

DRG dorsal root ganglion

EC50 half-maximum effective concentration

ECP eosinophil cationic protein

EGTA ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid

EPSC excitatory postsynaptic current

FcεRI high affinity Fc receptor for IgE

GIRK G-protein coupled inwardly rectifying K+ channel

GM-CSF granulocyte-macrophage colony-stimulating factor

GTP guanosine-5’-triphosphate

HEPES 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid

HETE hydroxyeicosatetraenoic acid

HOE 140 D-Arginyl-[Hyp3, Thi5, D-Tic7, Oic8]-bradykinin

5-HT 5-hydroxytryptamine

HVA high-voltage-activated

[Hyp3]-BK [Hyp3]-bradykinin

IC50 half-maximum inhibitory concentration

IgE immunoglobulin-E

IL-4 interleukin-4

IL-5 interleukin-5

I K(M) M-type K+ current

KATP ATP sensitive K+ channel

LO lipoxygenase

LTB4 leukotriene B4

MBP major basic protein

NGF nerve growth factor

NKA neurokinin A

NOP novel opioid receptor

NPo open probability

OP-D ophiopogonin-D

PIP2 phosphatidylinositol-(4,5)-bisphosphate

PKC protein kinase C

PLA2 phospholipase A2

PLC phospholipase C

PLD phospholipase D

PTG paratracheal ganglia

PTX pertussis toxin

RAR rapidly adapting stretch receptor

SAR slowly adapting stretch receptor

SP substance P

TG trigeminal ganglia

Tris-OH Tris( hydroxymethyl) aminomethane

TrkA tropomyosin-receptor kinase A

TRPV1 transient receptor potential vanilloid type 1

TTX tetrodotoxin

Th2 type 2 helper T cell

VH holding potential

VGCC voltage-gated Ca2+ channel

Contents Introduction ･･･････････････････････････････････････････････････････････････････････ 1 Part I Fundamental study ･････････････････････････････････････････････････････････････････ 5

1 Modulation of ACh responses by BK in PTG neurons ････････････････････ 6

1.1 Effects of BK on muscarinic ACh responses ･･････････････････････････ 7

1.2 Potentiation of nicotinic ACh responses by BK ･･･････････････････････ 10

1.2.1 Potentiation of nicotine-induced current by BK ･･･････････････････ 10

1.2.2 Effects of bradykinin B2 receptor antagonist and agonist ･･･････ 12

1.2.3 Possible contribution of pertussis toxin-sensitive G-protein

and phospholipase C to the BK-induced potentiation of INic ････ 14

2 EPSCs and their potentiation by BK in rat PTG neurons ･････････････････ 18

2.1 EPSCs in rat PTG neurons ･･････････････････････････････････････････････ 18

2.2 Potentiation of EPSCs by BK ････････････････････････････････････････････ 23

3 Discussion ･････････････････････････････････････････････････････････････････････ 27

Part II Applied study ････････････････････････････････････････････････････････････ 32

1 Effect of suplatast in single sensory and PTG neurons ･･･････････････････ 33

1.1 Effect of suplatast in sensory neurons ･････････････････････････････････ 38

1.2 Inhibition of nicotinic responses by suplatast in PTG neurons ･･････ 47

1.2.1 Effect of suplatast in PTG neurons ････････････････････････････････ 47

1.2.2 Inhibition of nicotinic response･･････････････････････････････････････ 50

1.2.3 Single channel analysis ･････････････････････････････････････････････ 56

2 Effects of suplatast on EPSCs in rat PTG neurons ････････････････････････ 65

2.1 Inhibition of EPSCs by suplatast ････････････････････････････････････････ 65

2.2 Effects of suplatast on EPSCs potentiated by BK ･････････････････････ 68

3 Discussion ･････････････････････････････････････････････････････････････････････ 70

Effects in sensory neurons ･････････････････････････････････････････････････････ 70

Inhibition of EPSCs in PTG neurons ･･････････････････････････････････････････ 71

Suplatast and chronic cough ･･･････････････････････････････････････････････････ 74

Summary and Conclusion ･･････････････････････････････････････････････････････ 78

Materials and Methods ･･･････････････････････････････････････････････････････････ 84

1 Preparations ･･･････････････････････････････････････････････････････････････････ 84

1.1 Animals ････････････････････････････････････････････････････････････････････ 84

1.2 Dissociation of PTG neurons ････････････････････････････････････････････ 84

1.3 Dissociation of trigeminal ganglion neurons ･･･････････････････････････ 85

2 Electrophysiological recordings･･･････････････････････････････････････････････ 86

2.1 Patch clamp recording and data analysis ･････････････････････････････････ 86

2.2 Fast drug application with the “Y-tube” ････････････････････････････････････ 89

3 Solutions and chemicals･･･････････････････････････････････････････････････････ 90

3.1 Solutions for cell dissociation and patch clamp recording ･･･････････････ 90

3.2 Chemicals ･･････････････････････････････････････････････････････････････････ 91

References ･･････････････････････････････････････････････････････････････････････････････ 93

Acknowledgments ･･････････････････････････････････････････････････････････････ 104

1

Introduction

The number of patients complaining the unprofitable chronic coughs is

recently increasing in clinic.1) Chronic cough is defined as the cough lasting for

more than eight weeks. Although numerous narcotic and non-narcotic

antitussives have been developed, the most effective antitussives currently

available are still narcotic antitussives. The beneficial value of them has limited

due to the associated unacceptable side effects. Even to such narcotic

antitussives, chronic coughs are resistant. Therefore, development of new

strategies to inhibit chronic coughs has been desired. Because cough

responses are usually triggered by the stimulation of peripheral sensory nerve

terminals in the airway and regulated by central and peripheral nervous system,

it seems to be important for developing new antitussive drugs to understand

pharmacological properties of the airway nervous system.

Elevation in cough sensitivity may result from chronic inflammation and

pathophysiological changes at several potential sites along the neural pathway

that mediates the cough reflex. Recent investigations on the pathogenic

mechanisms of chronic cough have focused mostly on the peripheral site of the

neural circuit, the ‘cough receptors’. These sensory receptors are known to be

susceptible to the abnormal changes in the microenvironment surrounding the

receptor terminals, which can be adversely altered by pathophysiological

conditions of the airways. Several major types of ion channels, both

ligand-gated and voltage-gated channels, potentially involve the hypersensitivity

of these sensory receptors caused by airway inflammation.2) Results of these

studies further suggest certain ion channels as potential targets for therapeutic

2

intervention. Electrophysiological data are supported by immunohistochemical

evidence illustrating the presence of the receptor proteins for the various

endogenous inflammatory mediators on the neuronal membrane.2) These

studies demonstrate that the excitability of both Aδ- and C-fiber afferents can be

elevated by autacoids. The potential involvement of these changes in the

enhanced sensitivity to cough during airway inflammation should gain additional

support if similar observations can be demonstrated in the specific subset of Aδ

afferents that have been identified by Canning et al.3) as the cough receptor.

In contrast, the predominant control of airway function is thought to be

exerted by cholinergic nerves arising from the paratracheal ganglia (PTG) that

localize on the serosal surface of the dorsal tracheal wall. The excitability of

PTG neurons is controlled by the preganglionic neurons via central vagal reflex.

In addition, it can be modulated by a peripheral reflex mechanism because the

collateral branches of neurokinin-containing C-fibers project to the PTG neurons

(Fig.1).4) Stimulation of the afferent C-fiber terminals probably releases

neurokinins from the C-fiber terminals and enhances cholinergic

neurotransmission in the PTG.5) Our group also found that bradykinin (BK), a

potent inflammatory peptide, depolarized the membrane potential via inhibition

of M-type K+ channels and induced action potential generation.6) Thus, the PTG

are thought to be not only a relay neuron of the parasympathetic nerve, but also

integrative sites for the neuromodulation of normal airway function and

important for pathogenesis in airway inflammation.

In addition, we previously reported that ophiopogonin-D (OP-D), an active

constituent of Bakumondo-to, a Chinese herbal medicine that is effective for

treating chronic coughs in clinic, hyperpolarize the membrane potential via

3

Fig.1. Vagal reflex and neurogenic inflammation in the lower airway.

See text in detail. CNS, central nervous system, PTG, paratracheal ganglia, SP, substance P, NKA, neurokinin A, CGRP, calcitonin gene related peptide.

activation of K+ current, reducing the cell excitability of PTG neurons.7) Ganglion

blocker also inhibits the allergic cough.8) On the other hand, codeine,

representative narcotic antitussives, did not affect on the function of PTG

neurons.9) Therefore, PTG might become a novel target for developing the new

class of antitussives for treating chronic coughs. However, the modulations of

synaptic transmission in PGT neurons by inflammatory substances have poorly

understood. Therefore, it is indispensable to know how the synaptic

Stimulants

Aδδδδ-fiber

C-fiber

PPTTGG

Vagal afferent nerve

Vagal efferent nerve

Smoothmuscle

Gland Capillary Mast cell

SP NKA CGRP

SP NKA CGRP

SP NKA CGRP

Collateral branch of afferent nerve

SP, NKA

CNS

4

transmission in PTG is modulated by inflammatory substances and whether any

drugs effective for chronic coughs other than OP-D inhibit the excitability of PTG

neurons.

In the part I of this study, I studied the effect of BK on the muscarinic and

nicotinic ACh responses and then the cholinergic synaptic currents in PTG

neurons, because 1) BK is a potent inflammatory mediator and 2) it has been

reported that BK stimulates cough responses resistant to codeine. In the part II,

I studied effect of suplatast on BK-induced and related responses and synaptic

currents in PTG neurons, because 1) suplatast had antitussive effect on

coughing resistant to codeine in guinea pigs10) and 2) it showed effective

antitussive effect on chronic coughs in humans.11,12) In addition, effect of

suplatast in rat sensory neurons was also studied.

The final objective of the present study is to establish a new strategy for

developing new antitussives effective for chronic coughs by clarifying

pharmacological significance of the airway nervous system for developing such

antitussives.

5

Part I Fundamental study

6

1 Modulation of ACh responses by BK in PTG neurons

BK is a potent inflammatory peptide that has been implicated as a potential

mediator of human airway diseases. In the lower airway, BK induces

bronchoconstriction, mucus secretion, microvascular leakage, and cough.13) The

mechanisms underlying BK actions in the airway are considered to be largely

indirect and to occur primarily through airway nerve activation.14)

BK stimulates afferent C-fibers15) and causes cholinergic reflex via brainstem

neurons. In addition, the excitability of PTG neurons can be modulated by a

peripheral reflex mechanism because the collateral branches of neurokinin-

containing C-fibers project to the PTG neurons.4) Stimulation of the afferent

C-fiber terminals probably releases neurokinins from the C-fiber terminals and

enhances cholinergic neurotransmission in the PTG.5)

In addition, inflammatory mediators may directly modulate the function of PTG

neurons because pro-inflammatory substances and antigen exposure affect their

function.16~20) If this is the case, it seems likely that alteration of the function of

PTG neurons by inflammatory mediators underlies the pathology of inflammation

in the airway. Recently, our group has reported that BK activates bradykinin B2

receptors and induces the action potential generation via the inhibition of the

M-type K+ current (IK(M)) in dissociated single PTG neurons.6) Furthermore, it has

been reported that BK stimulates cough responses resistant to codeine in guinea

pigs. However, the actions of BK on cholinergic synaptic transmission in the PTG

are still unclear. Therefore, I studied the effects of BK on the cholinergic

responses and excitatory postsynaptic currents (EPSCs) in acutely dissociated

PTG neurons.

7

1.1 Effects of BK on muscarinic ACh responses

When ACh and BK were applied to PTG neurons, they depolarized neurons at

the concentration range of 10-9 ~ 10-6 M and 10-10 ~ 10-7 M, respectively (Fig.2).

Fig.2. Concentration-response relationships for ACh- and BK- induced depolarization in rat PTG neurons.

A: representative membrane potential traces at various concentrations of ACh or BK in the presence of 3x10-7 M tetrodotoxin. ACh and BK were applied for the period indicated by the horizontal bar above each trace. Upper and lower traces were obtained from different neurons. B: concentration-response curves for ACh and BK (n=4 to 5). Continuous line was drawn in accordance with the formula (1) described in Materials and Methods.

A

B

10-10 10-9 10-8 10-7 10-6 10-50

3

6

9

12

Dep

olar

izat

ion

(mV)

ACh (M)10-10 10-9 10-8 10-7 10-6 10-5

0

3

6

9

12

Dep

olar

izat

ion

(mV)

BK (M)

5 m

V

30 sec

30 sec

5 m

V

10-9 ACh

10-10 BK

5x10-9 10-8 10-7 M

10-8 10-7 5x10-7 M

8

ACh-induced depolarization was completely inhibited by 10-6 M pirenzepine, a

muscarinic receptor antagonist (data not shown). For studying the effect of BK

on muscarinic receptor-mediated depolarization, ACh was applied for 30 sec at

least twice at 5 min interval. Thereafter, the neurons were treated with 10-9 M BK

for 3 to 8 min and then the mixture of ACh and BK was applied. ACh at 5x10-7 M

and BK at 10-9 M depolarized 6.8 ± 0.6 mV (n=5) and 1.1 ± 0.4 mV (n=5),

respectively (Fig.3A). After BK treatment, simultaneous application of ACh and

BK depolarized further 6.7 ± 0.5 mV (n=5), indicating the summation of

ACh-induced and BK-induced depolarization. A similar result was observed in

simultaneous application of 5x10-8 M ACh and 10-9 M BK (n=4, data not shown).

In voltage clamp mode, both ACh and BK inhibited IK(M). The IK(M) was

measured as the time-dependently relaxing current component during

hyperpolarizing voltage steps from a VH of -25 mV to -50 mV (Fig.3B).6,21,22)

When 3x10-7 M ACh or 3x10-9 M BK was applied for 40 sec, ACh and BK

inhibited IK(M) by 37.8 ± 4.7 % (n=6) and 15.4 ± 4.6 % (n=6), respectively. These

inhibitions were caused via muscarinic M1 and bradykinin B2 receptors

respectively.23,6) When neurons were pretreated with 3x10-9 M BK for 3 min and

then ACh and BK were simultaneously applied for 40 sec, IK(M) was inhibited by

51.8 ± 3.7 % (n=6, Fig.3B), indicating the additive effect of ACh and BK. A similar

result was observed in simultaneous application of 3x10-7 M ACh and 10-9 M BK

(n=6, data not shown).

BK caused strong desensitization at the concentrations higher than 10-8 M. BK

at 10-7 M induced the maximum response in concentration-response relationship.

Therefore, we further studied whether the strong desensitization caused by 10-7

M BK affects muscarinic receptor-mediated response. At a VH of –40 mV, both

9

ACh 3x10-7 M

60

0

20

40

% In

hibi

tion

of I K

(M)

ACh + BK

BK3x10-9 M

ACh3x10-7 M

ACh+BK 0.2 secBK BKACh ACh 100

pA

Dep

olar

izat

ion

(mV)

A a

B a

b

b30 sec

BK 10-9 M

5 m

V

ACh 5x10-7 M

50 p

A

1 min

BK 10-7 M

C a b

0

0.5

1.0

1.5

2.0

Rel

ativ

e C

urre

nt

ACh + BK

BK10-7 M

ACh3x10-7 M

0

2

4

6

8

ACh5x10-7 M(control)

BK10-9 M

ACh5x10-7 M(with BK)

10

Fig.3. Effect of BK on muscarinic M1 ACh receptor-mediated responses.

