hexamethonium, the effects of acetylcholine were on muscarinic receptors, probably

Transcription

1 J. Phyaiol. (1978), 279, pp With 8 text-figure8 Printed in Great Britain MODULATION OF CANINE ANTRAL CIRCULAR SMOOTH MUSCLE BY ACETYLCHOLINE, NORADRENALINE AND PENTAGASTRIN BY T. Y. EL-SHARKAWY AND J. H. SZURSZEWSKI From the Departments of Physiology, Biophysics and Pharmacology, Mayo Medical School, Rochester, Minnesota 5591, U.S.A. (Received 19 September 1977) SUMMARY 1. The effects of acetylcholine, noradrenaline and pentagastrin on the action potential of canine antral circular muscle were determined using the intracellular micro-electrode technique. 2. Acetylcholine increased the amplitude and duration of the plateau potential of the action potential. Since these effects were blocked by atropine but not by hexamethonium, the effects of acetylcholine were on muscarinic receptors, probably located on the smooth muscle cell. 3. Pentagastrin 2 x 1-1 M increased the size of the plateau potential and the frequency of the action potential; pentagastrin 1 x 1-9 M increased the frequency of the action potential complex and produced a marked diastolic depolarization between action potentials. The effect on the size of the plateau potential was biphasic. The amplitude and half-time duration of the plateau potential increased in the first 3 min, but thereafter, during steady-state conditions, they were the same as or slightly greater than those obtained in Krebs solution. 4. All the effects produced by pentagastrin were due to a direct action on the smooth muscle cell. 5. Noradrenaline decreased the size of the plateau potential but increased its frequency; high concentrations ( > 1-5 M) additionally produced a diastolic depolarization between action potentials. These effects were mediated primarily by a-adrenoceptors presumably located on the smooth muscle cell. 6. It was concluded that the substances studied primarily alter the size of the plateau potential in antral circular muscle. Since phasic contractions are associated with the plateau potential, it is suggested that agents which increase the size of the plateau potential increase the force of the contraction whereas agents which decrease the size of the plateau potential have the opposite effect. INTRODUCTION Vagal (Daniel & Sarna, 1976) and splanchnic nerve stimulation (Brown, McSwiney & Wadge, 193; Furness & Costa, 1974) and gastrointestinal hormones (Isenberg & Grossman, 1969; Sugawara, Isaza & Woodward, 1969; Szurszewski, 1975) alter gastric motor activity. The intracellular potential changes which underlie the effects of the neurotransmitters and hormones have not been previously studied. This paper describes the effects which acetylcholine, noradrenaline and pentagastrin have on

2 31 T. Y. EL-SHARKA WVY AND J. H. SZURSZEWSKI the action potential which occurs in circular muscle of the proximal canine antrum. We studied this region of the stomach because, unlike in more distal regions of the antrum and pylorus, spike potentials do not normally occur on top of the plateau potential (El-Sharkawy, Morgan & Szurszewski, 1978). Some of the results have been previously communicated (El-Sharkaway & Szurszewski 1976). METHODS Sixty-eight dogs of either sex weighing 1-22 kg were used. Under pentobarbitone (Fort Dodge Laboratories, Inc) anaesthesia, the abdomen was cut open and the whole stomach removed. The methods used to isolate strips of antral circular muscle and to record intracellularly from single muscle fibres have been described by El-Sharkawy et al. (1978). The standard solution used was a modified Krebs solution containing (mm): Na+, 137-4; K+, 5-9; Ca2+, 2-5; Mg2+, 1-2; Cl-, 134; HC3-, 15-5; H2P4-, P-2; dextrose, It was equilibrated with a 97 % 2-3 % CO2 gas mixture and at 37 C had a ph of The drugs used were acetylcholine chloride (Sigma), atropine sulphate (Sigma), hexamethonium bromide (Sigma) L-noradrenahne bitartrate (NA), phentolamine mesylate (CIBA), DL-propanolol hydrochloride (Sigma) and pentagastrin (Sigma). During an experiment, the change from one Krebs solution to another containing a hormone or drug was made by means of a Y-valve located on the upstream side of a flow control valve. RESULTS Effects of acetylcholine Fig. 1 illustrates the effect of 5 x 1-8 M-acetylcholine on the gastric action potential. Acetylcholine increased the amplitude and duration of the plateau potential from 23 to 29 and from 6-7 to 8-1 sec. respectively. In twenty-seven preparations this concentration of acetylcholine increased the amplitude of the plateau potential from to (mean, SE. of mean) and the duration from to sec (mean, S.E. of mean). These increases were statistically significant (P < -5). Observation through a dissecting microscope showed that phasic contractions occurred in association with the action potential and persisted while the concentration of acetylcholine was maintained constant in the bathing medium. In one experiment, we studied the effect of a prolonged exposure to acetylcholine. In this experiment, the effect of acetylcholine on the configuration of the action potential remained the same 1-5 hr after adding acetylcholine as it was soon after adding the agonist. The upstroke potential and resting membrane potential were not significantly affected (P > -5) by this or lower concentrations of acetylcholine (5 x 1-8 to 5 x 11 M). It proved difficult to study intracellularly the effect of higher concentrations of acetylcholine because the increased force of contraction dislodged the electrode from the cell immediately following the upstroke of the first action potential after the onset of action of acetylcholine. The effects of acetylcholine were unaffected by hexamethonium (1-8 M) but were completely blocked by atropine (Fig. 2). Effects of noradrenaline Noradrenaline (1-5 m) reduced the size of the action potential plateau and increased its frequency (Fig. 3). The mean decreases in the amplitude of the plateau potential and half-time duration were (S.E. of mean, n = 14) and sec

