5-HT1A receptor-mediated inhibition of acetylcholine release from guinea pig myenteric plexus: potential mechanisms.

The mechanisms through which presynaptic 5-HT1A receptors cause inhibition of acetylcholine release from the guinea pig myenteric plexus were investigated. The selective 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) and 5-hydroxytryptamine (5-HT) caused concentration-dependent inhibitions of the electrically evoked release of [3H]acetylcholine from myenteric plexus preparations that had been preincubated with [3H]choline. The inhibitory effects were not modified by the activator of adenylyl cyclase, forskolin (10 microM), the phosphodiesterase inhibitor, AH 21-132 (100 microM), or after pretreatment of the guinea pigs with pertussis toxin (60 micrograms/kg). In contrast, the protein kinase C activator 4 beta- phorbol-12,13-dibutyrate (0.1 microM) prevented the release-inhibiting effect of 8-OH-DPAT, whereas the inactive isomer 4 alpha-phorbol-12,13-dibutyrate (0.1 microM) was without effect. The results suggest that the presynaptic 5-HT1A receptor is not coupled to a pertussis toxin sensitive G protein or to adenylyl cyclase. However, protein kinase C seems to be involved in the mechanism of inhibition of acetylcholine release by presynaptic 5-HT1A receptors.

Nitric oxide-sensitive guanylyl cyclase inhibits acetylcholine release and excitatory motor transmission in the guinea-pig ileum.

This study examined the mechanism through which nitric oxide inhibits the release of acetylcholine and excitatory motor neurotransmission in the guinea-pig ileum. The selective inhibitor of nitric oxide-sensitive guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), concentration-dependently enhanced both basal release (-log EC50: 6.8) and electrically (10 Hz)-evoked release (-log EC50: 6.0) of [3H]acetylcholine from longitudinal muscle-myenteric plexus preparations preincubated with [3H]choline. The increase by ODQ of basal release appeared to be exocytotic since it was prevented by tetrodotoxin (300 nM) and absence of calcium from the superfusion medium. In addition, ODQ (1 microM) increased the electrically-evoked tachykininergic and cholinergic muscle contractions as measured in the presence of scopolamine (100 nM) or of the neurokinin-1 receptor antagonist CP 99994 (100 nM), respectively. The nitric oxide synthase inhibitor L-N(G)-nitro-arginine (100 microM) behaved similar to ODQ and increased cholinergic and tachykininergic motor neurotransmission. The nitric oxide-independent activator of soluble guanylyl cyclase, 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole, concentration-dependently inhibited the electrically evoked acetylcholine release (-log EC50: 6.0) and longitudinal muscle contractions (-log EC50: 5.7). ODQ (10 microM) antagonized the effects of 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole. The results suggest that endogenous nitric oxide tonically activates soluble guanylyl cyclase in myenteric neurons which leads to inhibition of the release of the excitatory transmitters acetylcholine and substance P. ODQ prevents the effects of nitric oxide and thus facilitates cholinergic and tachykininergic motor neurotransmission in the guinea-pig ileum.

Spontaneous release of endogenous 5-hydroxytryptamine and 5-hydroxyindoleacetic acid from the isolated vascularly perfused ileum of the guinea-pig.

The spontaneous release of 5-hydroxytryptamine and its metabolite 5-hydroxyindoleacetic acid from the enterochromaffin cells of the small intestine into the portal circulation was investigated in vitro using the vascularly perfused ileum of the guinea-pig. The release of 5-hydroxytryptamine decreased by 70% in a calcium-free medium and by 35% in the presence of tetrodotoxin. Inhibition of monoamine oxidase activity by pargyline (100 microM) had no effect on the spontaneous release of 5-hydroxytryptamine although it caused a 75% reduction in the outflow of 5-hydroxyindoleacetic acid. Imipramine (1 microM), an inhibitor of neuronal uptake of 5-hydroxytryptamine, reduced the 5-hydroxyindoleacetic acid outflow by 57% and increased the release of 5-hydroxytryptamine by 66%. The combination of both drugs showed no additional effect. The tissue content of 5-hydroxytryptamine and its metabolite was not changed after perfusion with the precursor L-tryptophan or monofluoromethyldopa, an inhibitor of the L-aromatic amino acid decarboxylase. The results show that the spontaneous release of 5-hydroxytryptamine and its metabolite reflects largely calcium-dependent exocytotic release of the amine. "Neuronal uptake" (into aminergic and/or enterochromaffin cells) followed by deamination appears to be the main pathway of 5-hydroxytryptamine catabolism in the guinea-pig ileum.

