Effects of bombesin and gastrin releasing peptide on catecholamine secretion from rat adrenal gland, in vitro

The effects of bombesin and gastrin releasing peptide (GRP) on the release of catecholamine were investigated by using isolated rat adrenal gland. Bombesin and GRP stimulated an epinephrine (E) release with dose-dependency. A half maximal effect of bombesin was observed at 1.2 X 10(-9) M, and a maximal release of E occurred at 1 X 10(-6) M of bombesin. The stimulatory effect of GRP on the E release was very similar to that of bombesin. Although both these peptides also stimulated a norepinephrine (NE) release, a significant effect was detected at concentrations of bombesin and GRP above 1 X 10(-7) M. Nicotine and pilocarpine stimulated both E and NE releases dose dependently, but the effect of pilocarpine on E and NE release was 1/100 or less potent than that of nicotine. Bombesin-induced catecholamine releases were not inhibited by hexamethonium or atropine that fully impeded the stimulatory effects of nicotine or pilocarpine. In addition, bombesin had additive effects on the nicotine- or pilocarpine-induced E and NE releases. These data strongly suggest that bombesin or GRP plays a physiological role as one of the important regulators in catecholamine secretion in the adrenal gland.     

Differential regulation of smooth muscle contraction in rabbit internal anal sphincter by substance P and bombesin.

Substance P and bombesin induce contraction of isolated IAS smooth muscle cells by different intracellular mechanisms. The cells contracted in a dose dependent manner to both peptides. The kinetics of contraction were different. Substance P induced contraction peaked at 30 seconds and declined in a time dependent manner while bombesin induced contraction peaked at 30 seconds and was maintained for up to 8 minutes. The absence of extracellular calcium in the medium (0 calcium and 2 mM EGTA) had no affect on substance P induced contraction while it blocked bombesin induced contraction. Substance P induced contraction was blocked by the calmodulin antagonist W7 (10(-9)M) and was not affected by the PKC antagonist H7 (10(-6)M). Bombesin induced contraction was blocked by the PKC antagonist H7 and was not affected by the calmodulin antagonist W7. Our data indicate that substance P induces a transient contraction utilizing intracellular calcium and a calmodulin dependent pathway, while bombesin induces a sustained contraction utilizing calcium from extracellular sources and a calmodulin independent pathway.     

Neural control of relaxation in cat airways smooth muscle

Functional innervation of cat airways smooth muscle was examined in isolated segments of trachea and bronchi using electrical field stimulation (EFS) techniques. Field stimulation caused contraction in tissues at resting tone and biphasic responses (contraction followed by relaxation) in tissues precontracted with 5-hydroxytryptamine (5-HT). Contractions were abolished by 10(-6) M atropine. Inhibitory responses were dependent on impulse voltage, duration, and frequency. At low voltages (less than or equal to 10 V) and pulse durations (less than or equal to 0.3 ms), EFS induced relaxations were abolished by 3 X 10(-6) M tetrodotoxin (TTX). Greater stimulus parameters elicited TTX-resistant relaxations. Pretreatment of the tissues with 10(-6) M propranolol and 10(-5) M guanethidine caused rightward shifts in relaxation frequency-response curves. These findings indicate that cat airways are innervated by excitatory cholinergic, inhibitory adrenergic, and inhibitory nonadrenergic noncholinergic (NANC) nerves. Pretreatment of the tissues with hexamethonium, cimetidine, indomethacin, or nordihydroguaiaretic acid did not affect NANC relaxation responses. It is concluded that NANC inhibitory responses in cat airway smooth muscle are mediated through intrinsic postganglionic nerve fibers and occur independently of histamine H2-receptor activation and without involvement of cyclooxygenase or lipoxygenase products of arachidonic acid metabolism.     