3 MODULATION OF GASTRIC SMOOTH MUSCLE 311 (s.e. of mean, n = 14), respectively. In addition, noradrenaline produced an afterhyperpolarization which followed the repolarization of the plateau potential. In most preparations (twenty-six of twenty-nine), neither the resting membrane potential nor the action potential upstroke were affected by noradrenaline (Fig. 3). In the three exceptions, noradrenaline hyperpolarized the membrane by up to 9 (Fig. 4), and although the plateau potential was reduced the increase in frequency was Krebs soluution I: A Acetylcholine, 5x1 R M. I B -5 C Krebs solution I -5 1 sec Fig. 1. Effect of acetylcholine on the configuration of the action potential obtained with intracellular micro-electrode in a single cell. A, action potential in Krebs solution; B, 5 min after adding acetylcholine (5 x 1-8 M); a, 1 min after washing out acetylcholine. In B, note increase in size of plateau potential. In this and subsequent figures, top of voltage calibration denotes zero potential. much less ( 1-9 action potential/min) than in preparations where the membrane potential was not affected. Also, in these preparations, the size of the upstroke potential increased by an amount equal to the hyperpolarization. Noradrenaline (2 to 5 x 1-5 M) initiated a diastolic deplorization between action potentials which rose at a mean rate of 15 /sec (n = 14) (cf. Fig. 5C). The ax-adrenoceptor blocker phentolamine or f8-adrenoceptor blocker propranolol reduced the effects of noradrenaline on the configuration and frequency of the action potential. This could be best illustrated with both antagonists studied together. A

4 312 T. Y. EL-SHARKAWY AND J. H. SZURSZEW}rSKI typical example is shown in Fig. 5. A high concentration of noradrenaline (2-5 x 1-5 M) markedly reduced the size of the plateau potential and increased the frequency of the action potential (Fig. 5A). Propranolol (2 x 1-5 M) alone reduced Krebs solution A _-L -5 Atropine, 3x1 6 M B I-5 Atropine+ACh, 5x 1 8 M -*1 C _ --5 Krebs solution -r D L5 1 sec Fig. 2. Effect of pretreatment with atropine on acetvlcholine-induced changes. A, action potential in Krcbs solution: B, 5 min after adding atropine (3 x 16 M);, 15 mm after adding atropine and 5 min after adding acetylcholine and D, 4 min after washing out both drugs. the increase in the frequency caused by noradrenaline and partially restored the size of the plateau potential (Fig. 5C). When phentolamine (1-5 M) was also added, the frequency of the action potential and the size of the plateau potential were restored

5 MODULATION OF GASTRIC SMOOTH MUSCLE 313 to those observed in normal Krebs solution (Fig. 5E). The duration of the action potential in the presence of both blockers (Fig. 5E) was longer compared to the duration in Krebs solution (Fig. 5A). This was observed in two of six experiments. The reason for this prolongation is not understood. When phentolamine was added alone, either before or during the action of noradrenaline, the effects of noradrenaline were completely blocked. The effect of phentolamine and propranolol on the membrane hyperpolarization observed in some cells was not studied because this effect was not commonly observed. BEU 15 -~~~~~~~~~~~~~~~~~~7 Noradrenaline, 1 >1 m c uiri 5 T _5 Fig. 3. Effect of noradrenaline on frequency and configuration of action potentials. A, Krebs solution; B, effect of noradrenaline (1-5 M); note decrease in size of plateau potential. C and D after washing out noradrenaline. Traces are continuous. Effects of pentagastrin In the longitudinal muscle of the canine antrum, pentagastrin acts directly on the smooth muscle cell membrane to increase the frequency of the gastric action potential (ED5O 2 x 1-1 M maximum effect at around 1-9 M; Szurszewski, 1975). In the present study, these two concentrations of pentagastrin were studied in the circular muscle layer. The effect of the low concentration of pentagastrin (2 x 11 M) is shown in Fig. 6. Pentagastrin increased the frequency of the action potential and the amplitude and duration of its plateau potential. In thirty-two preparations, the frequency of the action potential increased from to c/min (mean, + S.E. of mean), the plateau amplitude increased from to (mean, s.e. of mean) and the half-time duration increased from to see (mean s.e. of mean). Observation through a dissecting microscope showed that phasic contractions occurred in association with the action potential. These effects min