Dicyclomine discriminates between M1- and M2-muscarinic receptors in the guinea-pig ileum.

1. The affinity of the antagonist dicyclomine for subtypes of muscarinic receptors has been assessed in the myenteric plexus-longitudinal muscle preparation of the guinea-pig. 2. Dicyclomine had a high affinity (pA2 9.13) for the neuronal M1-receptor whose activation by pilocarpine causes an increase in acetylcholine release. Dicyclomine had a low affinity for both the prejunctional M2-receptor (pA2 7.61) mediating inhibition of the electrically-evoked acetylcholine release and the postjunctional M2-receptor (pA2 7.21). 3. It is concluded that dicyclomine distinguishes between M1- and M2-muscarinic receptors in functional experiments.

Enhancement of guinea-pig intestinal peristalsis by blockade of muscarinic M1-receptors.

1. The effects of pirenzepine and hyoscine on the peristaltic reflex were investigated in the guinea-pig isolated small intestine. Peristalsis was induced by raising the intraluminal pressure and the volume of fluid propelled was taken as a measure of the efficiency of peristaltic activity. 2. Low concentrations of pirenzepine (0.1-1 nM) and of hyoscine (0.01 nM) significantly enhanced peristalsis, whereas larger concentrations of both drugs caused inhibition. Pirenzepine was about 6 times less potent than hyoscine in increasing peristalsis, but was about 100 times less potent in inhibiting it. 3. Neither tolazoline (1 microM) nor naloxone (0.3 microM) affected the stimulatory action of pirenzepine on peristalsis. 4. Bicuculline increased the efficiency of peristalsis at concentrations of 1 microM and 10 microM; at 10 nM, bicuculline reduced significantly the increase of peristalsis by pirenzepine. gamma-Aminobutyric acid (GABA) did not affect peristaltic activity, but the stimulatory effect of pirenzepine was abolished in the presence of 100 microM GABA. 5. The results indicate that activation of neuronal M1-receptors causes inhibition of small intestinal peristalsis. Bicuculline-sensitive 'GABAergic' synapses are probably involved in this inhibition.

Differential effects of nitric oxide donors on basal and electrically evoked release of acetylcholine from guinea-pig myenteric neurones.

1. The effects of the nitric oxide (NO) donors, 3-morpholino-sydnonimine (SIN-1), S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside on basal and electrically evoked release of [3H]-acetylcholine were studied in myenteric plexus longitudinal muscle preparations of the guinea-pig small intestine preincubated with [3H]-choline. 2. The NO donors concentration-dependently increased basal release of [3H]-acetylcholine. The increase in release was calcium-dependent and was prevented in the presence of tetrodotoxin. Superoxide dismutase (150 u ml-1) potentiated the effect of SIN-1. The selective inhibitor of soluble guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ, 0.01-1 microM), antagonized the facilitatory effect of SNAP. 8-Bromo cyclic GMP and the cyclic GMP-specific phosphodiesterase inhibitor, zaprinast (both 0.1-1 mM), also enhanced basal [3H]-acetylcholine release. The effect of 10 microM SNAP was significantly enhanced in the presence of zaprinast. 3. The NO donors concentration-dependently inhibited the electrically evoked release of [3H]-acetylcholine, whereas 8-bromo cyclic GMP and zaprinast enhanced the evoked release. The inhibition of acetylcholine release by SNAP was not affected by ODQ (0.01-1 microM). 4. It is concluded that NO stimulates basal acetylcholine release from myenteric neurones through activation of guanylyl cyclase. In addition, NO inhibits the depolarization evoked release of acetylcholine by a presynaptic mechanism unrelated to cyclic GMP. The data imply that NO is not only an inhibitory transmitter to intestinal smooth muscles but also a modulator of cholinergic neurotransmission in the myenteric plexus.

Modulation of acetylcholine release in the guinea-pig trachea by the nitric oxide donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP).