A: effect of 10-9 M BK on muscarinic M1 ACh receptor-mediated depolarization. a: representative membrane potential traces in the presence of 3x10-7 M tetrodotoxin at resting potential of –60.7 mV. ACh and BK were applied for the period indicated by the horizontal bar and the hatched column above each trace. ACh was applied at 5 min interval. Tetrodotoxin was applied continuously during the recording. b: peak amplitudes of depolarization induced by 10-9 M BK, 5x10-7 M ACh and that in the presence of 10-9 M BK. The amplitudes shown by arrows were measured and summarized (n=4 to 8). B: effect of 3x10-9 M BK on 3x10-7 M ACh-induced inhibition of IK(M). a: representative current traces before (black) and during application (grey) of 3x10-7 M ACh, 3x10-9 M BK, or their mixture. Right two traces are after control. After termination of the BK or mixture application, neurons were washed with normal external solution for more than 15 min to eliminate BK-induced desensitization, because the effect of 3x10-9 M BK on IK(M) was almost completely recovered after 15 min of washout (95.5 ± 2.8%, n=5). Black traces were recorded just before drug application and grey ones were recorded when the inhibition became peak. b: percent inhibition of IK(M) by ACh, BK and the mixture (n=6). C: influence of BK-induced desensitization on 3x10-7 M ACh-induced inward current. a: representative current traces induced by 3x10-7 M ACh, 10-7 M BK and their mixture at a VH of -40 mV. b: relative inward current amplitude induced by 3x10-7 M ACh, 10-7 M BK and their mixture. All data were normalized to the average of inward current amplitudes induced by 3x10-7 M ACh before BK application (n=4). Left column shows the relative amplitude of ACh-induced current recorded just before BK application. ACh: acetylcholine, BK: bradykinin, IK(M): M-type current.

3x10-7 M ACh and 10-7 M BK induced inward currents via IK(M)-inhibition (Fig.3C).

Even after BK-induced current was desensitized completely, ACh at 3x10-7 M

was able to induce the inward current to the same extent as the control,

suggesting that BK and ACh are not cross-desensitized each other.

1.2 Potentiation of nicotinic ACh responses by BK

1.2.1 Potentiation of nicotine-induced current by BK

At a concentration range of 3x10-6 to 3x10-4 M, both ACh and nicotine induced

rapid inward currents at a VH of –50 mV (Fig.4A). EC50 and the Hill coefficient

were 1.32x10-5 M and 1.49 for ACh and 1.11x10-5 M and 1.78 for nicotine,

11

10-6 10-5 10-4 10-30

200400600800

100012001400

Nic ACh

I Nic

(pA

)

Nicotine (M)

A

B

a

a b

b

0

50

100

150

BK 10- 8 M + Nic

BK 10- 9 M + Nic

Nic 10- 5 M

% o

f Con

trol

∗

Nic

10 sec

200

pA

3x10-4 M10-43x10-5 10-5 3x10-6

Nic10-5 M

BK 10-8 M

10 sec10

0 pA

a1 a2

a3

12

Fig.4. BK-induced potentiation of INic.

A: ACh- and nicotine-induced rapid inward currents at a VH of -50 mV. a: representative nicotine-induced INic. Nicotine was applied for the period indicated by the horizontal bars above each trace. b: concentration-response relationships for ACh- and nicotine-induced currents (n= 4 to 5). Continuous line was drawn in accordance with the formula (1) described in Materials and Methods. B: potentiation by BK of the 10-5 M nicotine-induced INic. a: representative recording showing the potentiation of INic by 10-8 M BK at a VH of –50 mV. BK was applied for the period

indicated by the horizontal hatched column above the trace. b: concentration dependency of the BK-induced potentiation of INic. The current amplitudes shown by the arrows were measured, and all data were normalized to the average INic amplitudes recorded before BK application (n=12 to 10). The left-hand column shows the percentage of INic amplitude recorded just before BK application (2a2 / (a1+a2) x 100). In the presence of BK, INic was calculated as 2a3 / (a1+a2) x 100. *; P < 0.05 vs the control (nicotine 10-5 M).

respectively. To avoid the muscarinic effect of ACh, nicotine was used as a

nicotinic agonist for further study. When the neurons were pretreated for 1 to 1.5

min with 10-9 or 10-8 M BK and the mixture of BK and 10-5 M nicotine was then

applied, the nicotine-induced current (INic) was potentiated (Fig.4Ba). The

potentiation depended on the BK concentration. At 10-9 M, the potentiation was

15.6 ± 2.1% (n=12) and it became 35.1 ± 5.7% (n=10) at 10-8 M. The potentiation

at 10-8 M was statistically significant (P < 0.05, Fig.4Bb).

On the other hand, BK at 10-8 M did not affect the 3x10-4 M nicotine-induced

INic (data not shown), which is almost the maximum response in a concentration-

response relationship (Fig.4A).

1.2.2 Effects of bradykinin B2 receptor antagonist and agonist

We previously reported that BK inhibited IK(M) via bradykinin B2 receptor.6) To

clarify the receptor subtype mediating the BK-induced potentiation of INic, the

13

Fig.5. Involvement of bradykinin B2 receptor in the BK-induced potentiation of INic in PTG neurons.

A: representative recording of 10-5 M nicotine-induced INic in the presence of 10-8 M BK and 10-6 M HOE140, a bradykinin B2 receptor antagonist. B: representative recording of 10-5 M nicotine-induced INic in the presence of 10-7 M [Hyp3] -bradykinin, a bradykinin B2 receptor agonist.

C: effect of bradykinin B2 receptor antagonist and agonist. All current amplitudes were normalized to the average INic amplitude recorded before the application of bradykinin B2 antagonist and/or agonist (control) (n=9 and 3). *; P < 0.05 vs control (left-hand column).

[Hyp3] -BK 10-7 M

10 sec

100 pA

10-5 M Nic

BK 10-8 M A

10-5 M

10 sec 100 pA

C

B

0

50

100

150

[Hyp 3]-BK + Nic

HOE + BK + Nic

% o

f Con

trol

Nic 10 - 5 M

∗

Nic HOE 140 10-6 M

14

effect of bradykinin B2 receptor antagonist and agonist on BK potentiation was

examined. HOE 140, a bradykinin B2 receptor antagonist, did not affect INic but

blocked the potentiation of INic by BK (Fig.5A and C). On the other hand, a

bradykinin B2 receptor agonist, [Hyp3]-bradykinin at 10-7 M, significantly

potentiated the 10-5 M nicotine-induced INic (Fig.5B). The amplitude of the 10-5 M

nicotine-induced INic in the presence of [Hyp3]-bradykinin was 121.62 ± 5.21% of

the control (n=3, Fig.5C).

1.2.3 Possible contribution of pertussis toxin-sensitive G-protein and phospholipase C to the BK-induced potentiation of INic

Bradykinin B2 receptor is able to couple with both pertussis toxin-sensitive and

-insensitive G-proteins.24~27) Therefore, I studied the effect of pertussis toxin on

potentiation by BK. PTG neurons were incubated at 20°C for 12 to 24 hr in the

normal external solution with or without 500 ng/ml pertussis toxin. In neurons

treated with pertussis toxin, the 10-5 M nicotine-induced currents were

compatible with those in neurons not treated with pertussis toxin. BK at 10-8 M

induced a slow inward current even in neurons treated with pertussis toxin, as

reported previously.6) However, BK failed to potentiate INic (Fig.6). This effect was

observed in all neurons tested (n=13).

Bradykinin B2 receptor activates phospholipase C (PLC) and/or phospholipase

D (PLD) in neuronal and non-neuronal cells. At least the activation of PLC

occurs by Gβγ subunits associating with pertussis toxin-sensitive Gi/o proteins, as

well as the Gα subunit of Gq/11 .28~30) Therefore, I investigated the effect of

neomycin, a nonselective inhibitor of PLC and PLD, on the potentiation of INic by

BK. The potentiation was repeated when 10-8 M BK was applied at intervals of

15

Fig.6. Effect of pertussis toxin treatment on the BK-induced potentiation of INic.

A: representative current recording in the neuron treated with pertussis toxin for 14 hours. Note that BK induced inward current in the pertussis toxin-treated neuron, whereas it failed to potentiate INic. B: involvement of pertussis toxin-sensitive G protein. The data were calculated as described in Fig.4 (n=13). C: no correlation between the potentiation of INic and BK-induced current amplitude in the neurons treated with pertussis toxin.

of 30 min. However, BK failed to potentiate INic when 5 mM neomycin was

treated for 10 min after 30 min washout of the first BK application (data not

0

50

100

150

BK 10 - 8 M+

PTX Treatment

Nic 10 - 5 M

% o

f Con

trol

Nic 10 - 5 M

10 sec

100

pA

10-5 M A Nic BK 10-8 M

B

0 10 20 30 40 5050

75

100

125

150

% o

f Con

trol

IBK (pA)

C

16

shown). The application of 10-6 M U-73122, a PLC inhibitor, for 2 to 10 min also

inhibited the potentiation (Fig.7). Not only for the potentiation, neomycin and

U-73122 inhibited the BK-induced slow inward current in almost all neurons

tested. The example shown in Fig.7A is the only exception in which BK induced

a slow inward current even after pretreatment for 2 min with U-73122. On the

other hand, U-73433, an inactive analog of U-73122, had no effect on either the

potentiation of INic or the induction of a slow inward current in all three neurons

tested.

17

∗

Fig.7. Effect of PLC-inhibitor on the BK-induced potentiation of INic.

A: representative recording showing the effect of U-73122 on the BK-induced potentiation of INic. B: representative recording showing the effect of U-73433 on the BK-induced potentiation of INic. C: statistic analysis of the effects of U-73122 and U-73433 on the BK-induced potentiation of INic. All data were normalized to 10-5 M nicotine-induced INic amplitude recorded before the application of U-compounds and BK. The left-hand column indicates the current amplitude induced by the second application of 10-5 M nicotine without treatment with U-compound and BK (n= 3 to 17). *; P < 0.05, vs. control (left-hand column).

0

0.5

1.0

1.5

Nic+BK+U-73433

Nic+BK+U-73122

Nic+BK 10 -8M

Nic 10 -5 M

Rel

ativ

e I N

ic

A

B

C ∗

10-5 M Nic

BK 10-8 M

U-73433 10-6 M

10 sec

200

pA

10-5 M BK 10-8 MNic U-73122 10-6 M

18

2 EPSCs and their potentiation by BK in rat PTG neurons

In general, ACh and nicotinic ACh receptor work as principal neurotransmitter

and its receptor in autonomic ganglion, respectively. I indicated that BK

potentiated nicotinic INic in previous session. However, there are two kinds of

neuronal nicotinic ACh receptors are present in autonomic ganglia. One is

synaptic and the other is extrasynaptic.31) These two types might have different

subunit composition. It has also been indicated that pro-inflammatory mediators

modulate the transmission in PTG.32) However, the details of synaptic

transmission at single neuron level and its modulation by inflammatory

substances have remained to be clarified. Therefore, I tried to record EPSCs

and clarify their pharmacological properties in rat dissociated PTG neurons

attached with presynaptic buttons.

2.1 EPSCs in rat PTG neurons

In neurons mechanically dissociated after gentle enzyme treatment, I

succeeded to record the spontaneous transient inward currents at a VH of –60

mV in normal external solution containing 5 mM K+. Nystatin-perforated patch

clamp recording was used for the recording. Frequency of transient inward

currents was increased in 20 and 30 mM K+ external solution (Fig.8A). Increase

in frequency was statistically significant, whereas the peak amplitude were

almost the same at each external K+ concentration (Fig.8B). In 30 mM K+

external solution, cumulative probability distribution of inter-event interval, but

not peak amplitude, was shifted to the left (Fig.8C). The difference was

19

Fig.8. Spontaneous transient inward currents in dissociated PTG neurons

A: representative records showing spontaneous transient inward currents in 5, 20 and 30 mM K+ external solutions at a VH of –60 mV. B: effect of external K+ concentration on amplitude and frequency of spontaneous currents. All data were normalized to the respective control. Data were shown as mean±S.E.M. (n= 3 to 5). *; P<0.05 vs. 5 mM K+. C: cumulative probability plots for inter-event interval and amplitude of record shown in A. Circle and triangle indicate the record in 5 and 30 mM K+ solutions, respectively. D: activation and inactivation kinetics of transient inward currents. a: averaged current traces recorded in 5 and 30 mM K+ solutions shown in A. Activation and inactivation of averaged traces were fitted with single exponential function, respectively. b: superimposition of two current traces shown in a. Note that two traces were well superimposed. c: time constants of rising and decaying kinetics of averaged traces in 5 mM and 30 mM K+ solutions.

B C

D

5 mM K+

30 mM K+

400 ms 50 p

A

20 mM K+

5 mM K+

30 mM K+

A

a b

0

1

2

3

4

5 AmplitudeAmplitudeAmplitudeAmplitude

FewquencyFewquencyFewquencyFewquency

Nor

mal

ized

val

ue

Amplitude Frequency

5 mM 30 mM 20 mM

∗

∗

External K+ concentration 0

0

0.5

1

50 100 150Peak amplitude (pA)

00

1

0.5

3 6 9Inter-event interval (sec)

Cum

ulat

ive

prob

abilit

y

in 30 mM K+

in 5 mM K+

20 ms

20 p

A

c

0

6

12

18

24

Tim

e co

nsta

nt (m

s)

Rise τ

5 mM 30 mMExternal K+ concentration

Decay τin 30 mM K+

in 5 mM K+

20

significant in Kolmogorov-Smirnov Test (p<0.0001). Transient currents recorded

in 5 mM and 30 mM K+ external solutions were respectively averaged and their

activation and inactivation kinetics were analyzed (Fig.8D). When activation and

inactivation of current traces were fitted with exponential curves, both activation

and inactivation were well fitted a single exponential function. Rising and

decaying time constants were respectively 1.50±0.19 ms and 18.35±3.71 ms

in 5 mM K+ solutions, 1.52±0.26 ms and 19.35±4.11 ms in 30 mM K+ solutions,

respectively. There was no significant difference between the time constants in

5 mM and 30 mM K+ solutions.

Cd2+, a non-organic Ca2+ channel blocker, was concentration dependently

inhibited the transient inward current in 30 mM K+ external solution (Fig.9A and

B). Cd2+ did not affect on both activation and inactivation kinetics (Fig.9C). Both

activation and inactivation were well fitted with single exponential functions.

Rising and decaying time constants were respectively 1.19±0.17 ms and 27.79

±2.93 ms in control, and 1.23±0.19 ms and 22.38±0.09 ms in the presence of

Cd2+. Mecamylamine, a nicotinic ACh receptor antagonist, also inhibited the

transient inward current in 30 mM K+ external solution (Fig.10). Mecamylamine

at 10-5 M shifted the distribution of inter-event interval to the right and that of

amplitude to the left (Fig.10B). The differences were signif icant in

Kolmogorov-Smirnov Test (p<0.0001). Relative mean amplitude and frequency

of transient inward currents were 0.68±0.1 and 0.36±0.1 times of control,

respectively (Fig.10C). Transient currents recorded in 30 mM K+ external

solutions and co-application with 10-5 M mecamylamine were respectively

averaged and their activation and inactivation kinetics were compared (Fig.10D).