6 314 T. Y. EL-SHARKA WVY AND J. H. SZURSZEWSKI persisted for as long as pentagastrin was present. There was no change in the amplitude of the upstroke potential or in the resting membrane potential. The higher concentration of pentagastrin (1-9 M; Fig. 7) increased the frequency of the action potential to 6/min, caused a depolarization of 2 and produced a diastolic depolarization of up to 14 between action potentials. The upstroke r A 1-5 B --5 Noradrenaline, 1; 1 5 M 1- I C..1-5 I 5 D _L_ 5 1 min Fig. 4. Effect of noradrenaline on frequency and configuration of action potential during noradrenaline-induced hyperpolarization. A, Krebs solution; B, noradrenaline (1-5 M) added at arrow, withdrawn at arrow in C; D and E after washing out noradrenaline. Impalement lost at end of panel (E). For details, see text. potential was not altered. The mean increases in the amplitude of the plateau potential and half-time duration were (S.E. of mean, n = 11) and *3 see (S.E. of mean, n = 11), respectively. However, the effects of this higher concentration of pentagastrin on the size of the plateau potential were biphasic for 2-3 min after adding pentagastrin; the half-time duration and amplitude of the plateau progres-

7 MODULATION OF GASTRIC SMOOTH MUSCLE 315 sively declined; the amplitude became the same as or slightly higher than that in normal Krebs solution, whereas the duration was shorter. After washing out pentagastrin, particularly the higher concentration (Fig. 8), the rate of rise of the diastolic depolarization progressively slowed and the frequency of the action potential concomitantly declined. During some periods a diastolic A II --5 B IL C Noradrenaline, 2-5x1 5 M -5 4 Propranolol, 2 x 1 5 M D E 1T L_5 -*Phentolamine,1 x 1 5M --*ww@ 1 min Fig. 5. Effect of adrenoceptor blockade on response to noradrenaline. A, Krebs solution; B, noradrenaline (2.5 x 1O-5 M) added at arrow and maintained throughout experiment; C, propranolol (2 x 1-5 M) added at arrow and maintained throughout experiment; D, 5 min after adding propranolol; E, 5 min after adding phentolamine (1-5 M). Recordings from same cell. For details, see text. depolarization did not lead to an action potential (Fig. 8B). With return to normal, the diastolic depolarization disappeared and the membrane was isopotential between action potentials (Fig. 8 D). Pentagastrin and gastrin can act on the intramural cholinergic neurones to release acetylcholine in some preparations (Gregory & Tracy, 1964; Bennett, 1965; Jacoby &

8 316 T. Y. EL-SHARKA WY AND J. H. SZURSZEWSKI Marshall, 1969; Vizi, Bertaccini, Impicciatori & Knoll, 1973; Szurszewski, 1975). However, atropine (3 x 1-6 M) or tetrodotoxin (1 x 1- g/ml.) had no effect on the electrophysiological responses to pentagastrin. A l1-5 l Pentagastrin, 2x1 1 M T C 1_5 D 5 m 1 min Fig. 6. Effect of a low concentration of pentagastrin on the frequency and configuration of the action potential. A, Krebs solution; B, pentagastrin (2 x 1-1 M) added at arrow and maintained throughout C and D. Traces continuous. I DISCUSSION The main findings of this study were that acetylcholine and pentagastrin increased the size of the plateau potential of the gastric action potential whereas noradrenaline reduced the size of the plateau potentials. The electrical changes produced by acetylcholine were similar to those previously found in longitudinal antral muscle (Szurszewski, 1975) and were restricted to the plateau potential. It therefore seems reasonable to suggest that electromechanical coupling in canine antral circular muscle during stimulation by acetylcholine takes place via the plateau potential. The question arises as to whether the increase in the amplitude of the plateau potential causes contraction or whether it increases the force of contraction. In previous studies on the longitudinal muscle of the canine antrum in which the double sucrose gap technique was used to measure simultaneously potential and tension, it was found that spontaneous action potentials do not trigger contractions (Szurszewski, 1975). In the present study electrical activity of the circular muscle was studied using the intracellular microelectrode technique while mechanical