The effects of the nitric oxide (NO) donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) and the NO synthase inhibitor L-N(G)-nitroarginine (L-NOARG) on the electrically evoked [(3)H]-acetylcholine release were studied in an epithelium-free preparation of guinea-pig trachea that had been preincubated with [(3)H]-choline. SNAP (100 and 300 microM) caused small but significant increases of the electrically evoked [(3)H]-acetylcholine release (121+/-4% and 124+/-10% of control). Resting outflow of [(3)H]-ACh was not affected by SNAP. The increase by SNAP was abolished by the specific inhibitor of soluble guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ, 1 microM). The facilitatory effect of SNAP (100 and 300 microM) was reversed into inhibition of release (to 74+/-4% and to 78+/-2%) after pretreatment of the trachea with capsaicin (3 microM). ODQ prevented the inhibition. Capsaicin pretreatment alone did not significantly alter the release of [(3)H]-acetylcholine. A significant inhibition by SNAP (100 microM) of [(3)H]-acetylcholine release (78+/-3%) was also seen in the presence of the NK(2) receptor antagonist SR 48968 (30 nM). L-NOARG (10 and 100 microM) significantly enhanced the electrically-evoked smooth muscle contractions, but caused no significant increases of the evoked release from capsaicin pretreated trachea strips. This might indicate that the inhibitory effect of endogenous NO on acetylcholine release is too small to be detected by overflow studies. It is concluded that NO has dual effects on the evoked acetylcholine release. NO enhances release in the absence of modifying drugs, but NO inhibits acetylcholine release after blockade of the NK(2) receptor or after sensory nerve depletion with capsaicin. This suggests that NO and endogenous tachykinins act in series to produce an increase in acetylcholine release.

Adrenergic modulation of the release of 5-hydroxytryptamine from the vascularly perfused ileum of the guinea-pig.

1. Isolated segments of the guinea-pig ileum were vascularly perfused and the release of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) into the portal venous effluent was determined by h.p.l.c. with electrochemical detection. Test substances were applied via the arterial perfusion medium. 2. Isoprenaline (0.1 microM) increased the outflow of 5-HT and 5-HIAA maximally by about 75% and this was antagonized by propranolol (0.1 microM). Forskolin (1-10 microM) increased the outflow of 5-HT by approximately 105% and that of 5-HIAA by approximately 55%. The phosphodiesterase inhibitor AH 21-132 (0.1-1 microM) increased the outflow of 5-HT and 5-HIAA by about 70%. Isoprenaline (1 nM) and AH 21-132 (10 nM), which alone had no effect, increased the outflow of 5-HT and 5-HIAA by 75%, when applied in combination. 3. Clonidine (1 microM) reduced the outflow of 5-HT by 45%, an effect blocked by tolazoline (1 microM), but not by prazosin (0.1 microM). 4. The effects of isoprenaline, forskolin and clonidine were also observed in the presence of tetrodotoxin (1 microM) demonstrating a direct modulation of 5-HT release from the enterochromaffin cells. 5. In conclusion, the release of 5-HT from enterochromaffin cells is facilitated by activation of beta-adrenoceptors and inhibited via alpha 2-adrenoceptors. Enhancing intracellular cyclic AMP, by direct stimulation of adenylate cyclase with forskolin or by inhibition of phosphodiesterase, also facilitates the release of 5-HT. The beta-adrenoceptor-mediated effect on 5-HT release appears to involve an increase in cyclic AMP, as the effect of isoprenaline was potentiated after inhibition of phosphodiesterase.

Two types of receptors for 5-hydroxytryptamine on the cholinergic nerves of the guinea-pig myenteric plexus.

The effects of 5-hydroxytryptamine (5-HT) on spontaneous and electrically-evoked release of [3H]-acetylcholine (ACh) from guinea-pig myenteric plexus preparations preincubated with [3H]-choline have been investigated in the absence of cholinesterase inhibitors. 5-HT caused a transient increase in spontaneous release and an inhibition of the electrically-evoked release of [3H]-ACh. The 5-HT-induced contractions of the longitudinal muscle were clearly related to the increase in spontaneous release. The inhibitory effect was not due to activation of alpha-adrenoceptors since it was also observed in the presence of tolazoline and on strips from reserpine-pretreated guinea-pigs. After desensitization of the excitatory 5-HT receptors with 5-HT or metoclopramide the effects of 5-HT on spontaneous [3H]-ACh release were largely reduced. A variety of established antagonists at neuronal 5-HT receptors (i.e. metitepine 0.1-1 microM; methysergide 1 microM; ketanserin 0.1-1 microM; MDL 72222 0.1 microM; tropacocaine 1 microM) failed to block the excitation. The inhibition by 5-HT of the electrically evoked [3H]-ACh release was competitively antagonized by metitepine (pA2 7.6) and methysergide (pA2 7.0) but not by ketanserin. Tachyphylaxis to the inhibitory action of 5-HT did not occur. The results suggest that the excitatory 5-HT receptor ('M'-receptor) differs in its pharmacological properties from other neuronal 5-HT receptors. The presynaptically located inhibitory receptor may roughly correspond to the 5-HT1 receptor subtype but probably differs from the 5-HT autoreceptor.

