Naunyn-Schmiedeberg's Arch Pharmacol (1985) 328: 248 - 252 Naunyn-Schmiedeberg's

Archives of Pharmacology �9 Springer-Verlag 1985

A kinetic study of the release of noradrenaline by electrical stimulation: influence of presynaptic a-adrenoceptors*

Fernando Brandfio, R. Davidson, and J. Guilherme Monteiro Laborat6rio de Farmacologia, Faculdade de Medicina, P-4200 Porto, Portugal

Summary. Dog saphenous vein strips were incubated with 1.4 ~tmol/13H-(-)-noradrenaline for 60 rain, after inhibition of the noradrenaline-metabolizing enzymes and of extra- neuronal uptake. At the end of the incubation period the strips were perifused for 150 rain; cocaine (10 gmol/1) was added to the perifusion fluid from t = 75 min onwards. In some experiments either phentolamine (10~tmol/1) or clonidine (0.1 gmol/1) was also added at this time. Some strips were subjected to electrical stimulation from t = 100 to 150 rain ofperifusion (t = 0 being the start of perifusion), with frequencies ranging from 0.5 to 13.5Hz. A compartmental analysis of spontaneous or electrically-in- duced efflux of 3H-noradrenaline was made. The spon- taneous efflux had a long half time (t/2 = 124 min) and most of the 3H-noradrenaline which had accumulated in the strips did not participate in the efflux ("bound fraction", represent- ing 90% of tissue activity at t = 100 min of perifusion). Neither phentolamine nor clonidine modified the half time or the "bound fraction" observed for spontaneous efflux. Electrical stimulation ( > 0 . 5 H z ) mobilized only one compartment of noradrenaline, which represented about 50 % of the noradrenaline accumulated in the strips. The half time of 3H-efflux induced by electrical stimulation decreased when the frequency increased from 0.5 Hz up to 13.5 Hz. Phentolamine increased the rate of efflux for all frequencies of stimulation and decreased the half time of efflux. How- ever, the releasable pool of noradrenaline was only increased by phentolamine at 0.5 Hz, but not at higher frequencies. Clonidine was used only at two frequencies of stimulation, 1.5 and 4.5 Hz. For the low frequency clonidine decreased the releasable pool, but no change was observed at 4.5 Hz.

The results support the view that there is a noradrenaline pool which is resistant to electrical stimulation and that its magnitude is not dependent on the activity of presynaptic a-adrenoceptors.

Key words: Release of noradrenaline - Dog saphenous vein - Kinetic analysis - Presynaptic ~-adrenoceptors - Electrical stimulation

Send offprint requests to: F. Brandfio at the above address * Results presented in part to the 13th Annual Meeting of the

Portuguese Pharmacological Society (Porto, December 1982) and to the 5th Meeting on Adrenergic Mechanisms (Porto, October 1983)

Work supported by a grant from Instituto Nacional de In- vestigaq~o Cientifiea (FmP1)

Introduction

In a previous study (Brand~o et al. 1980) we have shown that the overflow of noradrenaline induced by sustained electrical stimulation was evanescent, but the efflux evoked by a long exposure to tyramine was sustained. This differ- ence was attributed to different mechanisms of action of tyramine and electrical stimulation: while electrical stimula- tion appeared to mobilize preferentially the vesicles located near the axonal membrane, tyramine would show no such specificity. This assumption was later supported by the find- ing that a high concentration of tyramine can mobilize the total content of noradrenaline existing in the adrenergic terminals (Brandgo et al. 1981 ; Rapoport et al. 1981). It was considered of importance to continue this investigation in order to obtain information about the kinetic parameters of the release of noradrenaline by electrical stimulation and the influence exerted by presynaptic c~-adrenoceptors.