Neurotransmitters in the intestine of the Atlantic cod, Gadus morhua

The effects of the putative neurotransmitters acetylcholine, adrenaline, adenosine, ATP, bombesin, 5-hydroxytryptamine, met-enkephalin, neurotensin, somatostatin, substance P and VIP have been investigated in the perfused intestine of the cod, Gadus morhua. The presence and distribution of the different types of nerves was investigated with immunohistochemistry and Falck-Hillarp fluorescence histochemistry. A spontaneous rhythmic activity of the perfused preparations usually occurred within a few minutes from the start of the experiment. This activity was diminished or abolished by addition of atropine, methysergide or tetrodotoxin to the perfusion fluid. Acetylcholine, 5-hydroxytryptamine or substance P caused a contraction of the intestinal wall. The response to acetylcholine was blocked by atropine but not by tetrodotoxin, while the response to 5-hydroxytryptamine was blocked by methysergide and usually also by tetrodotoxin. This indicates that the effect of acetylcholine is direct on the muscle cells, while the effect of 5-hydroxytryptamine may be at least partly via a second neuron. All adrenergic agonists (adrenaline, isoprenaline and phenylephrine) had a dominating inhibitory effect on the intestine. Experiments with antagonists showed that the inhibition is due to stimulation of both alpha-adrenoceptors and beta-adrenoceptors. ATP, adenosine and somatostatin also caused a relaxation of the intestinal wall, often followed by a contraction. Met-enkephalin produced variable responses, either a relaxation, a contraction or both. Bombesin caused a weak inhibition, if anything. Neurotensin and VIP did not visibly affect the intestinal motility. 5-HT-, substance P- and VIP-like immunoreactivity and catecholamine fluorescence were observed in the myenteric plexus, submucosa and muscle layers in all parts of the intestine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of VIP and NO on the motor activity of vascularly perfused rat proximal colon

The effects of vasoactive intestinal polypeptide (VIP) and nitric oxide (NO) on the motor activity of the rat proximal colon were examined in an ex vivo model of vascularly perfused rat proximal colon. VIP reduced motor activity and this inhibitory effect was not altered by either atropine, hexamethonium, tetrodotoxin (TTX) nor TTX plus acetylcholine (ACh), but was completely antagonized by NO synthase inhibitor N(G)-nitro-L-arginine (L-NA) and by VIP receptor antagonist, VIP(10-28). These results suggest that VIP may exert a direct inhibitory effect on the motor activity of the rat proximal colon via a VIP receptor located on the smooth muscle and this effect is mediated by NO but not by cholinergic pathways. Atropine and hexamethonium reduced but ACh stimulated motor activity and the effect of ACh was not changed by TTX, suggesting that the cholinergic pathway may exert a direct stimulatory effect on motor activity. Single injection of TTX, VIP(10-28) or L-NA induced a marked increase in motor activity, suggesting that the motor activity of rat proximal colon is tonically suppressed by VIP and NO generating pathways, and elimination of inhibitory neurotransmission by TTX may induce an abnormal increase of the motor activity. The interaction between VIP and NO in regulation of motor activity was further examined by a measurement of NO release from vascularly perfused rat proximal colon. Results showed that NO release was significantly increased during infusion of VIP and this response was reversed by L-NA. These results suggest that VIP generating neurons may inhibit colonic motility by stimulating endogenous NO production in either smooth muscle cells or nerve terminals.     

Bombesin potentiates the effect of acetylcholine on isolated strips of fish stomach.

