GastroenterologiaJaponica Vol. 21, No. 3 Copyright �9 1986 by The Japanese Society of Gastroenterology Printed in Japan

~ O r i g i n a l A r t i c l e w

C H O L I N E R G I C EFFECTS OF HISTAMINE-H2 RECEPTOR A N T A G O N I S T S P A R T L Y T H R O U G H I N H I B I T I O N

OF ACETYLCHOLINESTERASE

Mitsuru A O N 0 , M.D.* , Motoyuki M O R I G A , M.D.**, Kazuhiko M I Z U T A , M.D.** and

Hiroaki N A R U S A W A , M.D. * *

�9 Department o f Clinical Nutrition and **First Department o f Internal Medicine,

Kyoto University School o f Medicine, Kyoto 606,Japan

Summary

The effects of histamine Hz-receptor antagonists on acetylcholinesterase and pseudocholinesterase ac- tivity were studied. All H• tested inhibi ted both enzyme activities dose-dependently. The potency of inhibi tory activity of H~-antagonists on acetylcholinesterase estimated from median inhib i - tory dose were in the following order of decreasing activity: rani t idine > TZU-0460 > cimetidine > YM- 11170, whereas that on pseudocholinesterase were TZU-0460 > rani t idine > cimetidine > YM-11170.

As the effects derived from the inhibi t ion of acetylcholinesterase by H2-antagonists may affect intesti- nal motility, we studied i leal muscle contractions. Rani t id ine had the most potent st imulating effect on contraction, the pattern of which was similar to physostigmine and was blocked by atropine and mor- phine. YM-11170 had a weak action on muscle contraction and cholinesterase activities.

Key Words: A cetylcholine, Acetylcholinesterase, H2-antagonist, Guinea-pig ileum.

I n t r o d u c t i o n

Several h is tamine H2-receptor antagonists

have been developed and used clinically for the

t r ea tment of pept ic ulcers. They exert powerful

inhibi tory effects on gastr ic acid secretion in

animals and humans . They usually have no

effects on gastrointest inal moti l i ty , however, it

is possible that some of them may affect the

gastr ic empty ing and the lower esophageal

sphincter pressurel-S). Ber taccini et al.1) have

repor ted that ran i t id ine exerts cholinergic-l ike

Received October 11, 1985. Accepted January 13, 1986. Address requests for reprints to: Mitsuru Aono, M.D.,

Department of Clinical Nutrition, Kyoto University School of Medicine,'Sakyo-ku, Kyoto 606, Japan.

This study was supported in part by grant from the Japa- nese Ministry of Education, Science and Culture (No. 59570300).

effects. Rani t id ine provokes atropine-sensi t ive

cont rac t ion of smooth muscle p repara t ions of

several different an imal species. There are

some reports4-O tha t c imet id ine and ran i t id ine

inhibi t h u m a n erythrocytes and gastr ic acetyl-

cholinesterase (ACHE) as well as h u m a n se rum

pseudochol inesterase (PChE).

In the present study, the effects of Hg_-

antagonists on A C h E and PChE were ex-

amined . We c o m p a r e d the effects of new H2-

antagonists , TZU-04607) and YM-11170s,*),

with those of c imet id ine and rani t id ine . F rom

the result of ACHE, the effects of H2-antago-

nists on cont rac t ion of the ileal muscle p r epa ra -

t ion were invest igated.

M a t e r i a l s a n d Methods

Cholinesterase assay

214 M..40NO ET AL. Vol. 21, No. 3

Human erythrocyte AChE was obtained

from heparinized blood. The erythrocytes were

washed 5 times with saline and finally resus- pended in distilled water for hemolysis. Assays

were performed after dilution in 0.1 M phos- phate buffer, pH 8.0. Human serum was used

for PChE assay. Cholinesterase activities were measured at

25~ by a spectrophotometric method using acetylthiocholine as a substratel~ In the stan-

dard procedure, to 2 ml of enzyme solution in 0.1 M phosphate buffer, pH 8.0, 0.8 ml ali- quots of 0.33 mM 5,5'-dithiobis-(2-nitro- benzoic acid) (DTNB) in 0.1 M phosphate

buffer, pH 7.0 with or without 0.013 mM pro-

fenamine (Yoshitomi Pharmaceutical, Osaka)

as a PChE inhibitor were added. After a 15- rain preincubation at 25~ the reaction was

started by the addition of 0.2 ml of 1 mM

acetylthiocholine. The enzymatic reaction was

determined by the increase of absorbance at wave length 405 nm in a photometer equipped with a chart recorder.