Methods

Mongrel dogs ( 8 - 1 6 kg) were anaesthetized with sodium pentobarbital (30 rag. k g - I i.v. injected in the forelimb). Segments of both lateral saphenous veins proximal to the junction of the plantar and dorsal branches were removed, and helically cut strips prepared as described by Guimarges and Osswald 0969). Each strip, weighing about 100 mg and 4 cm long, was preincubated for 30 rain in Krebs' solution, at 37~ gassed with 95% 02 and 5% CO2, in the presence of 0.1 mmol/1 3,4-dihydroxy-2-methylpropiophenone (U-0521), 28 gmol/1 cortisol and 0.1 retool/1 pargyline, in order to block catechol-O-methyltransferase (COMT), ex- traneuronal uptake and monoamine oxidase (MAO), re- spectively. As shown in a previous study (Brand~o et al. 1981) this procedure markedly reduces the formation of metabolites. The strips were then incubated for 60 min in 3 ml of the same medium to which (-)-7-3H,noradrenaline (batch no. 1271-115 NEN with 74% of total tritimn in the 7 position and 26% in the 8 position, 4.5 Ci/mmol specific activity) was added; the final concentration of 3H-nor- adrenaline was 1.4 gmol/1. After the incubation the strips were continuously perifused for 150 rain in a l-ml glass organ bath; Krebs' solution (with U-0521 and cortisol as above) was pumped through the bath at a constant rate of 0.8 ml/min, and the overflow was collected. In all experi- ments cocaine (10 gmol/1) was added to the perifusion fluid from t = 75min onwards. In some experiments either phentolamine (10 gmol/l) or clonidine (0.1 gmol/1) were also added at this time. Field electrical stimulation (from

249

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Fig. 1 Outflow of 3H-noradrenaline from dog saphenous vein strips, from the t = 90 up to the t = 150 min of perfusion. Field electric stimulation was applied from t = 100 to t = 150 min. Shown are the average efflux curves. Ordinate: rate of effiux (in ng�9 g-1 �9 min-1; log scale); abscissae: time (in min) after the onset of washout�9 Compart- ment analysis (see Methods) was carried out by retrograde regression analysis (see solid line). Spontaneous effiux (�9 Field electric stimulation: 0.5 Hz (~), 1.5 Hz (e) , 4.5 Hz (11), and 13.5 Hz (A). A Control experiments; B phentolamine ex- periments (10 gmol/1); C clonidine experi- ments (0A ~tmol/1)

t = 100 to t = 150 min) was applied by means of thin plati- num electrodes attached to both ends of the preparation. The stimuli were rectangular pulses of alternating polarity, 2 ms duration and 100 V intensity, with frequencies from 0.5 up to 13.5 Hz (Hugo Sachs Elektronic Stimulator, model 215/II). The stimulus voltage was shown to be supramaximaly by testing voltages of 150 and 200 V, since no significant difference in the magnitude of the effiux was observed; these parameters were verified by an independent oscilloscope. The overflow was collected in 5- or 10-min fractions (see Results) and total radioactivity determined as previously described (Brandao 1977). To investigate whether our observations could have been due to excessive current flow through the Krebs' solution and not through the pre- paration, the following technique was also used. Strips of saphenous vein similar to and with the same pretreatment as those that were perifused were suspended between two platinum electrodes; Krebs' solution was made to flow in discrete drops around the tissue and collected at the bot tom electrode. The whole system was kept warm by a water jacket. Stimulus parameters and rate o f flow of Krebs' so- lution were identical to those in perifusion experiments. Under these experimental conditions no significant differ- ences were observed in the efflux parameters. The composi- tion o f the Krebs-Henseleit solution was (mmol/1): Na + 144, K + 4.7, Mg 2+ 1.2, Ca 2+ 2.6, C1- 130.3, HzPO4 1, HCO~-25, glucose 11.1, NazEDTA 0.04, ascorbic acid 0.11.