The interaction between bombesin and acetylcholine acting on smooth muscle of the stomach wall was investigated in two species of teleost fish. Oncorhynchus mykiss (rainbow trout) and Gadus morhua (Atlantic cod). Acetylcholine or bombesin alone has an excitatory effect on the stomach muscle. The effect on contraction amplitude of acetylcholine (10(-6)-10(-5) M) alone is about 10-times greater than the effect of bombesin (10(-9)-10(-7) M). In molar terms however, bombesin is more potent than acetylcholine. Bombesin (10(-8)-10(-7) M) added 0.5-3 min prior to acetylcholine potentiates the effect of acetylcholine in a dose-dependent manner. The potentiation is most pronounced in circular muscle preparations, but is present also in longitudinal muscle preparations. Bombesin affects the response to carbachol (10(-6) M) with a similar potentiation, indicating that the potentiation is not caused by inhibition of choline esterase activity. Atropine (10(-6)-10(-5) M) abolishes the response to bombesin plus acetylcholine as well as the response to acetylcholine alone. Tetrodotoxin (10(-6) M) does not block the effect of acetylcholine, bombesin or the combination acetylcholine plus bombesin. Substance P (10(-9)-10(-7) M) which has a similar excitatory effect on the stomach muscle as bombesin, does not potentiate the effect of acetylcholine. Immunohistochemistry has shown the presence of strong bombesin-like immunoreactivity in stomach nerves of the cod and weak bombesin-like immunoreactivity in rainbow trout nerves. In addition, bombesin-like immunoreactivity was demonstrated in endocrine cells in the gastric and intestinal mucosa of both species. It is concluded that bombesin, contained either in nerve fibres or in mucosal endocrine cells, specifically potentiates the effect of acetylcholine in the fish stomach.     

Immunohistochemical localization of substance P, vasoactive intestinal polypeptide and gastrin-releasing peptide in vas deferens and seminal vesicle, and the effect of these and eight other neuropeptides on resting tension and neurally evoked contractile activity

Immunohistochemical studies of the vas deferens and seminal vesicle of mouse, guinea-pig, and rabbit showed the presence of nerve fibres containing vasoactive intestinal polypeptide (VIP), substance P (SP), and gastrin-releasing peptide (GRP) supplying the smooth muscle layers as well as blood vessels. The nerve supply was better developed in the seminal vesicle than in the vas deferens. The motor activity of the vas deferens and seminal vesicle of the guinea-pig was studied in vitro. The vas deferens responded to transmural electrical stimulation with a twitch followed by a slow contraction. The twitch was blocked by guanethidine and tetrodotoxin, but not by atropine, propranolol, phenoxybenzamine, or fluphenazine. The slow contraction exhibited features of an alpha-receptor-mediated response. SP, physalaemin and eledoisin contracted the smooth muscle and also potentiated the twitch response to electrical nerve stimulation in a concentration-dependent manner. The SP blocking agent, (D-Pro2,D-Trp7,9)-SP, affected neither the resting tension nor the response to electrical stimulation. It is therefore suggested that the SP fibres act mainly prejunctionally. VIP, Leu-enkephalin, cholecystokinin octapeptide (CCK-8), angiotensin II, vasopressin, neurotensin, bombesin, and GRP had no effect on either the resting tension or the response to electrical nerve stimulation. The seminal vesicle responded to electrical stimulation with a contraction which was unimpaired by atropine, propranolol, phenoxybenzamine, and guanethidine, but abolished by tetrodotoxin. Hence, this contraction is mediated by a non-adrenergic, non-cholinergic neurotransmitter. Bombesin, GRP, SP, physalaemin and eledoisin contracted the smooth muscle and potentiated the response to electrical stimulation. VIP, Leu-enkephalin, CCK-8, angiotensin II, vasopressin, and neurotensin had no effect on the resting tension or on the response to transmural electrical stimulation. The SP antagonist abolished the contraction elicited by SP but did not influence the response to nerve stimulation. The results suggest that the SP and GRP nerves may have prejunctional and facilitating postjunctional effects in the seminal vesicle.     