Mecamylamine significantly fastened both the activation and inactivation

21

Fig.9. Effect of Ca2+ channel antagonist on spontaneous transient inward currents in

30 mM K+ external solution

A: representative record showing the effect of Cd2+ on spontaneous transient inward currents. First, 30 mM K+ external solution was applied for 1 min as a control. After washing out for 4 min with 5 mM K+ solution, neuron was treated with 10-6 M CdCl2 for 20 sec in 5 mM K+ solution and then CdCl2 10-6 M in 30 mM K+ solution was applied for 1 min. B: concentration-dependent inhibition by Cd2+ of amplitude and frequency of spontaneous transient inward current. All data were normalized to the respective control. Data were shown as mean±S.E.M. (n=3). **; P<0.01 vs. individual control in the absence of Cd2+. C: activation and inactivation kinetics of transient inward currents. a: averaged current traces recorded in the absence or presence of 10-6 M Cd2+ shown in A. Activation and inactivation kinetics were fitted with single exponential function, respectively. b: superimposition of two current traces shown in a. Trace in the presence of 10-6 M Cd2+ was normalized to the peak amplitude of the trace in the absence of Cd2+. c: Time constants of rising and decaying kinetics of averaged traces in the absence and presence of Cd2+, respectively. Data were shown as mean±S.E.M. (n=3).

A

B

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Nor

mal

ized

val

ue

AmplitudeFrequency

Control

Cd2+ 10-7 M 10-6 M 10-4 M

∗∗∗∗

∗∗

∗∗ ∗∗

C a b

Control (30 mM K+)

Cd2+ 10-6 M

400 ms

50 p

A

After control

c

0

10

20

30

Tim

e co

nsta

nt (m

s)

Rise ττττ

Decay ττττ

Control Cd2+

Control

20 ms

20 p

A in Cd2+

in Cd2+

Control

22

Fig.10. Effects of nicotinic ACh receptor antagonist on EPSCs in 30 mM K+ external

solution

A: representative record showing the effect of 10-5 M mecamylamine on EPSCs. First, 30 mM K+ external solution was applied for 1 min as a control. After washing out with 5 mM K+ external solution for 4 min, neuron was treated with10-5 M mecamylamine for 20 sec in 5 mM K+ external solution and then mecamylamine was applied for 1 min in 30 mM K+ external solution. B: cumulative probability plot for amplitude of record shown in A. Circle and triangle indicate the record in the absence and presence of 10-5 M mecamylamine, respectively. C: effect of 10-5 M mecamylamine on amplitude and frequency of spontaneous transient inward current. All data were normalized to the respective control. Data were shown as mean±S.E.M. (n=3). *; P<0.05, **; P<0.01 vs. individual control in the absence of mecamylamine. D: effect of mecamylamine on activation and inactivation kinetics of transient inward current. a: averaged current traces recorded in the absence and presence of 10-5 M mecamylamine shown in A. Activation and inactivation kinetics were fitted with single exponential function, respectively. b: time constants of rising and decaying kinetics of averaged traces in the absence and presence of 10-5 M mecamylamine.

0

5

10

15

20

25

Tim

e co

nsta

nts

(ms)

A

100

pA

200 ms

Mecamylamine10–5 M

Control (30 mM K+)

After control

B

Peak amplitude (pA)0

0

1

100 200 300

0.5

Control

Mecamylamine

400

20 p

A

20 ms

Control

C

Mecamylamine

Da b

0

0.4

0.8

1.2

Nor

mal

ized

val

ue

Amplitude Frequency

Control

∗

∗

Mecamylamine3

∗∗

Control Mecamylamine

Cum

ulat

ive

prob

abilit

y

Rise τDecay τ

∗

23

kinetics. Both activation and inactivation were well fitted with single exponential

functions. Rising and decaying time constants were respectively 1.28±0.17 ms

and 17.17±2.95 ms in control, and 0.63±0.06 ms and 7.41±0.57 ms in the

presence of 10-5 M mecamylamine. These results suggest that the transient

inward current recorded in 30 mM K+ external solution is cholinergic fast EPSC.

2.2 Potentiation of EPSCs by BK

When neurons were pretreated with 10-8 M BK for 20 sec in 5 mM K+ external

solution and then BK 10-8 M was applied for 1 min in 30 mM K+ external solution,

BK potentiated the EPSC amplitude recorded in 30 mM K+ external solution to

1.37±0.2 times of control and its frequency to 2.04±0.4 times (Fig.11A and B).

BK shifted the cumulative distribution of inter-event interval to the left and that of

amplitude to the right (Fig.11C). The differences were significant in

Kolmogorov-Smirnov Test (p<0.0001). Pretreatment of BK for 60 sec gave

similar effects on the EPSCs (data not shown). At 10-7 M BK, potentiation of

EPSC amplitude was similar to that at 10-8 M BK. However, its frequency was a

little more increased (Fig.11B). In the presence of BK, both activation and

activation were well fitted with single exponential function. BK did not affect the

activation and inactivation kinetics of EPSC (Fig.11D). Rising and decaying time

constants were respectively 0.79±0.19 ms and 14.07±0.85 ms in control, and

0.82±0.19 ms and 15.84±0.81 ms in the presence of BK. [Hyp3]-bradykinin, a

bradykinin B2 receptor agonist had similar effects on EPSCs at 10-6 M (Fig.12).

In the presence of [Hyp3] -bradykinin, relative mean EPSC amplitude and its

frequency were 1.49±0.36 and 2.16±0.44 times of control. [Hyp3]-bradykinin

also did not affect the activation and inactivation kinetics of EPSC. On the other

24

Fig.11. Effects of BK on EPSCs in 30 mM K+ external solution

A: representative record showing the effect of 10-8 M BK on EPSCs. B: concentration- dependent potentiation by BK of EPSC amplitude and its frequency. All data were normalized to the respective control. C: cumulative probability plots for inter-event interval and amplitude of records shown in A. D: activation and inactivation kinetics of averaged EPSCs in the absence and presence of BK. a: averaged EPSCs in the absence and presence of 10-8 M BK shown in A. Activation and inactivation kinetics were fitted with single exponential function, respectively. b: superimposition of two currents shown in a. Averaged EPSC in the absence of BK was normalized to the peak amplitude of EPSC in the presence of BK. Note that two current traces were completely superimposed. c: time constants of rising and decaying kinetics of averaged traces in the absence and presences of BK. Data were shown as mean±S.E.M. (n=3~4). *; P<0.05 vs. individual control in the absence of BK.

BK 10-7 M0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Nor

mal

ized

Val

ue

Frequency Amplitude

Control BK10-8 M

∗

∗

∗

∗

A

B C

400 ms

50 p

A

BK 10–8 M

Control (30 mM K+)

After control

Control

BK 10-8 M

D

20 ms

20 p

A

BK 10-8 M

Control ba

Inter-event interval 0

0

1

2000 4000

0.5

Cum

ulat

ive

prob

abilit

y

6000Peak amplitude (pA)

00

1

100 200

0.5

c

0

6

12

18

Tim

e co

nsta

nt (m

s)

Control BK 10-8 M

Rise τ Decay τ Control

BK 10-8 M

25

Fig.12. Effects of bradykinin B2 receptor agonist on EPSCs in 30 mM K+ external

solution

A: representative record showing the effect of 10-6 M [Hyp3]-bradykinin on EPSCs. B: effects of 10-6 M [Hyp3]-bradykinin on amplitude and frequency of EPSCs. The data of EPSC amplitude and frequency were normalized to the respective control. C: activation and inactivation kinetics of EPSCs in the absence and presences of 10-6 M [Hyp3]-bradykinin. a: averaged current traces recorded in the absence and presence of 10-6 M [Hyp3]-bradykinin shown in A. Activation and inactivation kinetics were fitted with single exponential function, respectively. b: time constants of rising and decaying kinetics of averaged traces in the absence and presence of 10-6 M [Hyp3]-bradykinin. All data were shown as mean±S.E.M. (n=3). *; P<0.05 vs. individual control in the absence of [Hyp3]-bradykinin.

400 ms

20 p

A

[Hyp3]-bradykinin 10–6 M

Control (30 mM K+)

After control

A

B C

20 p

A

ba

[Hyp3]-bradykinin

Control

20 ms0

1

2

3

Nor

mal

ized

val

ue

Amplitude Frequency

Control

∗

∗

[Hyp3]-BK 0

6

12

18

24

Tim

e co

nsta

nt (m

s)

Control [Hyp3]-BK 8

Rise ττττ Decay ττττ

26

Fig.13. Effects of bradykinin B2 receptor antagonist on EPSCs potentiated by BK in 30 mM K+ external solution

A: representative record showing the effect of 10-6 M HOE 140 on the potentiation of EPSCs by BK. B: normalized EPSC amplitude and its frequency in the absence or presence of BK and HOE 140. Data were shown as mean±S.E.M. (n=2).

hand, the potentiation of EPSCs by BK was abolished or somewhat suppressed

by 10-6 M HOE 140, a bradykinin B2 receptor antagonist (Fig.13). Relative mean

EPSC amplitude and its frequency in the presence of HOE 140 were 0.59±

0.19 times and 0.96±0.1 times of control, respectively. These values were not

significantly different from the control in the absence of BK and HOE 140.

0

0.2

0.4

0.6

0.8

1.0

1.2

Nor

mal

ized

val

ue

Amplitude Frequency

Control

A

B

400 ms 20 p

A

BK 10-8 M + HOE 140 10–6 M

Control (30 mM K+)

After control

BK + HOE 140

27

3 Discussion

In this study, I revealed that BK additively enhanced muscarinic responses

and potentiated INic in rat PTG neurons. BK also potentiated the amplitude of

spontaneous transient inward currents in 30 mM K+ external solution and

increased its frequency. Since increasing in external K+ concentration increased

only the frequency of spontaneous transient inward currents and Ca2+ blocker

inhibited this transient current without affecting its kinetics, this current was

considered to be EPSC. This EPSC must be nicotinic because mecamylamine

inhibited it at the considerable concentration. The effects of BK on airway

nerves are thought to be mediated by bradykinin B2 receptors.33) In this study,

Effects of BK on INic and EPSC were antagonized by HOE 140 and mimicked by

[Hyp3]-bradykinin, indicating the contribution of bradykinin B2 receptor.

BK-induced increase in EPSC frequency was much stronger than the

potentiation of its amplitude. Therefore, it is likely that bradykinin B2 receptors

modulate cholinergic transmission at both pre- and post-synaptic site.

Bradykinin B2 receptor has been reported to signal via the heterotrimeric

G-proteins of the Gi/o and Gq/11 families, to activate a number of targets including

PLC and PLD.30,34~37) Pertussis toxin inhibited the BK-induced potentiation of INic

(Fig.6). However, the BK-induced slow inward current or the inhibition of IK(M)

was not affected by pertussis toxin as previously reported.6) In addition, there

was no correlation between the amplitude of the 10-8 M BK-induced inward

current and the strength of the BK-induced potentiation of INic (Fig.6). Therefore,

the bradykinin B2 receptors in PTG neurons may activate dual signal

transduction pathways, pertussis toxin-sensitive and insensitive pathways. Such

28

multiple activation of different G proteins via bradykinin B2 receptor has been

reported in non-neuronal cells.27,38)

The significance of the multiple pathways in neuronal cells is unclear at

present. However, it should be pointed out that the potentiation of nicotinic

currents and the induction of slow inward currents were not always observed in

the same neuron. The neurons in which BK induced both the inhibition of IK(M)

and the potentiation of INic might play some special role in pathological

conditions.

In superior cervical ganglion neurons39) and N1E-115 neuroblastoma cells,40)

PLC inhibitors inhibit the BK-induced inhibition of IK(M). This effect was also

observed in the present study. Therefore, it was suggested that PLC is involved

in both the potentiation of INic and the inhibition of IK(M).

The effect of BK on EPSC frequency was strong. Therefore, there is no doubt

about the physiological significance of this phenomenon. On the other hand, the

potentiation of INic by 10-8 M BK was only 35%. Even at 10-7 M, BK potentiated

EPSC amplitude by only 38.5%. At present, it is difficult to determine whether

this potentiation is physiologically significant. However, the potentiation was

statistically significant. In addition, the potency of BK-induced potentiation of

EPSCs was almost the same as that of INic. Therefore, it is obvious that BK

potentiates nicotinic EPSCs at this potency. To consider the physiological role of

this potentiation, it is necessary to take into consideration the distribution of

synapses because it is a major factor that determines how synaptic inputs are

integrated in the postsynaptic neuron. Recent electron microscopic study has

revealed that the majority of synapses on PTG are axo-dendritic.41) This should

result in a high filter function of the ganglion, and indicates that airway

29

parasympathetic ganglia are not a simple relay station but the integrative center

of preganglionic input because excitatory postsynaptic potentials generated at

the dendrite reduce its amplitude in accordance with the cable properties of the

dendrite. Previous studies have shown that less than 50% of preganglionic

impulses reaching the airway ganglia result in the generation of an action

potential in the postganglionic neurons.42~44) Therefore, the potentiation of INic

would increase the probability of generating the action potential. This effect

must be supported by the BK-induced inhibition of IK(M)6) via the increase in

membrane resistance. Effect of BK on muscarinic response was small. However,

muscarinic increase in membrane resistance via inhibition of IK(M) might also

support the excitatory effect of BK at higher EPSC frequency.

Previously, it was reported that atropine did not affect BK-induced contraction

of tracheal smooth muscle in the guinea pig.45) However, ACh release from the

postganglionic nerve terminal in normal conditions is regulated in the inhibitory

direction by M2 muscarinic ACh receptors at the nerve terminals.46) This

negative auto regulation becomes dysfunctional due to airway inflammation.47,

48) Therefore, an increase in the excitability of PTG neurons would result in an

increase in ACh release from the postganglionic nerve terminals under

pathological conditions. In fact, normal subjects do not show any changes in

pulmonary function, even after high doses of BK are inhaled. However,

asthmatic subjects respond to BK inhalation dose-dependently.49,50) Airway

inflammation markedly enhances the airway reactivity of asthmatic subjects to

BK.51)

It has been reported that exogenously applied BK inhibited ACh release from

parasympathetic nerves by electrical stimulation of the isolated tracheal

30

preparation.52) However, it is also a fact that atropine significantly inhibits

BK-induced bronchoconstriction or reflex bronchospasm in normal guinea-pigs,

53~55) normal and allergic sheep56) and actively sensitized rats.57) In addition,

hexamethonium inhibits BK-induced increase in tracheal ciliary beat frequency,

suggesting that the stimulation of ciliary beat frequency by BK acts through

parasympathetic pathways.58) In the study reported by Fabiani et al.,52) electrical

stimulation excites not only cholinergic nerves but also other neuronal and

non-neuronal cells. Fabiani et al. themselves also showed in their report that α1

adrenergic receptor agonist inhibited electrical stimulation-induced ACh release.

BK may stimulate noradrenergic transmission.59,60) In addition, BK application to

the organ bath affects both bradykinin B1 and B2 receptors in many kinds of

cells. Chemical mediators must be released from some of them. However, the

authors did not take these nonselective effects of BK into consideration.

Therefore, the relation between the BK-induced potentiation of nicotinic

transmission in PTG neurons and the ACh release from postganglionic

parasympathetic nerves should be studied carefully in the future.

In conclusion, I demonstrated here that BK possibly potentiates

neurotransmission at the PTG via the potentiation of INic and increase in EPSC

frequency (Fig.14). This may aggravate pathological conditions of the lower

airway via enhanced ACh release from the postganglionic nerve terminal. The

present results suggest a new mechanism underlying the BK-induced

hyperreactivity of the vagal nerve in the airway.