Experiments with histamine-Hz-antagonists and other drugs were carried out by adding 0.5 ml of drug solution that had been diluted with

0.1 M phosphate buffer, pH 8.0, to 1.5 ml of enzyme solution. Ileal muscle preparation

Male guinea pigs, weighing 300 to 400 g, were killed by a blow on the head, and the ileum was isolated and placed in Tyrode solu-

tion. Tissue specimens about 3 cm in length

were suspended in a 10-ml organ bath with

Tyrode solution kept at 32~ and gassed with

95% 02 and 5% CO2. The Tyrode solution had the following composition (per liter): NaC1, 8.0 g; KCI, 0.2 g, CaC12, 0.2 g; MgCI2,

0.1 g, NaHzPO4, 0.05 g; NaHCO3, 1.0 g and glucose, 1.0 g. Response to drugs were re- corded isotonically under a tension of 1.0 g.

In some experiments ileal longitudinal muscle was gently separated from the under-

lying circular muscle. The dissected muscle strip was stimulated electrically by the method of Patonal). The electrodes were made of plati-

num and an intraluminal electrode was used as the anode. Rectangular pulses of 0.1 msec

duration were used at a frequency of 0.1 Hz

and at a voltage of 5 V. Agents used

Acetylcholine chloride (Daiichi, Tokyo),

morphine hydrochloride (Dainippon, Osaka), physostigmine sulfate (Wako, Osaka), hexa-

methonium chloride (Nakarai, Kyoto), beta-

zole hydrochloride and chlorphenylamine

malate (Shionogi, Osaka), diphenhydramine hydrochloride (Tanabe, Osaka), atropine sul- fate and pyrilamine malate (Sigma, St. Louis,

MO) were used. Cimetidine was a gift from SKF Fujisawa, Tokyo. Ranitidine was a gift

from Glaxo Japan, Tokyo. YM-11170 was a gift from Yamanouchi, Tokyo. TZU-0460 was a gift from Teikokuzoki, Tokyo. The chemical structure of Hz-blockers used are shown in Fig.

1.

Cimetidine

CH= -CH2SCH2CHzNHCNHCH=

I H ~ N N--CmN

Ranit, idine ,•-CH=SCH=CH=NHCNHCH= CHNO=

~NSO=NH= /==~CH=SCH=CH=C,~.NH

Y M - 1 1 1 7 0 ~ 'N ' (Famot.idine)

N=N~ ~N H=N/C

Fig. 1. The chemical structure of the histamine Hz- receptor antagonists used.

June 1986 Cholinergic Effects of H2-blockers 215

Results

Cholinesterase assay Cimetidine inhibited both AChE and PChE

activities in a dose-dependent manner. The

median inhibitory dose (ID-50) was 6.3 x 10-4 M for erythrocyte AChE and 2.6 x 10-3 M for

serum PChE shown in Fig. 4. In order to deter-

mine the type of cholinesterase inhibition by cimetidine and inhibition constant, saturation experiments with increasing substrate concen-

Fig. 2.

trations were carried out. The double recipro- cal plots obtained from the experimental data were linear in all cases examined, as shown in

Fig. 2. Both Km and Vmax were affected by the agent. These results are consistent with an inhibition of the mixed type, which presents characteristics c o m m o n to both competitive

and non-competitive inhibition. The inhibitory constants (Kt) were 1.5 x 10-4 M for AChE

and 1.9 x 10 -4 M for PChE, as calculated by

Dixon-plots. Fig. $ shows that histamine-

ACETYLCHOLINs PSEt,~OCHOLINESTERASE CI~ETI~INE ]oosf 6 10 .5~

Z/v 6.6 lO'4N CIMETIDoIflE DOSE 1/v 6.6 10"4M ~, IO-W~ 1 xlO'r 3. xlO'4N 1'6 10- M

IDIHE( ) 6 10"5M

10" 100

INE( ) 5 j

/ - / ~ / Ii " i ! I 1/10-3 1/10-~ 1/2.5x10-5 1/10-] 1/10-4 1/2.5x10-$ Iv[ "1

(l/S) (l/S)

Inhibition of erythrocyte AChE (left) and serum PChE (right) by cimetidine: double reciprocal plots. Acetylthiocholine in a range of concentrations (S): 0.025-1 mM; was used. V=A absorb- ance/min. The concentration of cimetidine are given at the range from 6.6 x 10- ] to 6.6 x 10- 2 mM. The points on the graph represent the mean values of 3 separate experiments performed in duplicate.