The effects of substance P and met5-enkephalin in dog ileum

Substance P initiated tonic contraction of dog ileum when administered in doses from 1 pg to 20 micrograms intraarterially (ED50 = 67 ng). Low doses acted to excite cholinergic postganglionic neurones since atropine or tetrodotoxin (TTX) increased the ED50 of substance P about 25-fold, while hexamethonium and local field stimulation had only a small effect to increase the ED50. Also atropine and tetrodotoxin effects were not additive. Higher doses apparently acted to stimulate smooth muscle directly, but no evidence was obtained that local field stimulation could release substance P to act on smooth muscle. Substance P tachyphylaxis prevented substance P actions on cholinergic nerves, but it did not affect responses to intraaterial acetylcholine or block distal inhibition from proximal distention or field stimulation. Met-enkephalin given intraarterially, was also excitatory in doses from 1 ng to 20 micrograms; the amplitude of tonic and phasic contractions produced was significantly decreased by TTX and atropine but was not diminished by hexamethonium or substance P tachyphylaxis. Partial tachyphylaxis to met-enkephalin was produced but was not diminished by hexamethonium or substance P tachyphylaxis. Partial tachyphylaxis to met-enkephalin was produced without affecting the ED50 for substance P. We conclude that substance P acts in small amounts on receptors in myenteric nerves to release acetylcholine by a mechanism, presumably involving postganglionic cholinergic nerves, while met-enkephalin also apparently may act at least in part through a similar TTX- and atropine-sensitive mechanism. These peptides also caused activation of other receptors, probably on smooth muscle by noncholinergic. TTX-insensitive mechanisms. Also the receptors for each peptide which are located on nerves were distinct and independent since tachyphylaxis could be produced to each without affecting the response to the other.     

Enteric opioid neurons modulate the basal tone of the isolated puppy ileum

We studied the role of enteric opioid neurons in the spontaneous motility of the longitudinal muscle in the isolated puppy ileum. Regular fluctuations in tone that rose above and returned to the basal level occurred at an interval of 4.7 +/- 0.3 min. Naloxone (10(-8) and 10(-7) M) reduced the spontaneous tonic contraction by 42.6 +/- 11.6% (p less than 0.02) and 77.0 +/- 3.6% (p less than 0.001), respectively. Tetrodotoxin (3.1 X 10(-7) M) and atropine (10(-7) M) terminated the fluctuations. Met- and Leu-enkephalins (10(-9)-10(-8) M) caused tonic contraction which was abolished by tetrodotoxin and atropine. The contractile response produced by transmural electrical stimulation was reduced by naloxone (10(-7) M). This response was also abolished by atropine and tetrodotoxin. These results suggest that enteric opioid neurons are spontaneously active and might operate, at least in part, to raise the basal tone of the longitudinal muscle in the puppy ileum through a cholinergic excitatory mechanism.     

Galanin: an inhibitory neural peptide of the canine small intestine

Galanin injected intraarterially during phasic activity of the canine small intestine in vivo produced inhibition. Fifty percent inhibition occurred at 1.5 +/- 0.5 X 10(-10) mols lasting for 0.7 min. The inhibitory response was not decreased by treatment with atropine, hexamethonium, yohimbine or naloxone, suggesting that muscarinic, nicotinic, alpha 2 adrenergic or opiate receptors were not being stimulated. Since tetrodotoxin blockade of nerves did not reduce the response and galanin at 10(-10) mols was able to eliminate the smooth muscle response to intraarterial acetylcholine, we suggest that galanin acts to inhibit smooth muscle directly. Galanin 10(-9) M added to the muscle bath also inhibited phasic activity of the canine ileum circular muscle in vitro in the presence of tetrodotoxin. These results suggest that the neural peptide galanin may be a non-adrenergic, non-cholinergic, non-opioid neurotransmitter in the canine small intestine.     

Inhibitory actions of eugenol on rat isolated ileum

The effects of eugenol (1-2000 microM) on rat isolated ileum were studied. Eugenol relaxed the basal tonus (IC50 83 microM) and the ileum precontracted with 60 mM KCl (IC50 162 microM), an action unaltered by 0.5 microM tetrodotoxin, 0.2 mM N(G)-nitro-L-arginine methyl ester, 0.5 mM hexamethonium, and 1 microM indomethacin. Eugenol did not alter the resting transmembrane potential (Em) of the longitudinal muscle layer under normal conditions (5.0 mM K+) or in depolarised tissues. Eugenol reversibly inhibited contractions induced by submaximal concentrations of acetylcholine (ACh) and K+ (40 mM) with IC50 values of approximately 228 and 237 microM, respectively. Eugenol blocked the component of ACh-induced contraction obtained in Ca(2+)-free solution (0.2 mM EGTA) or in the presence of nifedipine (1 microM). Our results suggest that eugenol induces relaxation of rat ileum by a direct action on smooth muscle via a mechanism largely independent of alterations of Em and extracellular Ca2+ influx.     