31

Fig.14. A schematic diagram of the signaling pathways that BK possibly potentiates

neurotransmission at the PTG via the potentiation of INic and increase in EPSC frequency.

PTG neuron

Preganglionic nerve terminal

ACh

Gααααq/11

PLC

(-)

ERCa2+

(-)

BK

M type K+

channels

Na+

Ca2+

K+

ACh

ACh

B2R

Giβγβγβγβγ

PLC

Gq/11

M1

IP3

nAChR

M type K+

channels

BK

B2R

(+)

(+)

32

Part II Applied study

33

1 Effect of suplatast in single sensory and PTG neurons

Suplatast (Fig.15) is a selective Th2 cytokine inhibitor61) that suppresses

synthesis of Interleukin-4 (IL-4) and IL-5 in vitro62~64) and allergen-induced

increases in peritoneal eosinophils in vivo in mice.65) As shown in Fig.16, IL-4 is

Fig.15. Chemical structure of suplatast tosilate (molecular weight 499.65)

pivotal in the pathogenesis of allergic disorders through its wide range of effects

including immunoglobulin-E (IgE) production and the development of mast

cells.66) Eosinophils are generally seen as a particularly harmful element in the

allergic inflammation. IL-5 seems to be the primary cytokine involved in vivo in

the production, differentiation, maturation and activation of the eosinophils.67)

IL-5-/- mice have decreased numbers of circulating eosinophils and fail to mount

a normal eosinophilic response to parasitic infections or to ovalbumin

challenge.68.69) Therefore, it is expected that suppression of IL-4 and IL-5

synthesis reduce the level of chemical mediators released from mast cells and

eosinophils.

It has been suggested that histamine, serotonin and tryptase released from

mast cell and eosinophil granule-derived cationic proteins such as eosinophil

cationic protein (ECP) activate or sensitize the sensory C-fibers (Fig.17).70~74) In

addition, NGF released from mast cell increases expression of substance P

CH3 SO3SCH3

CH3

CH2CH2CONH OCH2CHCH2OCH2CH3

OH

+· -

34

Fig.16. Molecular and cellular control of the major atopic diseases

Th2 (TH2) cells play a central role in orchestrating allergic inflammation that results in allergic syndromes. Following activation by antigen, Th2 cells produce the IL-4 required for B-cell maturation and IgE synthesis; the IL-3, IL-5, and GM-CSF required for eosinophil growth and differentiation; and IL-3, IL-4, and IL-9 required for mast cell development. IgE produced by

B-cells is captured at the cell surface by Fcε receptor I (Fcε RI) present on mast cells and eosinophils. Cross-linking of this receptor during subsequent encounter with antigen stimulates release of a variety of toxic products that together elicit atopic diseases. IL-4 and IL-13 may also elicit atopic diseases, especially asthma, through more direct effects on lung tissues. AHR,

airway hyperresponsiveness; FcεRI, high affinity Fc receptor for IgE; GM-CSF, granulocyte-macrophage colony-stimulating factor. From Foster et al. (2002).66)

(SP) in airway sensory neurons and change phenotype of vagal sensory

neurons.75) Therefore, suppression of the levels of such mediators might inhibit

cough response.

We recently found that suplatast inhibited the codeine-resistant coughs in

bronchitis model guinea pigs exposed to SO2 gas and the citric acid-induced

35

Fig.17. Interactions between inflammatory-cell products and airway nerves lead to

changes in a number of aspects of neural function.

(a) The eosinophil derived and mast-cell-derived neurotrophin NGF upregulates expression of the preprotachykinin gene in the nodose ganglia. Inflammatory-cell products also lead to central changes in the vagal nucleus. (b) Mast-cell products, including serotonin, histamine, tryptase and eosinophil proteins, can activate and sensitize airway sensory neurons. (c) Eosinophil MBP inhibits the function of muscarinic M2 receptors. Mast cell and eosinophil products may also influence muscarinic M2 receptor expression. CNS, central nervous system; ECP, eosinophil cationic protein; MBP, major basic protein; NGF, nerve growth factor. From Curran et al. (2002).81)

coughs in guinea pigs pretreated with ACE inhibitor (Fig.18).10) Also in clinic, it

has been reported that suplatast improved the cough score and the cough

threshold for capsaicin in patients with cough variant asthma (CVA), without

significant side effects.11) Moreover suplatast has been reported to be effective

36

in treating coughs persisting for more than two weeks,12) and allergic

eosinophilic airway inflammation.76) However, antitussive effect of suplatast

become significant within one week after the beginning of oral treatment,

whereas it need more than four weeks to become significant for treating

eosinophilic airway inflammation. Therefore, it is unknown whether the

antiallergic effects of suplatast contribute to mechanisms of its antitussive

effects.

In the animal models we used for studying the effect of suplatast, coughs

were resistant to codeine but inhibited by Bakumondo-to,77,78) which is effective

for treatment of intractable coughs that are resistant to treatment with other

antitussives.79,80) Interestingly, OP-D, an active constituent of Bakumondo-to,

inhibited the excitability of PTG neurons.7)

In order to elucidate the airway nervous system, in particular, PTG neurons

as a new target for new antitussives, it seems important to determine effect of

suplatast on PTG neurons, since this drug has an antitussive effect on

intractable cough in humans and in animal models.

Therefore, in this part, I studied effects of suplatast in PTG neurons,

especially on BK potentiation of nicotinic responses and EPSC in single PTG

neurons of rats. To compare the efficacy of suplatast, I also studied effects of

suplatast in sensory neurons.

37

Fig.18. Effects of suplatast and codeine on coughs caused by mechanical and

chemical stimulation of trachea in guinea pigs.

A: effect of suplatast and codeine on coughs caused by mechanical stimulation of trachea in SO2-exposed guinea pigs. In SO2-exposed guinea pigs, codeine did not suppress coughs caused by mechanical stimulation of trachea but suplatast significantly suppressed the coughs.

∗ ; P<0.05 vs corresponding saline group. B: effect of suplatast and codeine on coughs augmented by pretreatment with enalapril. Cough response was produced by inhalation of 0.5% citric acid solution. Enalapril was subcutaneously injected at 1 mg/kg before 30 min of challenge with 0.1 M citric acid solution. Suplatast significantly suppressed the coughs augmented by

enalapril, whereas codeine did not. ∗ ; P<0.05 vs. control group, #; P<0.05 vs. saline group.

∗ #

Saline Codeine SuplatastControl 1 mg/kg 10 mg/kg

Enalapril-pretreatment

0

2

4

6

8

10

Num

ber o

f cou

gh

A

B

CodeineSaline 1 mg/kg

Suplatast10 mg/kg

0

20

40

60

80

100 (%

of p

re-a

dmin

istra

tion)

C

ough

resp

onse

SO2-expossure

Codeine1 mg/kg

Suplatast 10 mg/kg

Saline

∗ ∗ ∗

38

1.1 Effect of suplatast in sensory neurons

We already revealed that suplatast inhibited the sensory afferent discharges

caused by the injection of BK or 4-aminopyridine (4-AP) into the closed artery of

the lower airway.10) Therefore, I studied the effects of suplatast on the sensory

neurons. When suplatast was applied at 10-4 M to the trigeminal ganglion (TG)

neurons for 10 sec to 5 min, it did not induce any responses at variety of

membrane potentials from 0 to -80 mV (n=5, data not shown). Therefore, I next

studied the effects on the C-fiber receptor responses, because airway

inflammation may underlie the chronic coughs. Transient receptor potential

vanilloid type 1 (TRPV1) channels predominantly distribute in sensory C-fibers

and its origin of small sensory neurons. TRPV1 is a ligand-gated cation channel

that is activated by heat, acid and capsaicin, a principal ingredient in hot

peppers, and possibly works as a polymodal molecular detector. TRPV1 is

activated by various endogenous lipids, such as anandamide, N-arachidonoyl-

dopamine, and various metabolic products of lipoxygenases. Its activity is

constitutively inhibited by PIP2 and modulated by various kinases such as

protein kinase A and C. BK potentiates TRPV1 receptor-mediated response via

multiple pathways as shown in Fig.19. Therefore, I studied the effects of

suplatast on capsaicin, acid and BK-induced responses at first.

Capsaicin concentration-dependently induced inward currents (Icap) in small

diameter TG neurons (< 30 µm) (Fig.20) at a VH of -60 mV. EC50 and the Hill

coefficient were 1.42x10-6 M and 0.9, respectively (n=3 to 6). When neurons

were pretreated with 10-4 M suplatast for 30 sec and then the mixture of

suplatast and capsaicin was applied, suplatast did not affect on the Icap.

Switching the external pH from 7.4 to 6.0 induced rapid transient inward

39

Fig.19. A schematic diagram of the signaling pathways that activate and sensitize

TRPV1 in an airway sensory neuron

BK can indirectly activate the TRPV1 receptor via the bradykinin B2 receptor. B2 receptor couples to a G protein to activate PLA2 and liberate arachidonic acid (AA), which is then

metabolized by LO to HETEs or LTB4, which then activate TRPV1. HETEs or LTB4 may also be released from lymphocytes or epithelial cells and penetrate the cell to act on TRPV1. These pathways, as well as direct activation by noxious heat or protons, activate the channel, allowing Na+ and Ca2+ influx and generation of action potentials in these vagal sensory (afferent) fibers.

In addition to the activation of TRPV1, BK is capable of sensitizing TRPV1 via two possible pathways. First, PLC and PKC generated by activation of bradykinin B2 receptors, NGF TrkA

receptors and maybe CysLT receptors can phosphorylate and sensitize TRPV1. Second, activation of PLC removes constitutive inhibition of TRPV1 by PIP2. In the sensitized state the proton and heat sensitivity of the channel may be increased such that physiological pH and body temperature can activate the channel and allow Na+ and Ca2+ influx. When excited — e.g.

via TRPV1 — action potentials are generated and propagated to the central nervous system, where transmitters are released from central terminals of the vagal afferents. Peripheral terminal of afferent fibers also releases tachykinins such as substance P (SP) when activated, which induce contraction of airway smooth muscle. PLA2, phospholipase A2; LO, lipoxygenase; HETE, hydroxyeicosatetraenoic acid; LTB4, leukotriene B4; PLC, phospholipase C; PKC, protein kinase C; NGF, nerve growth factor; TrkA, tropomyosin-receptor kinase A; CysLT, cysteinyl leukotrienes; PIP2, phosphatidylinositol-(4,5)-biphosphate. From Hwang, et al. (2002).82)

TRPV1TRPV1TRPV1TRPV1 TRPV1TRPV1TRPV1TRPV1

40

Table 1 Summary of effects of selected ionotropic and metabotropic activators on

guinea pig vagal afferent nerve subtypes

This summary pertains only to presumed direct activation of afferent nerves. Many chemicals can indirectly lead to afferent nerve activation secondary to events such bronchial smooth muscle contraction or vascular events. ASIC, acid sensing ion channel; RAR, rapidly adapting stretch receptor; SAR, slowly adapting stretch receptor. From Lee, et al. (2004).83)

current (Iacid; Fig.21). When neurons were pretreated for 30 sec with 10-4 M

suplatast at pH 7.4 and then it was applied at pH 6.0, Iacid was also not

significantly affected. Relative Iacid amplitude in the presence of suplatast was

0.94±0.06 times of control.

Pretreatment of 10-9 M BK for 1 to 1.5 min and its following application with

capsaicin potentiated Icap by 1.52±0.11 times of control (P<0.01, Fig.22). When

neurons were pretreated with the mixture of 10-4 M suplatast and 10-9 M BK and

then they were applied together with capsaicin, suplatast did not affect on Icap

potentiated by BK. Relative Icap amplitude in the presence of BK and suplatast

was 1.52±0.17 times of the control recoded in the absence of BK and

suplatast.

Histamine is one of major factors that contribute to airway inflammation.

Ionotropic

Metabotropic

41

Fig.20. Capsaicin-induced currents and the effect of suplatast on it in rat sensory

neurons.

A: representative record of capsaicin-induced current (ICap) at a VH of –60 mV. Bars indicate the period of capsaicin application. Capsaicin was applied at 5 to 6 min interval. B: concentration-response relationship for ICap (n=3~6). Continuous line was drawn in accordance

with the formula (1) described in Materials and Methods. C: representative record showing the effect of 10-4 M suplatast on 3x10-7 M capsaicin-induced current. Bars above the trace indicate the period of drugs application. D: statistic analysis of the effect of suplatast on Icap. All data were normalized to the average of amplitude recorded before suplatast application and left bar indicates the Icap amplitude just before capsaicin application (n=3).

Histamine at 10-6 M potentiated Icap caused by capsaicin 10-7 M to 2.16±0.5

times of control (response of capsaicin alone) (P<0.05, Fig.23). Suplatast at 10-4

10-8 10-7 10-6 10-50

0.2

0.4

0.6

0.8

1.0

1.2

Rel

ativ

e I c

ap

Capsaicin (M)

B

3x10-6

A

5 sec

200

pA

Capsaicin 10-6 10-5 M

C Capsaicin 10-4 M

0

0.2

0.4

0.6

0.8

1.0

1.2

Rel

ativ

e I ca

p

D

Capsaicin + Suplatast

Suplatast

3x10-7 M

10 sec

50 p

A

42

Fig.21. Effects of suplatast on acid-induced current

A: representative record showing the effects of 10-4 M suplatast on acid-induced current (Iacid) at a VH of –60 mV. Grey bars and hatched column above the traces indicate the period of application of external solution at pH 6 and suplatast, respectively. B: statistic analysis of the effects of suplatast on Iacid. Data were normalized to the average of Iacid amplitude recorded before suplatast application (n=4).

M had no effect on histamine potentiation of Icap. Even when suplatast was

pretreated for 5 min, it had no effect on the Icap potentiated by histamine.