From the plot o f in of rate of efflux vs. time, the regres- sion lines were calculated from t = 145 or t = 147.5 min of perifusion, proceeding backwards, according to the "peel- ing" technique (Henseling et al. 1976). F rom the regression lines the following values were obtained: slope (k), half time (t/2 = in 2/k), size of the compartment responsible for the

monophasically declining effiux of tritium at t = 150 min of perifusion (C15o = rate of efflux at t = 150 min/k) and "bound fraction" i.e. radioactivity not contributing to the effiux up to t = 150 min (BF = tissue content at 150 min - C~50). "Bound fraction %" is the ratio between B F a n d tissue activity at t = 100 min of perifusion (TA ~ o 0) multiplied

BF by 100 ( B F % - x 100).

TA l oo Differences between means were compared by Student's

t-test and those with a P-value of 0.05 or less were considered significant.

Drugs used were clonidine hydrochloride (0sterr. Stickstoffwerke AG, Linz, Austria), cocaine hydrochloride (Uquipa, Lisboa, Portugal), cortisol phosphate (Actocortin Vit6ria, Lisboa, Portugal), pargyline hydrochloride (Sigma, St. Louis, MO, USA), phentolamine hydrochloride (Regitin, Ciba, Switzerland), U-0521 (The Upjohn Co., Kalamazoo, MI, USA).

R e s u l t s

A. Spontaneousefflux

Under control conditions (tissue exposed to neither phentolamine nor clonidine) the rate of efflux from dog saphenous vein strips declined slowly between t = 100 and t = 150 rain (Fig. i and Table 1), with a half time of 124 min (k=0.00561 min-1). The "bound fraction" represented 90% of tissue content at t = 100 min of perifusion. Neither the half time nor the "bound fraction" was significantly altered by phentolamine or clonidine.

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Table 1. Influence of phentolamine (10 ~tmol/1) and clonidine (0.1 ~tmol/l) on tritium efflux induced by field electrical stimulation (between the 100th and 150th min of per• in dog saphenous vein strips. MAO, COMT, neuronal, and extraneuronal uptake were inhibited. Shown are arithmetic means _+ SE for TA (total activity) and BF% (bound fraction) and geometric means for k and t/2 values with 95% confidence limits

Experimental group n TA ~oo (rig - g- 1) BF% k (rain- 1) t/2 (rain)

Control

Spontaneous efflux 5 919.618 • 90.975 90.1 _ 1.5 (a) 0.00561 (n) 123.6 (0.00499; 0.00632)

0.5 Hz 5 1,053.369 _ 93.688 82.4 ___ 3.8 (b) 0.00494 (o) 140.3 (0.00396; 0.00617)

1.5 Hz 6 1,132.657 + 178.156 49.6 • 7.6 (c) 0.00454 (p) 152.7 (0.00364; 0.00567)

4.5 Hz 7 1,017.038 • 80.200 51.1 _+ 5.0 (d) 0.00940 (q) 73.7 (0.00868; 0.01017)

13.5 Hz 9 1,139.921 _+ 122.570 57.3 __+ 3.1 (e) 0.01267 (r) 54.7 (0.01147; 0.01400)

Phentolamine

Spontaneous efflux 5 981.429 _+ 191.958 89.5 _+ 1.4 (f) 0.00451 (s) 153.7 (0.00358; 0.00568)

0.5 Hz 6 1,055.011 + 122.700 52.9 _ 6.9 (g) 0.00719 (t) 96.4 (0.00471 ; 0.01097)

1.5 Hz 5 901.408 • 164.783 53.8 _+ 5.1 (h) 0.01384 (u) 50.1 (0.01109; 0.01727)

4.5 Hz 8 1,003.276 ___ 60.200 53.6 _ 4.3 (i) 0.02087 (v) 33.2 (0.01873; 0.02324)

13.5 Hz 7 1,154.332 • 121.769 50.5 _+ 2.2 (j) 0.02455 (w) 28.2 (0.02233; 0.02699)

Clonidine

Spontaneous effiux 8 725.802 _ 72.650 92.3 _+ 1.1 (k) 0.00720 (x) 96.3 (0.00659; 0.00785)