Cooling-induced contraction of guinea pig tracheal smooth muscle

Cooling of isolated guinea pig tracheal smooth muscle from 38 to 28 degrees C over 2.25 min produced a transient contraction followed by sustained relaxation. The cooling-induced contraction was blocked either by pretreatment with ouabain at concentrations of 10(-5) M or greater or by substitution of normal physiological salt solution with K-free solution. In contrast, the contractile response to cooling was not inhibited by pretreatment with phentolamine (10(-5) M), atropine (10(-5) M), tetrodotoxin (3 X 10(-7) M), diphenhydramine (10(-5) M), cromolyn sodium (10(-3) M), indomethacin (3 X 10(-7) M), nifedipine (10(-7) M), or verapamil (3 X 10(-6) M). Addition of NaHCO3 to the bath during cooling, preventing a change in pH of the physiological salt solution, did not affect the cooling-induced contraction. It is concluded that cooling of isolated guinea pig trachea produces a transient ouabain-sensitive contraction, and that the data suggest the contraction is mediated by inhibition of Na-K-ATPase in the smooth muscle rather than through neuronal stimulation or chemical mediator release.     

The mechanism of action of bombesin on cat lower esophageal sphincter

The effect of bombesin on the tone and the responses of strips from the lower esophageal sphincter (LES) to field electrical stimulation (FES) (2 Hz, 0.2 ms, supramaximal current intensity, 20 s duration) was studied. Bombesin dose-dependently increased the LES tone. The threshold for this effect was 10(-14) M and was particularly pronounced with a concentration of 10(-8) M. The response reached maximum between the 3rd and the 5th min after application, persisted for 15-20 min, and was followed by a slight time-dependent decrease. Bombesin increased FES-produced relaxation of LES by 39% as compared to the control. The potentiating effect of bombesin on the LES relaxation was also observed after cholinergic and adrenergic receptor blockade. It is concluded that bombesin may modulate the release of cholinergic, adrenergic and noncholinergic, nonadrenergic inhibitory neurotransmitters.     

Biphasic dose-response relationship for acetylcholine in the chick jugular vein

The pharmacological mechanism of biphasic dose-response relationship for acetylcholine (ACh), relaxation at low doses (1 nM to 0.3 μM) and contraction at high doses (1 μM to 30 μM), in the chick jugular vein was investigated. Neither relaxations nor contractions were affected by the treatment with tetrodotoxin, hexamethonium, d-tubocurarine, phentolamine, propranolol, reserpine, or ouabain. Besides, anoxia did not affect the biphasic pattern of dose-response curve. The contraction was attenuated by the treatment with aspirin or indomethacin, but only slightly. The dose-response curves for these responses to acetylcholine were shifted to the right by the treatment with atropine. Methacholine, carbachol, bethanechol, and arecoline caused similar biphasic responses, although contractions caused by highest doses of bethanechol or arecoline were very small in amplitude. On the other hand, pilocarpine and McN-A-343 only relaxed the strips. The dose-response curves for cholinomimetics were all shifted to the right by the treatment with atropine. It was demonstrated that the responses of the chick jugular vein to muscarinic agonists are different from those of mammalian veins. The mechanisms underlying the biphasic response are discussed.     

Involvement of Dihydropyridine-Sensitive Ca2+ Channels in Adenosine-Evoked Inhibition of Acetylcholine Release from Guinea Pig Ileal Preparation