Differential effects of anandamide on acetylcholine release in the guinea-pig ileum mediated via vanilloid and non-CB1 cannabinoid receptors.

1. The effects of anandamide on [3H]-acetylcholine release and muscle contraction were studied on the myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum preincubated with [3H]-choline. 2. Anandamide increased both basal [3H]-acetylcholine release (pEC(50) 6.3) and muscle tone (pEC(50) 6.3). The concentration-response curves for anandamide were shifted to the right by 1 microM capsazepine (pK(B) 7.5 and 7.6), and by the combined blockade of NK1 and NK3 tachykinin receptors with the antagonists CP99994 plus SR142801 (each 0.1 microM). The CB1 and CB2 receptor antagonists, SR141716A (1 microM) and SR144528 (30 nM), did not modify the facilitatory effects of anandamide. 3. Anandamide inhibited the electrically-evoked release of [3H]-acetylcholine (pEC(50) 5.8) and contractions (pEC(50) 5.2). The contractile response to the muscarinic agonist methacholine was not significantly affected by 10 microM anandamide. 4. The inhibitory effects of anandamide were not changed by either capsazepine (1 microM), SR144528 (30 nM) or CP99994 plus SR142801 (each 0.1 microM). SR141716A (1 microM) produced rightward shifts in the inhibitory concentration-response curves for anandamide yielding pK(B) values of 6.6 and 6.2. 5. CP55940 inhibited the evoked [3H]-acetylcholine release and contractions, and SR141716A (0.1 microM) shifted the concentration-response curves of CP55940 to the right with pK(B) values of 8.4 and 8.9. 6. The experiments confirm the existence of release-inhibitory CB1 receptors on cholinergic myenteric neurones. We conclude that anandamide inhibits the evoked acetylcholine release via stimulation of a receptor that is different from the CB1 and CB2 receptor. Furthermore, anandamide increases basal acetylcholine release via stimulation of vanilloid receptors located at primary afferent fibres.

Characterization of prejunctional muscarinic autoreceptors in the guinea-pig trachea.

1. The effects of ten muscarinic antagonists on electrically evoked [3H]-acetylcholine release and muscle contraction were compared in an epithelium-free preparation of the guinea-pig trachea that had been preincubated with [3H]-choline. 2. The M3-selective antagonists UH-AH 37, 4-diphenyl-acetoxy-N-piperidine methobromide and para-fluorohexahydrosiladiphenidol were more potent in reducing the contractile response than in facilitating the evoked [3H]-acetylcholine release. Hexahydrosiladiphenidol did not discriminate between pre- and postjunctional effects. The rank order of the postjunctional potencies of the ten antagonists as well as the postjunctional pA2 values obtained for hexahydrosiladiphenidol (7.95) and AQ-RA (7.08) identified the muscular receptor as an M3 subtype. 3. The M2-selective antagonists methoctramine, AF-DX 116 and AQ-RA 741 were more potent in facilitating the evoked [3H]-acetylcholine release than in inhibiting the contractile response. The increase in release by low concentrations of methoctramine, AF-DX 116 and AQ-RA 741 was paralleled by an enhancement of the stimulation-evoked contractions. 4. Comparison of the pre- and postjunctional potencies of the M1-, M2- and M3-selective antagonists suggests that autoinhibition of acetylcholine release is mediated via an 'M2-like' receptor which differs from the cardiac type M2 receptor in its relatively high affinity for hexahydrosiladiphenidol.