1.5 Hz 5 839.663 • 81.060 69.3 + 2.0 (1) 0.00585 (y) 118.5 0.00471 ; 0.00727)

4.5 Hz 5 974.681 + 56.780 59.4 _ 5.0 (m) 0.00950 (z) 73.0 (0.00816; 0.01105)

BF%, "bound fraction" % = BF/TA x 100; k, slope of the regression line for "In rate of efflux vs. time"; TA, total activity in the tissue at the 100th rain of per• t/2, half time. Significant were the differences between: (a) and (c); (a) and (d); (a) and (e); (b) and (d); (b) and (e); (f) and (h); (f) and (i); (f) and (j); (k) and (1); (n) and (r); (p) and (r); (s) and (v); (s) and (w); (t) and (v); (t) and (w); P < 0.001. (b) and (c); (f) and (g); (n) and (q); (o) and (q); (o) and (r); (p) and (q); P < 0.01. (s) and (u) P < 0.02. (c) and (1); (q) and (r); (t) and (u); (v) and (w) P < 0.05

B. Overflow evoked by f ield stimulation

Under control conditions (Fig. 1 A), electrical stimulation (from 0.5 Hz up to 13.5 Hz) markedly increased the rate o f efflux, the increase being dependent on the frequency of stimulation. The peak value of the rate of effiux was attained at about t = 110 min; thereafter the rate of efflux decayed monoexponentially until the end of per• The half time of effiux for 0.5 and 1.5 Hz was not statistically different from that for spontaneous efflux; with higher frequencies the half time decreased, the shortest value being obtained with 13.5 Hz. The rate of effiux increased with increasing frequency of stimulation. Electrical stimulation decreased the "bound fraction", to about 50% for all frequencies except for 0.5 Hz (Table 1).

Phentolamine increased the rate of effiux at all fre- quencies o f stimulation. After the peak value was attained, at about t = 110 min of per• a monoexponential decay of the rate of effiux was observed (Fig. 1 B). The half time of the effiux decreased with increasing frequency of stimulation from 96 min for 0.5 Hz, to 28 rain for 13.5 Hz (Table ~). Phentolamine also increased the rate o f efflux for all frequencies of stimulation. The "bound fraction" was similar

for all frequencies and about 50% of the TAloo value (Table 1). This value of the "bound fraction" was not statisti- cally different from the minimum value obtained under con- trol conditions.

With clonidine only two frequencies of stimulation were used (1.5 and 4.5 Hz). The rate of effiux was slightly decreased from the control value for either frequency. The "bound fraction" was increased for 1.5 Hz but not for 4.5 Hz when compared with control values at the same frequencies (Fig. 1 C and Table I). Thus no further stimulus frequencies were considered necessary.

Taken together, the results show that about 50% of 3H- noradrenaline could be mobilized when its release evoked by electrical stimulation exceeded a certain level, namely 2.5 ng�9 g - 1 . rain-1 in our experiments, which was reached at 1.5 Hz in controls, 0.5 Hz or less in the presence of phentolamine and 4.5 in the presence of clonidine (Fig. 1 and Table 1).

Discussion

Our results show that the spontaneous efflux o f 3H after the lOOth rain of perifusion (in the presence o f cocaine) had a

long half time (about 124 min) and most of the radioactivity (about 90%) accumulated in the strips did not contribute to the efflux (measured up to t = 150 min), i.e., remains as a "bound fraction". These kinetic characteristics are in agree- ment with those observed for 3H-noradrenaline of vesicular origin (Henseling et al. 1976; Eckert et al. 1976; Brandgo et al. 1981). Neither phentolamine nor clonidine modified significantly either the half time of spontaneous efflux or the "bound fraction"; similar results were obtained in the rabbit pulmonary artery (Starke et al. 1974).