The effects of adenosine and nifedipine on endogenous acetylcholine (ACh) release evoked by electrical stimulation from guinea pig ileal longitudinal muscle preparations exposed to physostigmine were evaluated using an HPLC with electrochemical detection (ECD) system. Resting ACh release, which was sensitive to tetrodotoxin (0.3 microM), was enhanced by Bay K 8644 (0.5 microM; a Ca2+ antagonist) or 4-aminopyridine (30 microM; a K+ channel blocker) but not by theophylline (100 microM; a P1 purinoceptor antagonist) or atropine (0.3 microM). The enhancement of the resting ACh release by Bay K 8644 was virtually unaffected by atropine. Electrically evoked ACh release was enhanced by around two- to fourfold in the presence of theophylline, atropine, Bay K 8644, 4-aminopyridine, or atropine. On the other hand, the evoked ACh release was reduced by adenosine (10-30 microM), nifedipine (0.1-0.3 microM; a dihydropyridine Ca2+ channel antagonist), or bethanechol (1-3 microM) in a concentration-related fashion. The reduction induced by adenosine or nifedipine was almost abolished by either theophylline or Bay K 8644, whereas that induced by bethanechol was virtually unaffected by these drugs. The inhibition by adenosine of ACh release was not influenced in the presence of 4-aminopyridine or atropine. However, this inhibition by adenosine was considerably enhanced by halving the Ca2+ concentration in the Krebs solution and was diminished by doubling the Ca2+ concentration. These findings suggest that adenosine produces a cholinergic neuromodulation presumably via modifying dihydropyridine-sensitive Ca2+ channel activities in the cholinergic neurons, and thus L-type Ca2+ channels may exist on the nerve terminals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of cholecystokinin octapeptide and somatostatin on the motility and release of [3H]acetylcholine in canine colon

1. The longitudinal and circular muscle layers of canine colon showed a different pattern of mechanical activity: regular rhythmic phasic contractions in the circular strips and irregular rhythmic prolonged contractions in the longitudinal strips.2. The spontaneous motility of both layers was suppressed by atropine (1 μM) or hexamethonium (1 μM), suggesting the involvement of ACh.3. Somatostatin (1 nM–1μM) decreased, while CCK8 (1–10 nM) increased the spontaneous and electrically-induced contractions of the colonic muscles, the circular layer being more sensitive as compared to the longitudinal layer.4. CCK8 enhanced both resting and electrically-induced [³H]ACh release, while SOM inhibited the electrically-stimulated [³H]ACh release.     

Motor effects of gastrin releasing peptide (GRP) and bombesin in the canine stomach and small intestine

Close intraarterial injections of synthetic porcine gastrin releasing peptide (GRP) or bombesin stimulated contractions in the stomach and inhibited ongoing contractile activity in the small intestine of anaesthetized dogs. Contractile activity of the circular muscle was recorded by serosal strain gauges and phasic activity when desired was elicited by local field stimulation or intraarterial motilin injections. In the stomach (corpus and antrum) following tetrodotoxin blockade of field-stimulated contractions, the contractile response to either peptide was not present, suggesting that stimulation of receptors on nerves initiated contractions in the stomach. Similarly, in the small intestine, the inhibitory response was eliminated by tetrodotoxin suggesting a neural receptor. Pre-treatment with reserpine did not alter the inhibitory response, either in the presence or absence of atropine, therefore, adrenergic inhibitory mechanisms did not appear to be involved. The concentration of bombesin producing 50% inhibition of field stimulation (ED50) was increased following treatment with the putative M1 muscarinic antagonist, pirenzipine suggesting activation of M1 cholinergic inhibitory receptors by bombesin. After blockade by atropine of field-stimulated contractions and the contractile response to intraarterial acetylcholine, the ED50 for bombesin inhibition of motilin contractions was increased. After muscarinic blockade, the residual inhibitory response of GRP/bombesin may involve activation of a neural non-cholinergic non-adrenergic inhibitory mechanism. These results suggest that GRP and bombesin act to alter motility in the dog in vivo by affecting neural activity.     