From the results described above, it was suggested that suplatast may not

affect TRPV1 receptor function. Therefore, I further studied the effects of

0

0.2

0.4

0.6

0.8

1.0

Rel

ativ

e I ac

id

Acid + Suplatast 10-4 M

A

B

1 sec

200

pA

Suplatast 10 –4 M

pH 6

43

Fig.22. Effects of suplatast on the potentiation of capsaicin-induced current by BK

A: representative record showing the effects of 10-4 M suplatast on 3x10-7 M capsaicin-induced Icap in the presence of 10-9 M BK at a VH of –60 mV. Bars indicate the period of drug application. B: statistic analysis of the effect of suplatast on the potentiation of capsaicin-induced current by BK. Data were normalized to the average of current amplitude recorded before drug application (n=4). ∗ and ∗∗ ; P<0.05 and P<0.01 vs. Icap in the absence of BK and suplatast, respectively.

suplatast on the other receptors capable of inducing generator potentials in

cough-associated afferent terminals in the airways. 5-HT3 and P2X receptors

have been found in airway sensory nerve terminals.84~86) 5-HT is a major

component of inflammatory chemical milieu. In this study, 5-HT concentration-

A

B

0

0.5

1.0

1.5

Rel

ativ

e I ca

p

Capsaicin 3x10-7 M

+ BK and Suplatast+ BK

∗∗ ∗

BK 10-9 M Capsaicin 3x10-7 M

10 sec

50 p

A

Suplatast 10-4 M

44

Fig.23. Effects of suplatast on the potentiation of capsaicin-induced current by

histamine

A: representative record showing the potentiation of Icap by 10-6 M histamine and the effect of 10-4 M suplatast on the potentiation of Icap by histamine at a VH of –60 mV. Bars indicate the period of drug application. B: statistic analysis of the effect of histamine on Icap and that of suplatast on the potentiation of Icap by histamine. Data were normalized to the average of current

amplitude recorded before drug application (n=3). ∗ ; P<0.05 vs. Icap in the absence of histamine and suplatast.

dependently induced inward currents (I5-HT) at a VH of –60 mV and its kinetics

was rapid at 10-4 M, suggesting the currents via 5-HT3 receptors (Fig.24). When

A

B

0

1

2

3

Rel

ativ

e I ca

p

Capsaicin 10-7 M + His and Suplatast+ His

∗ ∗

Suplatast 10-4 MHistamine 10-6 M

10 sec

100

pA

Capsaicin 10-7 M

45

Fig.24. Effects of suplatast on 5-HT-induced current

A: representative record showing 5-HT-induced current (I5-HT) at a VH of –60 mV. Bars indicate the period of 5-HT application. B: concentration-response relationship for I5-HT (n=3~6). Continuous line was drawn in accordance with the formula (1) described in Materials and Methods. C: representative record showing the effects of 10-4 M suplatast on I5-HT. Hatched column indicates the period of suplatast application. D: statistic analysis of the effect of suplatast on I5-HT. Data were normalized to the average of current amplitude recorded before drug application (n=4).

neurons were pretreated with 10-4 M suplatast for 30 sec and then the mixture

of suplatast and 10-4 M 5-HT was applied, I5-HT was not affected (Fig. 24C and

10-7 10-6 10-5 10-4 10-30

70

140

210

280

350

I 5-H

T (pA

)

5-HT (M)

10-5 10-6

B

50 p

A

2 sec

5-HT 10-4 M

A

C D

0

0.2

0.4

0.6

0.8

1.0

1.2

Rel

ativ

e I 5-

HT

5-HT 10-4 M + Suplatast

Suplatast 10–4 M

10-4 M 5-HT

46

Fig.25. Effects of suplatast on ATP-induced current

A: representative record showing the effects of 10-4 M suplatast on 10-4 M ATP-induced current (IATP) at a VH of –60 mV. Bars indicate the periods of drug application. B: statistic analysis of the effect of suplatast on IATP. Data were normalized to the average of current amplitude recorded before drug application (n=3).

D). Application of ATP at 10-4 M induced rapid inward currents (IATP) at a VH of

–60 mV. When neurons were pretreated with 10-4 M suplatast for 30 sec and

then the mixture of suplatast and ATP was applied, IATP was not affected

(Fig.25).

A

B

0

0.2

0.4

0.6

0.8

1.0

Rel

ativ

e I A

TP

ATP 10-4 M + Suplatast

ATP 10-4 M

Suplatast 10 –4 M

2 sec

100

pA

47

1.2 Inhibition of nicotinic responses by suplatast in PTG neurons

1.2.1 Effect of suplatast in PTG neurons

As described in previous session, it was suggested that suplatast might have

no direct effect on the sensory neurons. Therefore, I further studied the effect of

suplatast on PTG neurons, because OP-D, an active constituent of

Bakumondo-to which has been used for treating chronic coughs, opens K+

current and hyperpolarizes membrane potential, reducing the cell excitability in

PTG neurons.

Unlike with OP-D, suplatast did not induce any current at various membrane

potentials even at 10 -3 M (data not shown). In addition, 10 -4 M suplatast did not

inhibit BK-induced depolarization (Fig.26A). Treatment of neurons with 10-4 M

suplatast for 20 hr also did not affect the BK-induced depolarization. Amplitude

of BK-induced depolarization in neurons treated with suplatast was 7.9 ± 1.0 mV.

This value was compatible with that of 9.2 ± 2.7 mV in neurons not treated with

suplatast (Fig.26B). Further, I injected suplatast 50 mg/kg subcutaneously for 7

days. However, suplatast had no effect on BK-induced depolarization. Amplitude

of BK-induced depolarization in neurons dissociated from normal and

suplatast-injected rats were 8.1 ± 1.1 and 9.7 ± 2.1 mV, respectively (Fig.26C).

In PTG neurons, M1 and M2 muscarinic ACh receptors are located. They are

coupled with M-type K+ channels and high voltage activated (HVA) Ca2+

channels, respectively. Suplatast did not affect muscarinic depolarization/current

via M1 receptor (Fig.27A and B). Suplatast also did not affect or somewhat

reversed the inhibition of HVA Ca2+ current via M2 receptor (Fig.27C and D).

48

0

4

8

12

BK

-indu

ced

depo

lariz

atio

n (m

V)

0

4

8

12

BK

-indu

ced

depo

lariz

atio

n (m

V)

Fig.26. Effect of suplatast on BK-induced depolarization in rat PTG neurons

A: representative records showing no acute effect of 10-4 M suplatast on BK-induced depolarization without (a) or with (b) action potential generation. Bars upper traces indicate the period of drug application. B: effect of prolonged application of suplatast on BK induced depolarization. a: representative record showing BK-induced depolarization in neurons treated with 10-4 M suplatast for 20 hr at 20 in vitro. b: statistic analysis of the effect of prolonged application of 10-4 M suplatast on BK-induced depolarization. C: effect of in vivo administration of suplatast for 7 days on BK-induced depolarization. a: representative record showing BK-induced depolarization in neuron dissociated from rat subcutaneously administered suplatast (50 mg/kg/day) for 7 days. b: statistic analysis of the effect of in vivo administration of suplatast on BK-induced depolarization.

10-4 M

5 m

V

BK 10-8 M

30 sec

A

B

C

BK 10-8 M 5

mV

30 sec

BK 10-8 M

a b

a

a

b

b

Suplatast

Control

Saline Suplatast

Suplatast

49

Fig.27. Effects of suplatast on ACh-induced muscarinic responses in rat PTG neurons

A: representative record showing effect of 10-4 M suplatast on 3x10-7 M ACh-induced depolarization. Bars upper traces indicate the period of drug application. B: representative record showing effect of 10-4 M suplatast on 3x10-7 M ACh-induced pseudo slow inward current at a VH of –40 mV. C: representative record showing the effect of 10-4 M suplatast on 3x10-7 M ACh-induced inhibition of HVA ICa evoked by a depolarizing step from a VH of –50 mV to +20 mV.

D: statistic analysis of effect of 10-4 M suplatast on 3x10-7 M ACh-induced inhibition of HVA ICa.

10-4 M 10-4 M

50 p

A

3x10-7 M

30 sec 30 sec

A Suplatast

ACh Suplatast

3x10-7 MACh

B

C D

50 p

A

30 ms

0

10

20

30

40

% In

hibi

tion

of I H

VA C

a2+

ACh + Suplatast

ACh Control ACh 3x10-7 M

+ Suplatast10-4 M

- 50 mV 20 mV

50

1.2.2 Inhibition of nicotinic response

Suplatast inhibited 10-5 M nicotine-induced INic at a VH of –50 mV (Fig. 28A).

When neurons were pretreated with 10-4 M suplatast for a variety of time up to

60 sec, magnitude of the inhibition of suplatast did not change, indicating no

time dependency of the effect (Fig.28B). Therefore, suplatast was pretreated for

10 sec before simultaneous application of suplatast and nicotine in the following

studies.

Inhibition of INic by suplatast was concentration dependent. The IC50 and the

Hill coefficient were 9.86x10-5 M and 0.69, respectively (Fig.29). Even when 50

mg/kg suplatast was administered subcutaneously for 7 days in vivo, the

potency of inhibition was not changed. The effect of suplatast was not

use-dependent (Fig.30).

In the presence of 10-4 M suplatast, nicotine concentration dependently

induced INic and the hump responses were observed after wash out of nicotine

at higher concentrations (Fig.31). Suplatast inhibited the maximum response of

INic without affecting EC50 and the Hill coefficient of nicotine response. EC50, the

Hill coefficient and the relative maximum response for nicotine were 1.15x10-5 M,

1.45 and 1.14 in the absence of suplatast and 1.24x10-5 M, 1.17 and 0.65 in the

presence of suplatast, respectively. The Lineweaver-Burk plot of the result

showed that two lines were intersected at abscissa axis (1/[nicotine]), indicating

the non-competitive inhibition. The percentage of inhibition by suplatast was

depended on the membrane potential (Fig.32).

The similar results were obtained from the PTG neurons in guinea pigs (data

not shown).

51

Fig.28. Inhibition of INic by suplatast and no time-dependency on it

A: representative record showing the inhibition of INic by 10-4 M suplatast. Bars upper traces

indicate the period of drug application. B: statistic analysis of the effect of pretreatment on the inhibition of 10-5 M nicotine (Nic)-induced peak inward current. All current amplitudes were normalized to the control induced by 10-5 M nicotine alone. Data were shown as mean±S.E.M. (n=3~9).

10 sec

100

pA

10-5 M

A

B

0 5 10 30 600

0.2

0.4

0.6

0.8

Rel

ativ

e I N

ic

Suplatast-pretreated time (sec)

Nic 10-4 M Suplatast

52

Fig.29. Concentration-dependent inhibition of INic by suplatast

A: representative record showing the concentration-dependent inhibition of INic by suplatast at a

VH of –50 mV. Bars upper traces indicate the period of drug application. B: concentration- inhibition relationship for suplatast on peak INic. Data were normalized to the average of peak

amplitude induced by 10-5 M nicotine (Nic) alone before suplatast application. Data were shown as mean ± S.E.M. (n=3~7). Continuous line was drawn in accordance with the formula (2) described in Materials and Method.

10-5 3x10-4 M 10-5

10 sec

10-4

A

B

10-6 10-5 10-4 10-3 10-20

0.2

0.4

0.6

0.8

1.0

Rel

ativ

e I N

ic

Suplatast (M)

Nic Suplatast

50 p

A

53

Fig.30. No use-dependency on suplatast-induced inhibition of INic

A: representative record showing the effect of continuous application of 10-4 M suplatast on INic induced by successive applications of nicotine. Bars upper traces indicate the period of drug application. B: time course of the inhibitory effect of suplatast on peak INic. Data were shown as mean±S.E.M. (n=3~7).

Nic 10-5 M

A

0 5 10 15 20 25

100

200

300

I Nic

(pA

)

Time (min)

B

10 sec

Suplatast 10-4 M

100

pA

54

Fig.31. Non-competitive inhibition of INic by suplatast

A: representative INic traces at various concentrations of nicotine in the presence or absence of 10-4 M suplatast at a VH of –50mV. Bars upper traces indicate the period of drug application. Note the hump currents observed when the mixture of suplatast and nicotine 10-4 M or 3x10-4 M was washed out with normal external solution. B: concentration-response relationship for nicotine-induced peak currents in the presence or absence of suplatast. All current amplitudes were normalized to the peak amplitude induced by 10-4 M nicotine in the absence of suplatast. Data were shown as mean ±S.E.M. (n=3～6). Continuous lines were drawn by according to the formula (1) described in Materials and Methods. C: Lineweaver-Burk plots of data shown in B. Note that the plots shared a common intercept on 1/[Nic] axis.

-6 -4 -2 0 2 4 6 8 10

1

2

3

4

5

1/R

elat

ive

I Nic

3x10-6 10-5 3x10-5 10-4 3x10-4 M

A

B C

10-6 10-5 10-4 10-30

0.2

0.4

0.6

0.8

1.0

1.2

　

Rel

ativ

e I N

ic

Nicotine (M) 1 / [Nic] (x104 M-1)

3x10-6 10-5 3x10-5 10-4 3x10-4 M

200

pA

10 sec

Nic

Nic

10-4 M Suplatast

Nic Nic + Suplatast

Nic Nic + Suplatast

200

pA

10 sec

55

Fig.32. Voltage-dependent inhibition by suplatast of INic

A: representative record showing the effect of suplatast on INic at different holding potential. B: current-voltage relationship for INic in the absence and presence of suplatast. Data were shown as mean ± S.E.M. (n=3～8). C: voltage-dependent inhibition of INic by suplatast. Percentage of inhibition caused by suplatast was plotted against the holding potentials.

10-5 M 10-4 M

A

B

VH – 39.5 mV

VH – 79.5 mV

50 p

A

10 sec

Nic

100

pA

10 sec

-80 -60 -40 -20 20

-600

-400

-200

100

Nic Nic + Suplatast

INic (pA)

VH (mV)

-80 -60 -40 -20 0 200

20

40

60

80

100

% Ih

ibiti

on o

f I N

ic

VH (mV)

∗∗∗ ∗ ∗

C

Suplatast

56

1.2.3 Single channel analysis

For single channel analysis of nicotinic response, I first used a concentration

of 10-5 M of nicotine close to the EC50 for nicotinic response (Fig.4). But this

concentration of nicotine was inappropriate for the single channel analysis,

because this concentration activated multiple nicotinic ACh receptor/channel

complexes simultaneously in outside-out mode of patch clamp (Fig.33).

Fig.33. Single nicotinic channel currents recorded by using outside-out mode of patch

clamp in rat PTG neuron

A: single-channel currents induced by 10-5 M and 3x10-6 M nicotine at a VH of –50 mV. Bars indicate the period of nicotine application. B: expanded traces showing 3x10-6 M nicotine-induced single-channel currents (iNic) at different holding potentials. Dashed lines represent closed state.

A VH – 50 mV

1 sec

5 pA

Nic 10-5 M

VH –32.8 mV

- 52.8 mV

- 72.8 mV

- 92.8 mV

50 ms

2 pA

Nic 3x10-6 M

B

57

Therefore, 3x10-6 M nicotine was used for single channel analysis in outside-out

mode.

Single-channel current (iNic) amplitude became larger at lower holding

potentials (Fig.33B). All-points amplitude histograms at each potential were

prepared by using the bin width of 0.6 ms (256 bins), which provides the finest

resolution. The distributions were well fitted with two or in some case with three

Gaussian functions. Since the largest and the other peaks in the distribution

represent the base and the opened current levels, respectively, single channel

current amplitude at each potential was determined as the difference between

the current amplitudes of two peaks laying side-by-side. In Fig.34D, the mean

single channel current amplitude at each potential was plotted against the

holding potential. The result was fitted with the linear function and the

conductance of 23.7 pS was obtained from the slope of straight line which

reversed at around -10 mV.

When membrane patch was pretreated with 10-4 M suplatast for 30 sec and

then nicotine and suplatast was simultaneously applied, the open duration of

the channel was obviously shortened and the flickering of channel open was

observed (Fig.35). Suplatast reduced the numbers of open events without

affecting the unitary current amplitude in the amplitude histogram. Nicotinic

single-channel conductance was 23.2±0.47 pS in the presence of suplatast

(n=4). Frequency of channel open and the open probability (NPo) were also

reduced by suplatast (Fig.36 and Table 2).