9 MODULATION OF GASTRIC SMOOTH MUSCLE 317 A HI 1Pentagastrin, I x 1 9 M - r B I~~~~~~~~~ 1 min Fig. 7. Effect of pentagastrin (1-9 M) on the frequency and configuration of the action potential. In A, pentagastrin added at arrow and maintained through panel B. Traces continuous. Note increase in frequency associated with the occurrence of a diastolic depolarization between action potentials. A I MA MA W-A Pentagastrin, 1 x 1 M T o B -5 -L -5 C D l- 5 1 min Fig. 8. Change in frequency and configuration of action potential following exposure to pentagastrin (1-9 M). In A, pentagastrin withdrawn at arrow. In rest of panel and B to D, recovery in Krebs solution. During recovery, decrease in frequency associated with decrease in slope and amplitude of diastolic depolarization. Frequency in D same as frequency before adding pentagastrin.

10 318 T. Y. EL-SHARKA W Y AND J. H. SZURSZEWSKI activity was studied by visual observation through the dissecting microscope. Muscle shortening (contraction) was seen to occur with each action potential during the presence of either acetylcholine or pentagastrin. No muscle contraction was detected during spontaneous action potentials. Since shortening of the muscle strip was documented only by visual observation, only strong drug induced contractions were detected. In the absence of drugs there may have been weaker contractions which were undetectable. Therefore, it should not be inferred that the plateau potential of the spontaneous action potential in circular muscle strips does not trigger spontaneous contraction as is the case in longitudinal muscle. Although pentagastrin caused a sustained increase in the frequency of the action potential for the duration of contact, the increase in the plateau potential was maintained only with the lower concentration of 2 x 1-1 M. Recently, we have noticed that the amplitude and duration of plateau potential decreases when frequency increases above approximately 3/min (K. G. Morgan & J. H. Szurszewski, unpublished observations). Alternatively the decline might be due to autoinhibition. Gastric acid secretion becomes depressed after prolonged stimulation by pentagastrin (Prugh, Schorr, Vlahuvic & Maklouf, 1975) suggesting that the peptide at first binds to receptors with higher affinity and efficacy and then later to other receptors with lower affinity and efficacy. Although pentagastrin mimics the increase in size of the plateau potential produced by acetylcholine, pentagastrin did not seem to act by releasing acetylcholine or stimulating nerves since atropine or tetrodotoxin did not alter this effect. The ability of pentagastrin to contract the circular muscle directly is a major difference from the longitudinal antral muscle where pentagastrin stimulates release of acetylcholine from intramural cholinergic neurones (Szurszewski, 1975). Further studies are needed in the circular muscle to determine if the same receptor mediates both chronotropic and inotropic responses to pentagastrin. The diastolic depolarization between action potentials with the high concentration of pentagastrin (and noradrenaline) was seldom observed in normal Krebs solution (El-Sharkawy et al. 1978). The primary mechanism which initiates the gastric action potential is not known, but it is spontaneous (1-2/min) and voltage-sensitive (Szurszewski, 1975). Perhaps the diastolic depolarization acts as a trigger and induced changes in frequency depend upon the slope of the diastolic depolarization. The site of action of noradrenaline in the gastrointestinal tract is disputed. Gershon (1967) found that stimulation of sympathetic nerves at frequencies sufficient to relax the guinea-pig stomach did not reduce the output of acetylcholine but Vizi et al. (1973) obtained a reduction in acetylcholine output. It has been suggested that noradrenaline inhibits the release of acetylcholine from Auerbach's plexus by an action on az-adrenoceptors (Vizi, 1973, 1976). Since the effects of noradrenaline in our experiments were more sensitive to a-blockade than fl-blockade, a-adrenoceptors are primarily involved. These observations agree with in vivo data in the dog (Daniel, 1965). The a-adrenoceptors may be located postsynaptically. Since tetrodotoxin and atropine alone or in combination have no effect on the frequency or configuration of the spontaneous action potential (El-Sharkawy, et al. 1978), any acetylcholine released must be too low to effect the action potential. Therefore, noradrenaline could not act by inhibiting acetylcholine release. Alternatively, noradrenaline may release an inhibitory transmitter from other intramural nerve terminals (Stockley & Bennett, 1977).