Nonadrenergic inhibitory nerves attenuate neurally mediated contraction in cat bronchi

Effects of nonadrenergic and noncholinergic (NANC) inhibitory nerves on cholinergic neurotransmission were examined in isolated bronchial segments from cats in the presence of propranolol (10(-6) M) and indomethacin (10(-6) M) by use of electrical field stimulation (EFS) techniques. EFS caused contraction alone in tissues at the baseline tension and biphasic responses (contraction and relaxation) in tissues precontracted with 5-hydroxytryptamine. Contraction was abolished by atropine (10(-6) M), and relaxation was abolished by tetrodotoxin (10(-6) M). At the baseline tension, EFS at frequencies greater than 10 Hz inhibited the subsequent (4 min later) contraction induced by EFS at 1-5 Hz. EFS-induced inhibition was stimulus frequency dependent and reached maximum at 20 Hz. However, EFS at 20 Hz did not inhibit the subsequent contractile response to acetylcholine (10(-7) to 10(-3) M). Exogenously applied vasoactive intestinal peptide mimicked EFS-induced inhibitory effects, but substance P and calcitonin gene-related peptide did not. The inhibitory effect of EFS at 20 Hz was not altered by pyrilamine, cimetidine, naloxone, methysergide, phentolamine, BW755C, AF-DX 116, or removal of epithelium. These results imply that the NANC transmitter acts via presynaptic cholinergic receptors.     

Antagonists of cholinergic and serotonergic responses of Aplysia buccal muscle

Cholinergic and serotonergic receptors of Aplysia californica buccal muscles were characterized pharmacologically by determining compounds that effectively inhibited contractile responses to acetylcholine (ACh) and modulatory effects of serotonin (5-HT), respectively. pA50 for ACh to elicit contraction averaged 4.7 ± 0.1 (mean ± SE, equivalent to 2 × 10⁻⁵ M). Both hexamethonium bromide and atropine inhibited ACh-elicited contractions, but neither inhibited the response completely, nor were the two together able to antagonize the response completely. Curare caused inhibition only at low ACh doses, and muscarinic antagonists pirenzapine and 4-diphenylacetoxy-N-methylpiperidine methiodide caused partial inhibition. The most effective blocker of ACh-elicited contractions was the nicotinic antagonist mecamylamine. 10⁻⁴M mecamylamine completely blocked the cholinergic response. ACh contractions were inhibited 90% within 10 min and took >40 min to recover from mecamylamine. Specificity was indicated by the lack of effect of mecamylamine on potassium-elicited contraction. NAN-190 blocked the potentiating effect of 5-HT without having inhibitory or potentiating effects by itself on ACh-elicited contractions. NAN-190 blocked the potentiating effect of 8-OH-DPAT. Cholinergic receptors on Aplysia buccal muscles are most effectively inhibited by mecamylamine and may have mixed nicotinic/muscarinic character. Serotonergic receptors have pharmacological similarities to vertebrate 5-HT_1A receptors and may be closely related to the gastropod 5-HTlym receptor.     

Cholinergic stimulation of skeletal muscle alanine and glutamine formation and release. Evidence for mediation by a nicotinic cholinergic receptor and guanosine 3':5'-monophosphate.

The mechanism of cholinergic stimulation of alanine and glutamine formation and release from skeletal muscle was studied using rat epitrochlaris preparations. The increased alanine and glutamine release produced by carbamylcholine (10(-6) M) was reproduced by tetramethylammonium (10(-6) M) but not by pilocarpine (10(-6) M) and was blocked by hexamethonium (10(-4) M) but not by atropine (10(-7) M). This increased alanine and glutamine release was not associated with altered muscle cAMP levels. However, carbamylcholine (10(-6) M) and tetramethylammonium (10(-6) M) did not increase levels of cGMP, 134% and 101%, respectively, and these increments in cGMP were blocked by hexamethonium but not by atropine. Carbamylcholine produced a concentration-dependent increase in cGMP levels. Methylisobutylxanthine and theophylline augmented the increased amino acid release and increased cGMP levels produced by carbamylcholine. Neither xanthine derivative alone altered alanine and glutamine release or cyclic nucleotide levels. Added cGMP increased amino acid release and the uptake of [U-14C]alanine and alpha-amino[14C]isobutyric acid. Carbamylcholine did not alter muscle phosphorylase a activity, glycogen levels, or basal adenylate cyclase activity. These data indicate that cholinergic stimulation of muscle alanine and glutamine formation and release involves a nicotinic cholinergic receptor and may be mediated by increased levels of cGMP, which in turn may result from a cholinergic stimulation of muscle guanylyl cyclase.