A I-r

50 -

Fig. 8.

loo - o - - ~ t r ~ ~ - ; ~ ~ - - ~ - - +

~ " . " " , ~ . . Hlstid,n. (0) �9 ". ~ Hlstltrnine (&)

�9 " . ".~\ Eetezole (~) \~ ~\ Oiphenhydrarnine

~ ~, on AChE (0) ~ (~ PChE (0)

', ~ Chlorpheflylarnine on AChE (FI)

o., , I{ . . . . . . .

0 i0 -8 i0 -7 i0 -6 10 .5 10 -4 i0 -3 i0 -2

DOSE (MOL)

Inhibition of erythrocyte AChE and serum PChE by histamine-related compounds.

216 M. A O N O E T /IL. Vol. 21, No. 3

100.

50.

Cimetidine

n e

o �9 P ~ O O A O L ~

I() -7 10-* 10 -= 10 -4 10-* 10 -= 10 -~ r oo~ (MOL)

T Z U - 0 4 6 0 ETC't'M( m'rmTY (~)

100 ~--II " ~ ~ m ' r v

50 L ~ SE

0 6 I~_. 10-" 16-" 10-" 1~-' 10-" 1~-" Tzu-o~o oo~ (MOL)

Fig. 4.

% Ranitidine

1

6 I I 16-, lg '-o lo-, 16-" lo-' lo-,

YM-11170 100 o If

0 ~ rt 10~? 10~,

5 0

F ~ t X X ~ 0 t . I ~ S ~

I to'-" 1~" to" 10 ~"

W4-1W/0 ~ OWL)

Inhibitin of erythrocyte AChE and serum PChE by several Hz antagonists. ID-50 of agents on AChE is shown by the dotted line.

related compounds such as histidine, histamine

and betazole hydrochloride, which have an

imidazole ring, had no influence on either cho-

linesterase. Histamine Hi-blocking agents,

such as chlorphenyramine and diphenhydra-

mine, inhibited this activity, especially that of

PChE.

Other Hz antagonists such as ranitidine,

TZU-0460 and YM-11170, also inhibited ac-

tivities of both cholinesterases (Fig. 4). Raniti-

dine was the most potent inhibitor of AChE among them. Its ID-50 was 1.3 x 10-6 M. YM-

11170 was a weak inhibitor of both AChE and

PChE.

Ileal muscle prepara t ion

Acetylcholine (0.05 aM) elicited an immedi-

ate phasic contractile response followed by

tonic contraction, whereas 29 aM of ranitidine

increased contractile activity gradually, and

enhanced acetylcholine-induced contractions

as shown Fig. 5. Cimetidine, TZU-0460 and

YM-11170 at a concentrat ion of 26 and 30/zM

respectively had no effect on acetylcholine-

induced contraction. However, at a concentra-

tion of 300 pM they slightly stimulated the con-

traction; the effect was identical to that seen

with 2.9 aM of ranitidine. Ranitidine and

physostigmine showed similar patterns of con-

traction.

Fig. 6 shows the effect of Hi-antagonists and

atropine sulfate on ranit idine-induced contrac-

tion. Diphenhydramine inhibited the contrac-

tion induced by ranitidine. Another HI-

blocker, pyrilamine, had no effect. The gan-

glionic blocker, hexamethonium, had no effect

on ranit idine-induced contractile activity.

Prior t reatment with morphine abolished the

ranit idine-induced activity. Exogenous acetyl-

choline-induced contractile activity was com-

pletely mainta ined despite the presence of mor-

phine. Fig. 7 shows the effects of H2-antago-

nists on the contract ion of the longitudinal

muscle preparat ion of isolated guinea-pig

ileum using electric field stimulation. H v

June 1986 Cholinergic Effects of H=-blockers 217

Ach Ach Ach Ach

Ranitidine( 29 )

Ach Ach ACh Ach Ach

Cimetidine TZU-OZ,60 ( 40 ) ( 26 )

Ach

Ranitidine ( 2 9 )

Fig. 5.

5 rain.

Omm

t Ranitidine( 29 ) Physostigmine( 0.1z )

Ach Ach Ach Ach Ach

,r

YI,6-11170 ( 3 o ) ( 2 9 )

10mm

J ~anitidine C~metid~r~

( z.9 ) (4o0) (26o)

~ 0mm

I~an~idine ~M -11170 ( z 9 ) (3oo)

Contraction of isolated guinea-pig ileal preparation by H2 antagonists was measured isotonically. ACh: Acetylcholine 0.05 ltM, parentheses indicated the concentration of agents (pM). Asterisks indicate the washing out of incubated solution in the preparation.