To investigate the open and close kinetics of nicotinic ACh receptor/channel

complex, open and closed durations were analyzed in patches in which the total

numbers of events exceeded 1000. Open duration was compiled in

58

Fig.34. Single-channel conductance for nicotine in PTG neurons

A. B. C: representative all point current amplitude histograms in the presence of 3x10-6 M nicotine at different holding potentials (black lines). The histograms were well fitted with 2 Gaussian functions (gray curves). D: single channel current-voltage relationship for nicotine. Single channel current amplitudes calculated from the all points current amplitude histograms were plotted against the holding potentials.

conventional histograms and fitted by simplex least square method using the

bin width of 0.06 ms (Fig.37A). The bin width was selected as an integer

multiple of the sampling rate (0.06 ms) to scale a linear histogram and correct

the binning and sampling promotion errors. The distribution of open duration for

Poi

nt-c

ount

s

0- 2 - 4- 6 - 8 0

8000

16000

32000

24000

Amplitude (pA)

Poi

nt-c

ount

s

0 - 2 - 4- 6- 8Amplitude (pA)

0

6000

12000

24000

18000

- 52.8 mV

- 72.8 mV VH – 92.8 mV

Poi

nt-c

ount

s

0 - 2 - 4 - 6 Amplitude (pA)

0

3000

6000

12000

15000

9000

A B

DC -120 -90 -60 -30 0

-4

-3

-2

-1

0

Ampi

ltude

(pA)

Holding potential (mV)

59

Fig.35. Effects of suplatast on nicotinic single channel current

A: representative record showing 3x10-6 M nicotine-induced single-channel currents in the absence and presence of 10-4 M suplatast at a VH of –50 mV. Note that suplatast caused flickering of channel open in right traces. B: representative all point current amplitude histograms showing the effect of suplatast. Note that suplatast reduced the height of second to fourth peaks but did not change the position of peaks in Gaussian distribution.

nicotine was well characterized by two open time constants (τO1 and τO2).

Suplatast shortened these two open time constants (Table 2). Since the closed

duration was distributed in a wide range of 0.3 ms to 58 ms, it was compiled in

A Nic 3x10-6 M Nic + Suplatast 10-4 M

B 100 ms

2 pA

0- 4- 6 - 8 - 2

Amplitude (pA)

6000

12000

18000

0

Nic

Nic+ Suplatast

Poi

nt-c

ount

s

60

Fig.36. Effect of suplatast on channel opening frequency and the open probability of

nicotinic channel in PTG neuron

A: determination of channel opening frequency. a: the frequency (f) of opening for each event is determined by the reciprocal of the open + closed time (T) since the last event. b: changes in nicotinic channel opening frequency along the time axis in the absence and presence of 10-4 M suplatast. B: changes in open probability (NPo) of nicotinic channel along the time axis in the absence and presence of 10-4 M suplatast. In this patch, NPo was reduced from 0.092±0.17 (n=3740) to 0.035±0.22 (n=3240).

Nic 3x10-6 M Nic + Suplatast 10-4 M

A

Open

Closed

T

0Time (sec)

105 15

0.6

0.4

0.2

020 25 30

1

0.8

B

0 Time (sec) 10 5 15 20 25

0.6

0.4

0.2

0

1

0.8

NP

o

Time (sec)10 5 15 20 25

8000

4000

0

16000

12000

Freq

uenc

y (H

z)

0 0Time (sec)

105 15 20 25 30

8000

4000

0

16000

12000

a

b

61

logarithmic histograms and fitted by maximum likelihood method using the bin

width of 0.06 ms (Fig.37B). In maximum likelihood method, data may be binned

into the variable width in a logarithmic histogram. The distribution of closed

duration for nicotine was well characterized by two closed time constants (τC1

and τC2). Suplatast prolonged these two closed time constants (Table 2).

Since the flickering of channel open was observed in the presence of

suplatast, burst analysis was further performed. Bursts were defined as groups

of openings separated by interburst interval. Interburst interval, i.e., the

minimum value of a closed duration which separates the burst was determined

as shown in Fig.38A. When the test interburst interval becomes identical to the

‘true’ interburst interval, and if the bursts are reasonably well-delineated, the

number of closings per “burst” will be relatively insensitive to further changes in

the test interburst interval.87) This point is the optimal interburst interval. In the

case of Fig.38A, this value was 64 ms.

Suplatast significantly reduced the open probability within bursts (Table 2).

Open duration in burst was compiled in conventional histograms and fitted by

simplex least square method using the bin width of 0.06 ms (Fig.38B). The

distribution for nicotine of open duration in burst was well characterized by two

open time constants (τBO1 and τ BO2). Suplatast prolonged τBO1, but significantly

shortened τBO2 (Table 2). On the other hand, closed duration in burst was

compiled in logarithmic histograms and fitted by maximum likelihood method

using the bin width of 0.06 ms (Fig.38C). The distribution for nicotine of closed

duration in burst was well characterized by two closed time constants (τΒC1 and

τΒC2). Suplatast prolonged τBC1 but significantly shortened τBC2 (Table 2).

62

Fig.37. Effects of suplatast on open and closed time of nicotinic channel

A: distributions of single-channel open time in the absence and presence of 10-4 M suplatast.

Each distribution was fitted with two exponential functions. Estimated open time constants τO1 and τO2 for 3x10-6 M nicotine were 1.09 ms and 2.31 ms and their proportions were 76 % and 24 %, respectively. In the presence of suplatast, these became 0.50 ms (41 %) and 1.06 ms (59 %), respectively. B: distributions of single-channel closed time in common logarithmic scale in the absence and presence of 10-4 M suplatast. Each distribution was fitted with two Gaussian

functions. Estimated closed time constants τC1 and τC2 (and their proportion) were 1.21 ms (31 %) and 14.1 ms (69 %), respectively. In the presence of suplatast, these became 1.54 ms (53 %) and 22.82 ms (47 %), respectively.

40

20

0

60

0Open time (ms)

42 6 8 10 12

Log Closed time (ms) 0-1-2 1 2 3

0

20

40

60

Num

ber o

f Eve

nts

Num

ber o

f Eve

nts

120 Open time (ms)

4 2 6 8 10

40

20

0

60

Nic 3x10-6 M Nic + Suplatast 10-4 M

Log Closed time (ms) 0 -1-2 1 2 3

0

20

40

60

B

A

63

Fig.38. Effects of suplatast on burst of nicotinic channel

A: representative analysis for determining the proper interburst interval. The point indicated by arrow is the optimal interburst interval. B: distributions of open time during burst in the absence and presence of 10-4 M suplatast. Each distribution was fitted by two exponential functions.

Estimated open time constants τΒO1 and τΒO2 (and their proportion) in burst for 3x10-6 M nicotine were 0.52 ms (67 %) and 4.28 ms (33 %), respectively. In the presence of suplatast, these became 0.49 ms (55 %) and 1.45 ms (45 %), respectively. C: distributions of closed time during burst in common logarithmic scale in the absence and presence of 10-4 M suplatast. Each

distribution was fitted by two Gaussian functions. Estimated closed time constants in burst τΒC1 and τΒC2 (and their proportion) for nicotine 3x10-6 M were 0.6 ms (38 %) and 42.7 ms (62 %), respectively. In the presence of suplatast, these became 0.99 ms (27 %) and 31.55 ms (74 %), respectively.

Duration of opening within burst (ms) 0 3 6 9 12 15

20

40

60

0Num

ber o

f Eve

nts

Duration of opening within burst (ms) 0 3 6 9 12 15

20

40

60

0

A

B Nic 3x10-6 M Nic + suplatast 10-4 M

0

10

20

30

0 -1 -2 -3 1 2 3Log Duration of closings within bursts (ms)

Num

ber o

f Eve

nts

0

10

20

30

0-1-2-3 1 2 3 Log Duration of closings within bursts (ms)

C

2000

3000

1000

4000

20 40 80 100 0 60Test interval (ms)

Num

ber o

f clo

sing

s

per b

urst

64

Table 2 Effects of suplatast on single channel properties

Data were presented as mean±S.E.M.

17.27±2.02 ∗ (47.9±5.60)

∗; P < 0.05

Conductance (pS)

Open probability

τC1 (proportion)

τC2 (proportion)

Closed time [ ms (%) ]

Closing duration within bursts [ ms (%) ]

τBC1 (proportion)

τBC2 (proportion)

Opening duration within bursts [ ms (%) ]

τBO1 (proportion)

τBO2 (proportion)

Open probability within bursts

Open time [ ms (%) ]

τO2 (proportion)

Nicotine 3x10-6 M

23.7±0.74

0.13±0.06

1.31±0.13 (34.28±2.27)

10.81±1.09 (65.72±2.27)

0.67±0.08 (32.98±5.76)

41.71±5.70 (67.02±5.76)

4.52±1.03 (25.05±7.58)

0.55±0.09 (74.95±7.58)

0.74±0.04

3.13±0.47 (21.4±5.86)

1.29±0.07 (78.6±5.86) τO1 (proportion)

16.54±3.64 ∗ (68.3±2.21)

1.87±0.25 ∗ (50.28±11.76)

1.02±0.02 ∗ (45.07±16.89)

0.77±0.16 ∗ (66.20±16.42)

Nicotine + Suplatast 10-4 M

23.2±0.47

0.09±0.03

2.0±0.27 (52.02±5.60)

1.21±0.31 (31.70±2.21)

0.75±0.24 (49.72±11.76)

0.54±0.04 ∗

65

2 Effects of suplatast on EPSCs in rat PTG neurons

As described in the previous session, suplatast inhibited INic in rat PTG

neurons, suggesting that suplatast may inhibit the vagal reflex at the PTG level.

However, two kinds of neuronal nicotinic ACh receptors have been known in

autonomic ganglion neurons.88) One is sensitive to alpha-bungarotoxin and the

other is insensitive. The former mainly presents at extra synapses and the later

at both synaptic and extra synaptic site.31) Therefore, I further studied whether

suplatast really inhibit synaptic transmission in PTG neurons.

2.1 Inhibition of EPSCs by suplatast

At first, EPSCs were recorded in the external solution containing 30 mM K+

for 1 min. After washing out of 30 mM K+ external solution with normal external

solution for 4 min, the neurons were pretreated for 30 sec with 10-4 M suplatast

dissolved in normal external solution and then the suplatast dissolved in 30 mM

K+ external solution was applied for 1 min. In this protocol, suplatast

concentration-dependently inhibited the EPSC amplitude and its frequency

(Fig.39). IC50s for the amplitude and frequency were 6.59x10-5 M and 1.09x10-5

M, respectively. Hill coefficients for them were 0.66 and 0.48, respectively.

Inhibition of the frequency was significantly potent against that of the amplitude

at 10-6, 3x10-6 and 10-5 M (Fig.39B).

At 10-4 M of suplatast, cumulative distribution for inter-event interval of

nicotine response was shifted to the right, whereas those for EPSC amplitude

were shifted to the left (data not shown). When EPSCs in the presence of

suplatast were averaged respectively, activation and inactivation of EPSCs were

66

Fig.39. Concentration dependent inhibition of EPSCs by suplatast

A: representative record showing EPSCs in 30 mM K+ external solution in the absence or presence of suplatast at a VH of –60 mV. B: concentration-inhibition relationship for suplatast on EPSC amplitude and its frequency. Continuous lines were drawn in accordance with the equation (2) described in Materials and Methods. Data were normalized to the individual control

recorded in the absence of suplatast (n=3~6). ∗; P<0.05 vs. EPSC amplitude at each concentration.

10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 10 -20

0.2

0.4

0.6

0.8

1.0

Rel

ativ

e va

lue

Suplatast (M)

∗∗

∗

50 p

A

500 ms

Suplatast 10-5 M

Suplatast 10-6 M

A

B

Control (30 mM K+)

After control

Amplitude

Frequency

67

Fig.40. Effects of suplatast 10-4 M on EPSCs

A: representative record showing the effect of 10-4 M suplatast on EPSCs in 30 mM K+ external solution at a VH of –60 mV. B: effect of 10-4 M suplatast on amplitude and frequency of EPSCs. All data were normalized to the respective control. Data were shown as mean±S.E.M. (n=3~6). *; P<0.05, **; P<0.01 vs. EPSC in the absence of 10-4 M suplatast. C: averaged EPSC traces recorded in the absence and presence of 10-4 M suplatast shown in A. EPSC activation were fitted with single and double exponential functions. D: time constants of rising and decaying kinetics of averaged EPSCs in the absence and presence of 10-4 M suplatast.

500 ms 50 p

A

Suplatast 10-4 M

20 ms

20 p

A

D

CControl (30 mM K+)

After control Suplatast

Control

B

A

0

0.2

0.4

0.6

0.8

1.0

1.2

Nor

mal

ized

val

ue

Frequency

∗∗∗

Control

Amplitude

Suplatast0

10

20

30

Tim

e co

nsta

nt (m

s) Rise τ Decay τ

Control Suplatast

68

well fitted with single and double exponential functions, respectively. Rising and

decaying time constants were respectively 1.92 ± 0.3 ms and 28.3 ± 6 ms in

control, and 1.75 ± 0.32 ms and 1.84 ± 0.34 ms and 10.84 ± 3.62 ms in the

presence of 10-4 M suplatast (Fig.40C and D). Suplatast did not affect the

activation kinetics of EPSC, but facilitated the EPSC decay, corresponding to

the result of single channel analysis.

2.2 Effects of suplatast on EPSCs potentiated by BK

As described in Part I, BK potentiated EPSC amplitude and its frequency to

136.8 ± 19.8 % and 203.5 ± 41.8 %, respectively. When neurons were

pretreated with 10-5 M suplatast for 20 sec and then with the mixture of BK 10-8

M and suplatast for 20 sec, EPSC amplitude in 30 mM K+ external solution

containing BK and suplatast was almost the same (101.98 ± 4.05 %) as that in

30 mM K+ external solution in the absence of these drugs. EPSC frequency was

slightly but significantly inhibited to 73.1 ± 8.9 % of control (Fig.41B). At 10-4 M

of suplatast, both the EPSC amplitude and the frequency were inhibited to 61.2

± 1.4 % and 41.2 ± 4.6 % of control, respectively. When all the data were

normalized to the value potentiated by 10-8 M BK, suplatast 10-5 M reduced the

EPSC amplitude to 74.5 ± 3.0 % and its frequency to 35.9 ± 4.4 % (Fig.41C). At

10-4 M, suplatast reduced the EPSC amplitude and its frequency to 44.8 ± 1.1 %

and 20.2 ± 2.2 %, respectively. These percentages of inhibition were similar to

those obtained in the absence of BK (Fig.39B).

69

Fig.41. Effects of suplatast on BK-induced potentiation of EPSCs

A: representative record showing the effect of suplatast on the potentiation of EPSCs by BK at a VH of –60 mV. B: inhibition by suplatast of EPSC amplitude and its frequency in the presence of BK. All data were normalized to the individual control recorded in 30 mM K+ external solution in the absence of any drug. Data were shown as mean±S.E.M. (n=3). C: inhibitor potency of suplatast on the EPSC amplitude and its frequency in the presence of BK. Data shown in B were normalized to the values in the presence of BK alone (right-hand neighbor set of the control in B).

A

BK 10-8 M + Suplatast 10-4 M

50 p

A

400 ms

Control (30 mM K+)

After control

0

0.2

0.4

0.6

0.8

1.0

1.2

Nor

mal

ized

val

ue

0

0.5

1.0

1.5

2.0

2.5

Nor

mal

ized

Val

ue

C

∗∗

∗∗ ∗∗ ∗∗

∗∗

∗

∗

B

∗

Suplatast

BK 10-8 M

Control 10-4 M

BK 10-8 M

10-5 M

Amplitude

Frequency

∗

Control Suplatast 10-4 M 10-5 M

70

3 Discussion

In Part II, I found that suplatast inhibited the cholinergic EPSCs in PTG

neurons via both presynaptic and postsynaptic mechanisms. On the other hand,

it may have no direct effect on various responses caused by capsaicin and

other chemicals in the sensory neurons, at least in nociceptive neurons.