11 MODULATION OF GASTRIC SMOOTH MUSCLE 319 Transmural field stimulation of canine antral muscle indicates that the intramural nerve network consists of cholinergic and noradrenergic nerve terminals and nerve terminals which contain a powerful inhibitor of antral contractions (P. Schmalz & J. H. Szurszewski, unpublished observations) which might be a purine nucleotide, ATP (Burnstock, 1972). We did not study the effects of exogenously added ATP but if 'purinergic' nerves are present in the canine antrum, then our data would predict that like noradrenaline, ATP would inhibit antral motility by decreasing the size of the plateau potential. In summary, the data suggest that in canine antral circular muscle, changes in the size of the plateau potential can be used to predict the corresponding effects on motility. An increase in size of the plateau potential should increase the force of contraction whereas a decrease should result in weaker gastric contractions. Further studies using substances which alter gastric motor activity are needed to establish the validity of this hypothesis. This work was done during the tenure of an Established Investigatorship of the American Heart Association (J. H. S.) and was supported by Research Grant AM from the National Institutes of Health, Public Health Service. Dr El-Sharkawy was supported by a Mayo Foundation Fellowship. The authors express their appreciation to Cynthia Schmidt for technical assistance. REFERENCES BENNETT, A. (1965). Effect of gastrin on isolated smooth muscle preparations. Nature, Lond. 28, BROWN, G. L., MCSWINEY, B. A. & WADGE, W. J. (193). The sympathetic innervation of the stomach. J. Physiol. 7, BURNSTOCK, G. (1972). Purinergic nerves. Pharmac. Rev. 24, DANIEL, E. E. (1965). Electrical and contractile responses of the pyloric region to adrenergic and cholinergic drugs. Can. J. Physiol. Pharmacol. 44, DANIEL, E. E. & SARNA, S. K. (1976). Distribution of excitatory vagal fibers in canine gastric wall to control motility. Ga8troenterology 71, EL-SHARKAWY, T. Y., MORGAN, K. G. & SZURSZEWSKI, J. H. (1978). Intracellular electrical activity of canine and human gastric smooth muscle. J. Phy8iol. 279, EL-SHARKAwY, T. Y. & SZURSZEWSKI, J. H. (1976). Spontaneous electrical activity of canine antral smooth muscle and its modulation by neurotransmitters. Fedn. Proc. 35, 33. FuRWNESS, J. B. & COSTA, M. (1974). The adrenergic innervation of the gastrointestinal tract. Ergebn. Phy8iol. 69, GERSHON, M. D. (1967). Inhibition of gastrointestinal movement by sympathetic nerve stimulation: The site of action. J. Phygiol. 189, GREGORY, R. A. & TRACY, H. J. (1964). The constitution and properties of two gastrins extracted from hog antral mucosa. I: The isolation of two gastrins from hog antral mucosa. II. The properties of two gastrins isolated from hog antral musoca. Gut 5, ISENBERG, J. I. & GROSSMAN, M. I. (1969). Effect of gastrin and SC on gastric motility in dogs. Gaatroenterology 56, JACOBY, H. I. & MARSHALL, C. H. (1969). Gastric motor-stimulating activity of gastrin tetrapeptide in dogs. Gastroenterology 56, PRUGH, M. F., SCHORR, B. A., VLAHUVIC, Z. R. & MAKLOUF, G. M. (1975). Intravenous pentagastrin as a partial agonist of gastric secretion in man: evidence in favour of the existence of hormonal inhibitory sites. Gastroenterology 68, STOCKLEY, H. L. & BENNETT, A. (1977). Relaxations mediated by adrenergic and non-adrenergic nerves in human isolated taenia coli. J. Pharm. Pharmac. 29, SUGAWARA, K., ISAZA, J. & WOODWARD, E. R. (1969). Effect of gastrin on gastric motor activity. Gastroenterology 57,

12 32 T. Y. EL-SHARKA WY AND J. H. SZURSZEWSKI SZURSZEWSKI, J. H. (1975). Mechanism of action of pentagastrin and acetylcholine on the longitudinal muscle of the canine antrum. J. Physiol. 252, Vizi, E. S. (1973). Acetylcholine release from guinea-pig ileum by parasympathetic ganglion stimulants and gastrin-like polypeptides. Br. J. Pharmac. Chemother. 47, Vizi, E. S. (1976). The role of a-adrenoceptors situated in Auerbach's plexus in the inhibition of gastrointestinal motility. In Physiology of Smooth Muscle ed. BULBRING, E. & SHUBA, M. F., pp New York: Raven Press. Vizi, S. E., BERTACCINI, G., IMPIccIATORE, M. & KNOLL, J. (1973). Evidence that acetylcholine released by gastrin and related polypeptides contributes to their effect on gastrointestinal motility. Gastroenterology 64,