.5 rain . .~

Diphenhydramine( 039 ) DPH( 039 ) Pyritamine Pyrilamine [ ~ ( o'~)PH~ ) I ~ ( " Pyrilam~ ) Atropine sulf. (DPH> . 1 * I ( 0.1S ) I (0~) [ ~ * I "'1 ..a~t 0.�88 ,

I ] lOmm

I I I | I | ~anitidine Ranitidine Ranitidine Ranitidine Ranitidine Ach Ach ( ~Z7 ) ( a27 )

Hexarr~thoniumic6( 37 ) , (C6:37 )1

t I Ranitidine Ranitidine

i , , l] I

Ach( 0.05 ) Ranitidine Ranitidine Ach( 0.05 ) Ranitidine Ach( o.os )

Fig. 6. Contraction of isolated ileal preparations by ranitidine. Concentration of ranitidine was 29 ,uM. Contraction by ranitidine was blocked by atropine, diphenhydramine and morphine, but not by hexamethonium or pyrilamine. Parentheses indicate the concentration of agents (,uM). Asterisks indicate the washing out of incubated solution in the preparation.

218 M. A O N O E T .'IL. Vol. 21, No. 3

Fig. 7.

Contraction

%

200" o-- Ranitidine o-- Cimetidine * - TZU-0460

/ o- u

/ / t

100 o ........................................................................................

0 1 '101 102 103 pM

Dose-response contraction of electrically stimulated longitudinal muscle strip (0.1 msec, 0.l Hz, 5 V) by H2-antagonists. Basal contraction is expressed as one hundred percent. Data are presented as mean +- S.E. of five experiments.

antagonists increased the contractile activity

dose-dependently. We compared the contrac- tile action of each H2-antagonists. Basal con- tractile activity was expressed as 100%. Raniti-

dine stimulated the contraction dose-depen- dently, and was 10 times more potent than the

other H2-antagonists. YM-11170 was a very weak stimulant. The highest dose of YM- 11170

used did not elicite a further increase of con-

tractile activity.

D i s c u s s i o n

The present study demonstrated that AChE and PChE were inhibited by H2-receptor antagonists. Ranitidine is the most potent in- hibitor of AChE among the H2-antagonists ex- amined. A previous observation s J2) that the

anticholinesterase activity of ranitidine is rather selectively directed toward AChE was confirmed. H2-antagonists may interfere with acetylcholine catabolism. Inhibition of AChE by H~-antagonists might follow the accumula- tion of acetylcholine at cholinergic nerve termi-

nals, resulting in cholinergic excitation. As a

consequence, several organ functions con- trolled by acetylcholine could be influenced.

Histamine as well as acetylcholine receptors are

important in the control of gastric secretion.

Inhibition on gastric secretion by H2-antago- nists might be far more greater than stimula-

tion of acetylcholine. Thus, the gastrointestinal functions other than gastric secretion may be affected.

Furthermore, we observed the action of these drugs during contraction of the isolated guinea-pig ileum. Ranitidine per se induced contraction of the ileum, the pattern of which was similar to that of physostigmine but differ- ent from that of acetylcholine. Doses of cimeti-

dine, TZU-0460 and YM-11170 100 times

higher than that of ranitidine were needed to

elicite even slight contraction. The mode of action of ranitidine-induced

contraction was examined by using Hi-antago- nists and atropine. Diphenhydramine inhibited

the contraction induced by ranitidine. Raniti- dine might have had histamine-Hx activity, however, diphenhydramine inhibited the con-

traction induced by acetylcholine. The Hi- blocker, pyrilamine, had no effect. This sug- gests that diphenhydramine acts as an anticho- linergiclS,x4). Pyrilamine did not inhibit the contraction induced by ranitidine, thus raniti- dine has no histamine-Hx activity. Atropine

June 1986 Cholinergic Effects of H~-blockers 219

sulfa te i nh ib i t ed the c o n t r a c t i o n i n d u c e d by

ran i t id ine , i n d i c a t i n g tha t r an i t i d ine has cho-

l inerg ic act ion.

T h e gang l ion ic b locker , h e x a m e t h o n i u m ,

had no effect on con t r ac t i l e effect of r an i t i d ine .

p r io r t r e a t m e n t wi th m o r p h i n e , a b locker o f

endogenous ace ty lcho l ine 11), abo l i shed the

r a n i t i d i n e - i n d u c e d c o n t r a c t i o n , while exoge-

nous a c e t y l c h o l i n e - i n d u c e d con t r ac t i l e act ivi ty

was comple t e ly m a i n t a i n e d despi te t he pres-

ence o f m o r p h i n e . T h i s suggests that m o r p h i n e

blocks the ac t ion of e n d o g e n o u s ace ty lchol ine .