Characterization of the muscarinic receptor subtype(s) mediating contraction of the guinea-pig lung strip and inhibition of acetylcholine release in the guinea-pig trachea with the selective muscarinic receptor antagonist tripitramine.

1. The muscarinic receptor subtypes mediating contraction of the guinea-pig lung strip and inhibition of the release of acetylcholine from cholinergic vagus nerve endings in the guinea-pig trachea in vitro have previously been characterized as M2-like, i.e. having antagonist affinity profiles that are qualitatively similar but quantitatively dissimilar compared to cardiac M2 receptors. The present study sought to establish definitely the identity of these receptor subtypes by using the selective muscarinic receptor antagonist, tripitramine. Guinea-pig atria and guinea-pig trachea (postjunctional contractile response) were included for reference. 2. It was found that tripitramine antagonized methacholine-induced contractions of the guinea-pig lung strip with pKB value of 8.76 +/- 0.05. Both the parallel shifts of the concentration-response curves and the slope of the Schild plot begin not significantly different from unity (when antagonist preincubation was for 2 h) indicated the involvement of a single population of receptors in the contractile response. From the pKB values obtained with tripitramine and a range of other selective muscarinic receptor antagonists (cf. Roffel et al., 1993), this single population of receptors can only be classified as M2-like. 3. Tripitramine antagonized methacholine-induced chronotropic and inotropic responses in guinea-pig right and left atria with apparent pKB values of 9.4-9.6. However, such values were only obtained when antagonist preincubation was relatively long and/or antagonist concentration relatively high (e.g with 1 h at 100 or 300 nM but 3 h at 30 nM). It thus appears that low concentrations of tripitramine do not readily equilibrate with M2 receptors in guinea-pig atria nor with M2-like receptors in the guinea-pig lung strip. 4. Tripitramine increased electrical field stimulation-induced cholinergic twitch contractions in guinea-pig trachea in concentrations of 0.3-100 nM, by blocking prejunctional muscarinic inhibitory autoreceptors; with higher concentrations, twitch contractions were progressively diminished, as a result of blocking postjunctional M3 receptors (apparent pKB value 6.07 +/- 0.15). The pEC20 value (-log concentration that increases twitch by 20% maximum) was 8.29 +/- 0.08, which would suggest that M4 receptors are involved in this response. 5. Oxotremorine-induced inhibition of the release of prelabelled [3H]-acetylcholine from guinea-pig trachea, under conditions where there is no auto-feedback, was blocked by tripitramine (2 h preincubation) with a pKB value of 8.56 +/- 0.06. The slope of the corresponding Schild plot was not significantly different from unity, which together with the parallel shifts of the concentration-response curves indicated the involvement of a single muscarinic receptor subtype. 6. Since the pKB value for tripitramine at prejunctional receptors in guinea-pig trachea is in between the affinities towards M2 and M4 receptors, correlation plots were constructed to compare the pKB values obtained with tripitramine and a range of other selective muscarinic receptor antagonists (cf. Kilbinger et al., 1995) to reported affinities at M1-M4 receptors. This showed rather similar distribution patterns of the data points around the line of equality in the case of M2 and M4 receptor subtypes. However, the correlation coefficient was markedly better for M2 (0.9667) than for M4 (0.5976). Since recent evidence suggests that M4 receptors are not expressed in cholinergic nerves from guinea-pig trachea, it is concluded that prejunctional muscarinic autoinhibitory receptors in this tissue exhibit an atypical M2 type character, with a pharmacological profile distinct from cardiac M2 receptors.

Release of non-neuronal acetylcholine from the isolated human placenta is mediated by organic cation transporters.

1. The release of acetylcholine was investigated in the human placenta villus, a useful model for the characterization of the non-neuronal cholinergic system. 2. Quinine, an inhibitor of organic cation transporters (OCT), reduced acetylcholine release in a reversible and concentration-dependent manner with an IC(50) value of 5 microM. The maximal effect, inhibition by 99%, occurred at a concentration of 300 microM. 3. Procaine (100 microM), a sodium channel blocker, and vesamicol (10 microM), an inhibitor of the vesicular acetylcholine transporter, were ineffective. 4. Corticosterone, an inhibitor of OCT subtype 1, 2 and 3 reduced acetylcholine in a concentration-dependent manner with an IC(50) value of 2 microM. 5. Substrates of OCT subtype 1, 2 and 3 (amiloride, cimetidine, guanidine, noradrenaline, verapamil) inhibited acetylcholine release, whereas carnitine, a substrate of subtype OCTN2, exerted no effect. 6. Long term exposure (48 and 72 h) of villus strips to anti-sense oligonucleotides (5 microM) directed against transcription of OCT1 and OCT3 reduced the release of acetylcholine, whereas OCT2 anti-sense oliogonucleotides were ineffective. 7. It is concluded that the release of non-neuronal acetylcholine from the human placenta is mediated via organic cation transporters of the OCT1 and OCT3 subtype.