Effects in sensory neurons

Previously we revealed that suplatast effectively depressed BK-induced

discharge of the vagal afferent nerves of the lower airway. Because BK-induced

discharges did not occur in capsaicin-treated guinea pigs, it has been

suggested that BK induces the discharges via activation of capsaicin-sensitive

fibers (probably C fibers).89) However, in the present study, suplatast did not

inhibit the currents induced by three major stimulants of nociceptors (capsaicin,

acid and BK, see Table 1) in dissociated sensory neurons. In addition, suplatast

had no effect on the currents induced by 5-HT, histamine or ATP. Therefore, it

was suggested that suplatast may have no direct effect on the sensory fibers, at

least the nociceptive fibers (C- and nociceptive Aδ-fibers).

Histamine potentiated the capsaicin-induced current (Fig.23). This histamine

concentration, 10-6 M, is comparative to the EC50s of 2x10-6 M for blocking the

leak K+ current and after hyperpolarization in ferret vagal afferents.90) Recently,

it was also suggested that histamine at 10-5 M induced Ca2+ influx in DRG

neurons via activation of TRPV1 receptor.91) In my study, histamine did not

induce any current at resting level. Therefore, histamine seemed to sensitize but

not activate the TRPV1 channel in sensory neurons. Since histamine is a major

71

chemical mediator for airway inflammation92,93) and capsaicin-sensitive fibers

may have roles for induction of chronic cough,94,95) further studies should be

continued as to whether histamine potentiation of capsaicin responses are

involved in augmentation of cough in airway inflammation.

Mast cells and sensory nerves appear to be closely associated with the

epithelium in the airway mucosa.96) Three major etiologies of chronic coughs,

cough variant asthma, eosinophilic bronchitis and atopic cough, are all

associated with eosinophilic airway inflammation, which is similar to that seen in

non-cough predominant asthma. However, evidence of activated mast cells and

increased concentrations of mast cell products appears to be confined to the

conditions associated with cough, suggesting a role for mast cell degranulation

in the superficial airway structures in the pathogenesis of cough. Since it has

been reported that suplatast suppressed the antigen-induced degranulation of

mesenteric mast cells and histamine release from peritoneal exudate cells of

rats,97) suplatast might indirectly inhibit sensitization of TRPV1 channel in

nociceptive fibers and coughing via reduction of histamine release.

Inhibition of EPSCs in PTG neurons

In PTG neurons, suplatast inhibited the amplitude of EPSCs (Fig.39 and 40).

The EPSCs appear to be cholinergic because mecamylamine inhibited them

(Fig.10). IC50 and the Hill coefficient for the inhibition of EPSC amplitude were

6.59x10-5 M and 0.66, respectively. On the other hand, IC50 and the Hill

coefficient for the inhibition of INic amplitude were 9.86x10-5 M and 0.69,

respectively. They were close each other. Therefore, it was suggested that

suplatast inhibits cholinergic synaptic transmission via the inhibition of

72

postsynaptic nicotinic ACh receptors. Similar inhibition of nicotinic response was

also observed in guinea pig PTG neurons.

Slightly higher concentrations were necessary to inhibit EPSC amplitude.

However, a high dose such as 300 mg/day is treated for treating chronic cough

in clinic. Consequently, plasma concentration (50 ng/ml) is also higher than

other medicines (for example 50 µg/day and 100 pg/ml in the case of

procaterol). In addition, suplatast at 10-4 M decreases the IL-4 production by

50 % in D10G4.1 cells, a Th2 clone.63) Recent study also indicated that

suplatast inhibited the production of cytokines including IL-4 and IL-5 with IC50s

of more than 10-4 M in both mouse splenocytes and human peripheral blood

mononuclear cells.64) Therefore, the concentrations required to inhibit EPSC

amplitude are considered to be acceptable.

Suplatast also facilitated the EPSC decay (Fig.40). This may be caused by

the channel block, because suplatast caused obvious flickering of nicotinic

channel opening. The results also suggested that the inhibition may be the open

channel block. Further, inhibition of INic was in noncompetitive and

voltage-dependent manners (Fig.31 and 32). The hump current was also

observed after wash out of nicotine at higher concentrations in the presence of

suplatast. These findings also support the idea described above (Fig.31).

The effect of suplatast did not depend on the pretreatment time (Fig.28) and

was not use-dependent (Fig.30). In addition, two straight lines in the

Lineweaver-Burk plot were crossed on the abscissa axis (Fig.31C). Therefore,

these results suggest the noncompetitive inhibition.

In addition, suplatast inhibited the EPSC amplitude potentiated by BK.

However, percentage of inhibition of EPSC amplitude normalized to the

73

amplitude potentiated by BK was similar to that obtained in the absence of BK

(Fig.39 and 41). Therefore, it was suggested that suplatast does not affect the

potentiation process of INic by BK.

Furthermore, suplatast reduced the EPSC frequency. Interestingly, the effect

on the EPSC frequency was observed at lower concentrations. IC50 for EPSC

frequency was about 10 times lower than that for EPSC amplitude (Fig.39).

However, the Hill coefficients for inhibition of EPSC amplitude and reduction of

EPSC frequency were different. Therefore, it seems unlikely that the reduction

of EPSC frequency may come from the inhibition of EPSC amplitude. The

reduction of EPSC frequency may work as a main effect of suplatast on the

function of PTG neurons.

As in the case of the EPSC amplitude, suplatast had little effect on the

facilitation of EPSC frequency by BK (Fig.41). However, it is noted that suplatast

at a lower concentration inhibited EPSC frequency in PTG neurons. Since

suplatast inhibits the compound 48/80-induced histamine release in mast cells

at the concentrations of 2x10-5 M and 2x10-4 M,97) the idea is plausible that

suplatast may affect the presynaptic terminals to inhibit the release of a

neurotransmitter in PTG neurons. Increase in intraterminal or intracellular Ca2+

play a crucial role in stimulation-release/secretion coupling. However, suplatast

did not affect on the HVA Ca2+ current inhibited by M2 muscarinic receptor

(Fig.27). Opening HVA Ca2+ channels are critical for transmitter release.98,99) On

the other hand, Ca2+ release from endoplasmic reticulum and following

capacitative Ca2+ influx via store-operated Ca2+ channels have critical roles for

exocytosis in mast cell.100,101) Therefore, some Ca2+-dependent process lying

the downstream might be a target of suplatast in nerve endings. Further studies

74

are necessary to understand the mechanism.

Suplatast and chronic cough

As described above, a relatively long time such as weeks after administration

is needed to cause antiallergic effect of splatast in humans. On the other hand,

a short time such as hours after dosing is enough to cause the antitussive effect

of suplatast. This fact means that antitussive mechanism of suplatast is different

from its antiallergic mechanisms. Suplatast showed inhibitory effect on

cholinergic responses and EPSCs in PTG neurons. The following has a merit to

discuss.

It has been reported that inhalation of capsaicin aerosols causes

dose-dependent increases in the number of coughs. Intra peritoneal application

of atropine shifts the dose-response curves to capsaicin to the right in a

dose-dependent fashion. On the other hand, carbachol aerosols shift the curve

to the left at the maximal concentration that does not cause

bronchoconstriction.102) Thus, it was suggested that the excess ACh release

from cholinergic neurons in pathological condition may sensitizes the cough

induction. At present, it is hard to conclude whether inhibition of PTG function

causes reduction of chronic cough. However, bronchodilators have been

recommended in clinic to treat the cough variant asthma, one of three major

causes of chronic cough.1) Hexamethonium, a ganglionic blocker, abolishes

vagally mediated bronchoconstriction but has no significant effect on

ACh-induced bronchoconstriction.103) In addition, hexamethonium inhibited

allergic cough.8) Therefore, inhibition of synaptic transmission in PTG neurons

might contribute to antitussive effect of suplatast in patients with cough variant

75

asthma.11) The fact that suplatast did not affect on the M2 receptor function in

PTG neuron also support the hypothesis.

In Japan, atopic cough and sinobronchial syndrome are other two causes of

chronic cough. Coughs with sinobronchial syndrome should not be reduced

aggressively by antitussives because it causes wet cough. On the other hand,

atopic cough is nonproductive dry cough and recommended to treat with

histamine H1 antagonists in the guideline for treatment of cough. At present, it is

unknown whether H1 receptor contributes the potentiation of capsaicin-induced

current. However, a H1 receptor blocker inhibits TRPV1 receptor mediated Ca2+

increase in DRG neurons.91) Functional H1 but not H2 receptor presents in

trigeminal ganglion neurons containing substance P or calcitonin gene-related

peptide.104) In addition, it has been reported that the cough induced by the

mechanical stimulation of tracheal bifurcation is resistant to the treatment by

codeine.105) But the codeine-resistant cough was reduced by H1 antagonists

(unpublished data). Interestingly, suplatast also inhibits this codeine-resistant

cough.10) Therefore, suplatast might be also effective for treating atopic cough.

Finally, I would like to propose a strategy for developing new antitussives,

describing pharmacological significance of the findings obtained from the

present study. The central dogma of pharmacological study of antitussives is

that cough is a biological reflex which is produced by activation of the cough

reflex arc consisting of the nervous systems. Therefore, it is very reasonable to

consider that the site of action of antitussives is on the neurons. In the present

study, I found that suplatast inhibited nicotinic responses in both pre- and

post-synaptic sites of PTG neurons. This means that suplatast inhibits the

excitability of PTG neurons. It has been reported that ophiopogonin-D, active

76

component of Bakumondo-to which is a Chinese herbal medicine and effective

to intractable coughs, inhibited the excitability of PTG neurons, although

inhibition mechanisms were different between the two substances.

On the other hand, suplatast had little or no effect on the responses in

sensory neurons studied. Therefore, it seems to be important to determine the

effect on PTG neurons, when antitussive effect and its mechanism of certain

compounds are investigated. Further, in this study, I succeeded in recording the

EPSCs in single PTG neurons attached with synaptic boutons. By using this

preparation, I found that suplatast had more potent inhibitory effect on the

frequency than the amplitude of EPSCs. Although this effect undoubtedly

contribute to reduction of apparent excitability of PTG neurons, this effect may

have more important pharmacological significance in depressing the activity of

the airway nervous systems. Suplatast is known to inhibit histamine releases

from the mast cells. Suplatast also inhibited the frequency of EPSCs in PTG

neurons, suggesting inhibition of releases of neurotransmitters. It is generally

known that release of chemical mediators and neurotransmitters has often

common mechanisms such as Ca2+ signals. Therefore, it seems to be possible

that suplatast may inhibit releases of neurotransmitters and chemical mediators

in the airway. Probably, this effect might contribute to inhibitory effect of

suplatast on BK- and 4-AP-induced discharges in the vagal afferent discharges

of the lower airway, since suplatast had little or no effect on sensory neurons

studied.

In conclusion, PTG neurons associated with synaptic boutons are a novel

preparation for studying and developing new antitussives which are effective for

chronic coughs. Further, I would like to present a working hypothesis that if

77

chemicals have inhibitory effects on both the function of nociceptive sensory

fibers and PTG neurons, it may become useful antitussives which are effective

in treating chronic coughs.

78

Summary and Conclusion

Cough is among the most common complaints for which patients seek

medical attention. Chronic cough is defined as cough lasting for more than 8

weeks.1) Cause of chronic chough is not simple and currently available

nonspecific antitussive therapy is often ineffective. In some chronic coughs,

specific therapy aimed at the underlying aetiology of cough is successful for

specialist. However, coughs ineffective for such therapy still remains and it is

still hard for general physicians to control the chronic cough. Therefore, the

greatest current need is for more effective nonspecific antitussive therapy,

whose purpose is to suppress the cough reflex and provide symptomatic relief

regardless of the underlying mechanism. To elucidate a novel strategy for

developing such nonspecific and effective antitussives, I considered that it is

important to understand pharmacological properties of the nervous system,

because cough reflex is triggered by the stimulation of peripheral sensory nerve

terminals and regulated by central and peripheral nervous system.

Recently, the following receptors and channels in sensory and central

neurons have been considered as the possible targets for developing novel

antitussives; novel opioid receptor family NOP1, TRPV1, neurokinin receptors,

BK receptors, GABAB receptor, cannabinoid receptors, large conductance Ca2+

activated K+ (BKCa) channel, ATP sensitive K+ (KATP) channel and G-protein

coupled inwardly rectifying K+ (GIRK) channel. However, we must not forget that

autonomic nervous system has a role as a part of cough reflex arc and

parasympathetic nerves predominantly control the airway function. Airway

inflammation appears to underlie chronic coughs and inflammatory substances

79

modulate the function of PTG neurons as well as sensory fibers/neurons. OP-D,

an active constituent of Chinese herbal medicine Bakumondo-to which is

effective in treating chronic coughs in clinic, reduce the excitability of PTG

neurons.7) In addition, bronchodilators have been recommended to treat cough

variant asthma.1) Therefore, I considered that PTG may become a novel target

for novel antitussives. However, much remains to be studied about physiology

and pharmacology of PTG neurons, although it is indispensable to know how

PTG function is modulated by chemical mediators. Furthermore, it seems to be

important to determine whether any other drugs effective for chronic coughs

inhibit the excitability of PTG neurons. Based on the idea described above, in

the part I, I studied effect of BK on cholinergic responses and EPSCs in PTG

neurons of rats. In part II, I studied effects of suplatast on BK potentiation of

cholinergic responses and EPSCs in PTG neurons, and on the function of the

sensory neurons, because suplatast has antitussive effect on cough variant

asthma in humans and codeine-resistant coughs in animal models. The results

obtained are summarized as follow:

1. There were no interaction between bradykinin B2 receptor and M1

muscarinic ACh receptor in PTG neurons, although these receptors couple

with PTX-insensitive G protein and M-type K+ channel. However, bradykinin

B2 receptor-mediated response was additive to M1 muscarinic ACh

receptor-mediated response. This result must conclusively affect

pathological condition via increment in length constant of membrane which

helps electrotonic spreading of the postsynaptic potential to axon hillock in

PTG neurons.

80

2. On the other hand, BK at low concentrations potentiated INic. Following

pathway is considered as the mechanism: bradykinin B2 receptor →

PTX-sensitive G protein → PLC. Downstream of PLC is remained to be

clarified. Since BK did not potentiate maximum nicotinic response, BK may

affect the affinity of nicotinic ACh receptor to its agonist. Our results

suggested that bradykinin B2 receptor couples with two distinct G proteins,

PTX-sensitive and PTX-insensitive. Electrotonic membrane potential in

physiological condition follows Ohm’s Law. Taken together with the effect

on muscarinic response, BK increases both the membrane resistance (r)

and the nicotinic current (i). Thus, BK probably facilitates the synaptic

transmission in PTG.

3. In PTG neurons attached with presynaptic boutons, I could record the

nicotinic fast EPSCs. BK potentiated not only amplitude but also frequency

of EPSCs via bradykinin B2 receptor. Thus, it was suggested that BK

stimulates bradykinin B2 receptors at both presynaptic and postsynaptic

sites and facilitate synaptic transmission in PTG neurons via three different

mechanisms, depolarization of membrane potential and potentiation of INic

at postsynaptic site and increase in ACh release at presynaptic site.

4. Suplatast did not affect directly sensory neurons, at least nociceptive

fibers/neurons. Interestingly, I found that histamine potentiated

capsaicin-induced response in rat sensory neurons. Since it has been

known that suplatast suppresses histamine release from mast cells, it is

possible that suplatast inhibits the activation of nociceptive fibers in

pathological condition via prevention of histamine-induced potentiation of

TRPV1 receptor-mediated currents.

81

5. Suplatast inhibited INic in noncompetitive- and voltage-dependent manners.

In outside-out mode of patch clamp, suplatast reduced the open time of

nicotinic receptor/channels and caused flickering in channel openings.