Under control conditions (without phentolamine or clonidine) electrical stimulation at 0.5 Hz failed to affect the "bound fraction". At frequencies of 1.5 Hz or higher there was an increase of the releasable compartment, and the "bound fraction" decreased to a minimal value of about 50%. Under control conditions, this minimal value was reached with 1.5 Hz. Phentolamine increased the mobiliza- tion of releasable noradrenaline, the minimum of the "bound fraction" being already reached with 0.5 Hz. Clonidine, on the other hand, impaired the mobilization, so that 4.5 Hz were needed to attain the minimum of the "bound fraction". These results are in agreement with previous studies in which it was shown that the stores of adrenergic transmitter cannot be exhausted by prolonged electrical stimulation (Davies 1963; Chang and Chang 1965; Brand~o et al. 1980). Under our experimental conditions an increase of the frequency increased the rate of effiux and decreased the half time of effiux.

Results obtained in different tissues have shown that the release of noradrenaline induced by short periods of electrical stimulation was subjected to modulation via pre- synaptic e-receptors; antagonists increase and agonists re- duce the release (for review see Langer 1977, 1980; Starke 1977, 1981). This was also previously shown in the dog saphenous vein (Lorenz and Vanhoutte 1975; McGrath 1977; Guimarges et al. 1978; Lorenz et al. 1979) and was confirmed by our results, since phentolamine markedly in- creased the rate of effiux and clonidine produced a small opposite effect. Our results also showed that the presynaptic e-receptors function at all frequencies of stimulation used.

In contrast to the marked effect of phentolamine on the rate of efflux, the maximum size of the releasable compart- ment was not significantly modified by c~-adrenoceptor an- tagonism. In fact, at frequencies higher than 0.5 Hz the blockade of presynaptic e-receptors only decreased the half time of the efflux. Clonidine decreased the size of the releas- able compartment at 1.5 Hz of stimulation, but not at 4.5 Hz.

Thus, since the size of the releasable compartment is not modified by the presynaptic e-receptors, on what does it depend?

The following hypotheses should be considered: a) Perhaps a certain population of nerve tenrfinals was not stimulated due to preferential electrical current flow through the perifusion medium and not through the tissue. This hypothesis does not seem to apply as voltage increase above 100 V or a drastic reduction of the fluid volume around the tissue (see Methods) did not alter the "bound fraction". b) Since isolated preparations can synthesize noradrenaline (Stj/irne and Wennmalm 1970; Farnebo and Lidbrink 1971 ; Muscholl and Spira 1982), and since the newly synthesized transmitter is selectively released by electrical stimulation (Kopin et al. 1968), perhaps under our experimental con- ditions some 3H-noradrenaline would remain in the prepara-

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tion as a "bound fraction", c) Bearing in mind the critical role of calcium in the release of noradrenaline (Hukovi6 and Muscholl 1962; Kirpekar et al. 1972) perhaps under our experimental conditions the calcium concentration in the Krebs solution was insufficient for total mobilization of 3H- noradrenaline, d) Hughes (1973) suggested that electrical stimulation preferentially mobilizes the vesicles near the axonal membrane. In this case, a long period of electrical stimulation could deplete the peripheral vesicles if there is not enough time for migration of the more centrally located vesicles to the axonal membrane; this central pool of vesicles could be the "bound fraction". Further work is needed to distinguish between the last three possibilities.

In conclusion, these results show that the kinetic characteristics of the release of 3H-noradrenaline by electri- cal stimulation are remarkably different from those obtained with tyramine (Brand~o et al. 1981). In fact, while there is no tyramine-resistant pool of 3H-noradrenaline, with electrical stimulation about 50% of 3H-noradrenaline is not available for release. Bearing this in mind we are tempted to consider that while tyramine in sufficient concentrations can mobilize all stored 3H-noradrenaline, electrical stimulation cannot.

Acknowledgements. The authors are indebted to Dr. G. Johnson, The Upjohn Company, Kalamazoo, MI, USA, for his generous gift of U-0521. The technical assistance of Miss Maria Manuela Moura is gratefully acknowledged.

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Received June 12, 1984/Accepted October 1, 1984