T h e effects o f r a n i t i d i n e m a y be r e l a t ed to the

e n d o g e n o u s release o f ace ty lchol ine , tha t is,

the re m a y be an i nd i r ec t r e l a t ionsh ip wi th the

inh ib i t ion of ace ty lcho l ines te rase .

S t a n d a r d d r u g levels o f these H2-blockers in

serumXS, 16) c a n n o t r e a c h levels sufficient to in-

h ib i t ace ty lchol ines te rase . In t he case o f musc le

p r e p a r a t i o n , c o n t r a c t i o n i n d u c e d by r a n i t i d i n e

b e g a n at the level of 0 . 8 6 / 2 M . It has b e e n re-

p o r t e d tha t s e r u m levels o f r an i t i d ine r e a c h e d

1 . 1 - 1 . 7 / 2 M w h e n the agen t was oral ly admin is -

t r a t e d at a dose o f 150 mgX6). Gas t ro in tes t ina l

mo t i l i t y in m a n m a y increase at t he usual

dosage level. T h e r e h a v e b e e n several repor t s

tha t r an i t i d ine affects the gas t r ic e m p t y i n g a n d

the lower e sophagea l s p h i n c t e r pressure in the

ratS), bu t not in manlT). It was not d e t e r m i n e d

if t he ex ten t o f the effects o f H~-hlockers ob-

served here are s imi la r to those in the h u m a n

body .

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3) Bertaccini G and Scarpignato C: Histamine H2- antagonists modify gastric emptying in the rat. Brit J Pharmaco177: 443, 1982

4) Hansen WE and Bertl S: The inhibition of acetylcho- linesterase and pseudocholinesterase by cimetidine. Arzneim-Forsch 33: 161, 1983

5) Hansen WE and Bertl S: Hemmung der Azetylcho- linesterasen-Ein relevanter Nebeneffekt yon Rani- tiding" Z Gastroenterologie 21: 164, 1983

6) Aono M, et al: Inhibition of acetylcholinesterase by histamine-H~ receptor antagonists. Jpn J Gastro- enterol 81 : 1653, 1984 (]pn)

7) Hasegawa Y, et al: Phase I study of TZU-0460, a novel histamine Hz-receptor antagonist. (1) Single and one day dose study. Yakuri to Chiryo 12: 637, 1984 (Jpn)

8) Takagi T, et al: Effect of a new potent H2-blocker on gastric secretion induced by histamine and food in conscious dogs. Arch Int Pharmacodyn 256: 46, 1982

9) Pendleton RG, et al: Studies on MK-208 (YM-11170) a new, slowly dissoeiable H2-receptor antagonist. Arch Int Pharmacodyn 266: 4, 1983

10) Ellman GL, et al: A new and rapid colorimetric deter- mination of acetylcholinesterase activity. Biochem Pharmacol 7: 88, 1961

11) Paton WDM: The action of morphine and related substances on contraction and on acetylcholine output of coaxially stimulated guinea-pig ileum. Brit J Phar- macol 12: 119, 1957

12) Galli A, et al: Effect of ranitidine on ileal myenteric plexus preparation and on acetyl- and butyrlcho- linesterase. Biochem Pharmacol 33: 1845, 1984

13) Rocha e Silva M and Antonio A: Bioassay of anti- histaminic action. In, Histamine II and anti- histaminics: Chemistry, Metabolism and Physiological and Pharmacological Action. (Rocha e Silva M, ed) Handbuch der Experimentellen Pharmakologie, Vol 18, Pt 2. Springer-Verlag, Berlin, 1978, p 381

14) Douglas WW: Histamine and 5-hydroxytriptamine (serotonin) and their antagonists. In, The Pharmaco- logical Basis of Therapeutics. 7th ed (Gilman AG, Goodman LS, Rall TW and Murad F eds) Macmillan, New York, 1985, p 605

15) Gaginella TS and Bauman JH: Ranitidine hydrochlo- ride drug intelligence. Clin Pharm 17: 873, 1983

16) Somogyi A and Gugler R: Clinical pharmacokinetics of cimetidine. Clin Pharmacokinetics 8: 463, 1983

17) Baldi F, et al: Oesophageal function after oral raniti- dine: An acute double blind study in normal subjects. Hepato-Gastroenterol 31 : 38, 1984