These suggest that suplatast works as a nicotinic channel blocker.

6. Suplatast also inhibited the EPSC amplitude and its frequency in PTG

neurons attached with presynaptic boutons. EPSC frequency was higher

sensitive to suplatast than EPSC amplitude. IC50 for EPSC frequency was

1.1x10-5 M, being similar to the effect on histamine release from mast cell

97) and lower than the inhibitory effect on cytokine production.62,64,65)

Suplatast also inhibited the EPSCs potentiated by BK, but it did not affect

the potentiation process itself by BK.

It has been well known that BK activates and sensitizes TRPV1

receptor/channel in sensory neurons under the airway inflammation (Fig.19). In

this study, I found novel effects of BK in PTG neurons that probably contribute

airway inflammation and aggravation of airway function. Suplatast had no

effect on BK-induced responses in both sensory and PTG neurons. However, it

is important that suplatast inhibited the function of PTG neurons at presynaptic

and postsynaptic sites (Fig.42). I showed that suplatast is the second example

of medicine that has antitussive effect and inhibits the function of PTG neurons.

Since suplatast, but not the first example of OP-D and others, inhibits both

codeine-sensitive and insensitive cough77,78)(Table 3), suplatast might become

a good seed for searching the novel antitussives for chronic cough. Here, I

would like to propose a novel working hypothesis that if chemicals have

inhibitory effects on the functions of both nociceptive sensory fibers and PTG

82

neurons, those may become useful antitussives which are effective in treating

chronic coughs caused by variety of causes. Finally, PTG neuron, in particular,

that attached with synaptic boutons, is a useful preparation for studying effects

of peripherally-acting antitussives and become a new target of novel

antitussives effective for chronic coughs.

Table 3 Effects of various antitussives or chemicals that have antitussive effects on

various cough models and vagal afferent discharges

stimulus codeine suplatast OP-D KATP channel opener

larynx normal animal

bifurcation

larynx n.d. n.d. SO2–exposed animal

bifurcation n.d.

ACE-I-treated animal Citric acid n.d.

BK Vagal afferent discharge

4-AP

～ n.d.: no detected : inhibition : tendency of inhibition : no action

83

Fig.42. A schematic diagram showing the effects of BK and suplatast on PTG.

(+)

ｎｎｎｎAChR

ACh

B2

Suplatast

(+)

(-) B2

BK

BK

(-)

PLC

84

Materials and Methods

1 Preparations

1.1. Animals

The animals were purchased from Kyudo Pharm (Kumamoto, Japan), and

were housed in the animal house of the Graduate School of Pharmaceutical

Sciences, Kumamoto University, at a room temperature of 22 ± 2 °C. All

experiments were conducted in accordance with the Guidelines of the Japanese

Pharmacological Society and Kumamoto University for the Care and Use of

Laboratory Animals.

1.2. Dissociation of PTG neurons

Both male and female Wistar rats (10- to 18-day-old) were anesthetized with

pentobarbital (50 mg/ml, i.p.). The rat was positioned on the back and cut along

the anterior median line from the neck to the abdomen, and then the liver and

heart were removed. The trachea and bronchus with esophagus and lung were

taken out from the body, and transferred to a Silicone-lining culture dish with

normal external solution. Under a stereoscopic microscope (MS-5, Leica,

Wetzlar, Germany), esophagus and lung were removed. The trachea and

bronchus were cut along the ventral top from the trachea to the distal bronchus,

turned to the dorsum, and pinned as a sheet over the surface with insect pins in

this dish. The preparation was stained with 0.02 % neutral red dissolved in

normal external solution for 30 min until PTG situated on the serosal surface

become red. Thereafter, the overlying connective tissue was removed with a

85

fine forceps carefully and PTG were picked out. PTG were treated with 0.3 %

trypsin and 0.3 % collagenase dissolved in normal external solution for 60 min

at 34 °C. For synaptic current recording, PTG were treated for only 20 min. After

enzyme treatment, PTG were transferred to another 35 mm culture dish filled

with normal external solution, and PTG neurons were mechanically dissociated

with a fine glass pipette (tip of 100 µm inner diameter) by gently pipetting the

ganglia. Then neurons were cultured in an incubator for 15 to 30 hr at 20 °C.

After most of glia cells covering on the neuronal surface fell out, the neurons

(Fig.43A) could be used for patch clamp recording.

To get single PTG neurons of guinea pigs, almost the same procedure is

employed. Briefly, both male and female Hartley guinea pigs (2- to 5-day-old)

were anesthetized with pentobarbital (50 mg/kg, i.p.) and the PTG were picked

up under stereomicroscope, followed by treatment with 1.4 % collagenase and

1.4 % trypsin for 60 min at 34 °C. Thereafter, PTG neurons were mechanically

dissociated with fine pipette and cultured in an incubator for 15 to 30 hr at

20 °C.

1.3. Dissociation of trigeminal ganglion neurons

The procedure is similar to that described elsewhere.106) Both male and

female Wistar rats (10- to 18-day-old) were anesthetized with pentobarbital (50

mg/kg, i.p.), and decapitated. Trigeminal ganglion was dissected and collected

in cold Krebs solution. After washing several times in Krebs solution, the ganglia

were dissected into small pieces and treated with 0.1% collagenase dissolved in

Krebs solution for 15-20 min at 37 °C, followed by 0.05% trypsin at the same

condition. After enzyme treatment, the ganglia were transferred to 35 mm

86

culture dish filled with DMEM, and neurons were mechanically dissociated with

a fine glass pipette (tip of 100 µm inner diameter) by gently pipetting the ganglia

under an inverted phase-contrast microscope. Then, dissociated neurons were

incubated in DMEM for at least 30 min (Fig.43C).

2 Electrophysiological recordings

2.1. Patch clamp recording and data analysis

Electrical measurements were performed by using the conventional whole cell,

nystatin-perforated whole cell or outside-out mode of patch-clamp recording

technique (Fig.44).107~110) Patch pipettes were made from borosilicate glass

tubes with a two stage vertical pipette puller (PP-83, Narishige, Tokyo, Japan).

The resistance of the recording electrode filled with the internal solution and the

reference electrode in the normal external solution was 5-8 MΩ.

Neurons were visualized on an inverted microscope with Hoffman modulation

(TE-300, Nikon, Tokyo, Japan). Membrane current and voltage were amplified

by an Axopatch 200B or 1D patch-clamp amplifier (Axon Instruments, Foster

City, CA, USA) and filtered at 1 or 2 kHz with a facility of the amplifier or a

three-pole low-pass Bessel filter (5610B, NF Electronic Instrument, Tokyo,

Japan) for whole cell recording. Then the output was digitized at 5 kHz with a

Digidata 1200B (Axon Instruments). The signals were acquired with pCLAMP 7

data acquisition software and stored for subsequent analysis on the computer.

All experiments were carried out at room temperature (21-24 °C). Recordings

were conducted in the neurons that had resting membrane potentials of –55 mV

or less. Data analysis was performed using Excel 2000 (Microsoft, Redmond,

87

Fig.43. Acutely dissociated rat PTG and trigeminal ganglion neurons

A: rat PTG neurons for whole cell and single channel recordings. B: rat PTG neurons for synaptic current recordings. C: rat trigeminal ganglion neurons.

20 µµµµm

A

B

C

88

Fig.44. Modes in patch clamp

WA, USA), Origin 5 (Microcal, Northampton, MA, USA) and pCLAMP 7 or 9

(Axon Instruments). The results are given as mean ± S.E.M. Statistical

significance was determined by the use of unpaired Student’s t-test or

Kolmogorov-Smirnov Test (for EPSC distribution). In all instances, P < 0.05 was

considered significant.

IK(M) was recorded by applying hyperpolarizing voltage steps from a VH of –25

to –50 mV. The inhibition of IK(M) was measured as described elsewhere.6) To

measure the voltage-dependent Ca2+ current, linear leak and capacitative

currents were subtracted. To study the effect of BK in PTG neurons, only one

neuron was used in each culture dish to eliminate the unexpected effect of

BK-induced long desensitization.

The concentration-response relationship was fitted with the following

equation,

Whole-cell recording

Single channel recording

89

nn

n

ECCCAA

50max +

= ………………………. (1)

where, A the current or voltage amplitude, Amax the maximum current or

voltage amplitude, C the agonist concentration, n the Hill coefficient, EC50 50 %

effective concentration.

The concentration-inhibition relationship was fitted with the following equation,

nn

n

ICCICAA

50

50max +

= ………………………. (2)

where, IC50 concentration need for 50 % inhibition, A, Amax, C and n same as

above.

For single-channel recording, patch currents were digitized at 100 kHz and

filtered at a cutoff frequency (f1) of 5 kHz using the Bessel filter and then at 16.7

kHz (f2) during data analysis. These settings yielded a final filter frequency (fc) of

4.8 kHz (fc-2=f1-2+f2-2) allowing channel openings ≧139 µs (twice the filter rise

time, tr=0.3321/fc) to be resolved.111,112)

Single channel events were detected by visual inspection of the transitions

from the base current and manually selected using Fetchan software of

pCLAMP 7.0. Thereafter, events were compiled into all-points histograms using

0.6 ms bin width and were fitted with Gaussian functions using a simplex

least-squares algorithm in pSTAT of pCLAMP 7.0. The frequency of opening for

each event was determined by the reciprocal of the open + closed time since

the last event. The probability of a channel being open (NPo) was measured

over specific time intervals, thus providing a quantitative description of the

activity of the channel vs. time. For determination of mean open and closed time,

open and closed duration was respectively compiled in conventional and

90

logarithmic histograms and then fitted by simplex least square method for open

time and maximum likelihood method for closed time.

2.2. Fast drug applications with the “Y-tube”

Drug application was carried out by the "Y-tube" (Fig.44). With this technique,

external solution could be completely exchanged within 30 ms.113) A major part

of the system was a Y-shaped tube. A polyethylene tubing (1 mm i.d. and 100

mm in length) was bent in a V-shape and a small hole (100 µm in diameter)

made at the curved end, into which a fine polyethylene tubing (50 µm i.d. and

10 mm in length) was inserted, and affixed by a silicon-based glue, making up

the tip of the “Y-tube”. One end of the V-shaped tubing was led to the test

solution by a silicon tubing (1 mm i.d. and 400 mm in length). The test solution

Fig.44. Schematic drawing of patch clamp recording and fast drug application with the

“Y-tube”

91

was held 20 cm above the level of the “Y-tube” tip, so that the solution flowed

out from the “Y-tube” tip by the difference of hydrostatic pressure. Another end

of the V-shaped tubing was led to a drain bottle through an electric valve, which

is normally closed. The drain bottle was kept at a negative pressure of

approximately 40 mmHg.

3 Solutions and chemicals

3.1. Solutions for cell dissociation and patch clamp recording

The composition of the normal external solution was (in mM): NaCl 131.7, KCl

5, MgCl2 1.2, CaCl2 2.5, HEPES 10 and glucose 11.5. For 30 mM K+ external

solution, 25 mM NaCl was replaced by equimolar KCl. The pH was adjusted to

7.4 with 1N NaOH. The ionic composition of the Krebs solution was (in mM):

NaCl 121.7, KCl 4.7, MgCl2 1.2, CaCl2 2.5, glucose 11.5, NaHCO3 15.5 and

KH2PO4 1.2. The solution was bubbled with 95% O2 and 5% CO2. The

composition of the external solution for recording Ca2+ currents was (in mM):

N-methyl-D-glucamine-Cl 140, CsCl 5, CaCl2 5, MgCl2 1, HEPES 10 and

glucose 10. The pH was adjusted to 7.4 with Tris-OH.

Patch pipette solution for nystatin-perforated patch-clamp recording contained

(in mM) KCl 80, K-gluconate 70 and HEPES 10. The pH was adjusted to 7.2

with 1N KOH. Nystatin was dissolved in methanol at 10 mg/ml and added to the

internal solution at a final concentration of 400 µg/ml just before use. Pipette

solution for conventional patch-clamp recording contained (in mM) KCl 70,

K-gluconate 70, MgCl2 5, CaCl2 1.03, HEPES 10, EGTA 5, ATP-2Na 4 and GTP

0.3. The pH was adjusted to 7.2 with 1N KOH. The composition of the pipette

92

solution for the single-channel recording was (in mM): NaCl 20, KCl 50,

K-gluconate 70, MgCl2 3, CaCl2 0.246, HEPES 10, EGTA 5 and ATP-2Na 2.

The pH was adjusted to 7.2 with 1N KOH.

3.2. Chemicals

ATP, ATP-2Na, capsaicin, collagenase, EGTA, GTP, mecamylamine,

neomycin, neutral red, nicotine, nystatin, tetrodotoxin (TTX), trypsin, U-73122

and U-73433 were purchased from Sigma (St. Louis, MO, USA). ACh, CdCl2,

histamine and serotonin were purchased from Nacalai Tesque (Kyoto, Japan).

Bradykinin, [Hyp3]-Bradykinin, HOE 140 and substance P were purchased from

Peptide Institute (Osaka, Japan). DMEM was purchased from Gibo BRL (Grand

Island, NY, USA). Pertusistoxin (PTX) was purchased from Seikagaku Kogyo

(Tokyo, Japan). Suplatast tosilate was synthesized and supplied by Taiho

Pharmaceutical Co. Ltd. (Tokyo, Japan). Capsaicin and nystatin were at first

dissolved in 98 % ethanol at 0.1 M and 99.8 % methanol at 10 mg/ml,

respectively. Other solutions were at first dissolved in deionized and distilled

water at 0.1 to 10-4 M. Stock solutions were stored at –20 °C and dissolved in

the optimum external or internal solutions just before use.

93

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Acknowledgements

From my deep heart I sincerely appreciate Professor Kazuo Takahama, my graduate

study supervisor, who has been constantly providing me his precious directions and

inspiring encouragements during the years of my graduate study. It is him who has

given me the opportunity to study in Kumamoto University of Japan and has provided

his warm and continuous advice on my family in Japan.

I am enormously grateful to Professor Takeshi Miyata for reviewing my doctoral thesis

and providing his earnest encouragement.

I would like to express my greatest gratitude to Associate Professor Tetsuya Shirasaki

for giving me his valuable guidance and careful advice during the years of my graduate

study and providing his detailed revising and correcting on my thesis.

I thank Associate Professor Keitarou Suzuki, Hidetoshi Arima and Yoichiro Isohama

for kind reviewing my doctoral and master thesis, the data of which compose one part of

my current doctoral thesis.

I am greatly grateful to Assistant Fumio Soeda for giving me his kind and patient help.

My study is a fruit of the collaborations between the group members and me. I thank

all of the current and previous members of Environmental and Molecular Health

Sciences, who have been giving me their warm helps during these years.

I sincerely appreciate to the Researching/Teaching Assistant Foundation from

Graduate School of Pharmaceutical Science, Kumamoto University, the Foreign Student

Scholarship Foundation of Kumamoto Province, the Kounan Asian Scholarship

Foundation and the Japan Foundation of Scholarship for their financial support to my

graduate research work and my personal life.

I thank Taiho Pharmaceutical Co. Ltd. for providing suplatast tosilate.

I am deeply indebted to my husband, who has done his best to support my study. I

would like to say “sorry” to my son; I seldom accompanied with him for reading a book

on evening as like as an ordinary mother. I devote this work and my deepest thanks to

them and all other family members, who have been giving me their endless love,

support and understanding no matter when and where I am.