Inhibition by nitric oxide and cyclic GMP of 5-hydroxytryptamine release from the vascularly perfused guinea-pig small intestine.

The effects of nitric oxide (NO) on the spontaneous release of 5-hydroxytryptamine (5-HT) were studied in the in vitro vascularly perfused guinea-pig small intestine. The NO donor SIN-1 concentration-dependently decreased 5-HT release with an EC50 of 1.34 microM, whereas the NO synthase inhibitor N(G)-nitro-L-arginine (100 microM) was without effect. The inhibition by SIN-1 of 5-HT release was enhanced by superoxide dismutase (150 U/ml) and antagonized by the selective inhibitor of soluble guanylyl cyclase, ODQ (1 microM). Tetrodotoxin (1 microM) prevented the inhibition by SIN-1 of 5-HT release, which suggests that the effect of SIN-1 is indirectly mediated via release of an inhibitory neurotransmitter. Substance P could be excluded as inhibitory transmitter because the effect of SIN-1 remained unchanged in the presence of the NK1 receptor antagonist CP 99994 (100 nM). The cyclic GMP analogue, 8-bromo cyclic GMP (300 microM), also decreased basal release of 5-HT, but this decrease was not tetrodotoxin-sensitive. It is concluded that NO inhibits the release of 5-HT from enterochromaffin cells via release of an enteric neurotransmitter. Acetylcholine (via nicotinic receptors) and substance P (via NK1 receptors) are not involved in the NO-mediated inhibition. The inhibition of 5-HT outflow by NO is due to the activation of soluble guanylyl cyclase. 8-Bromo cyclic GMP inhibited 5-HT release by a direct effect on the enterochromaffin cells.

Functional relevance of presynaptic muscarinic autoreceptors.

Pre- and postsynaptic muscarinic receptors have been characterized in the isolated trachea and ileal circular muscle of the guinea pig. The muscarinic autoreceptors mediating inhibition of acetylcholine release in the circular muscle belong to the M1 subtype, whereas those inhibiting acetylcholine release in the trachea are M2 or M4 receptors. In both tissues the postsynaptic muscarinic receptors are M3 receptors. Blockade of the autoreceptors by selective M1 and M2/M4 receptor antagonists leads to facilitation of cholinergic neurotransmission.

Regulation of 5-HT release from enterochromaffin cells.

Large amounts of 5-HT are present in the mammalian intestine where the amine is concentrated in the enterochromaffin cells (ECs) of the mucosa. ECs have the enzymes to synthesize 5-HT, are endowed with a specific, imipramine-sensitive 5-HT uptake mechanism and can store 5-HT in specific secretory vesicles. ECs can secrete 5-HT in a calcium-dependent manner. In particular, calcium influx through voltage-regulated channels and receptor-mediated liberation of intracellular calcium can evoke 5-HT release. 5-HT secretion from ECs occurs predominantly at the interstitial side and is controlled by a complex pattern of receptor-mediated mechanisms. Stimulatory receptors (beta-adrenoceptors, muscarine, nicotine and 5-HT3 receptors) and inhibitory receptors (alpha 2-adrenoceptors, histamine H3, GABAA- and GABAB-, A2 and P2y alpha purine and 5-HT4 receptors as well as receptors for vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase stimulating peptide (PACAP) and somatostatin) have been shown to be involved in the control of 5-HT release from the ECs.

Muscarinic modulation of acetylcholine release evoked by dimethylphenylpiperazinium and high potassium from guinea-pig myenteric plexus.

The effects of oxotremorine and atropine on the ACh release evoked from the guinea-pig myenteric plexus by dimethylphenylpiperazinium (DMPP) or by high potassium were investigated. DMPP caused an output of ACh by stimulating nicotine receptors that are probably situated on the soma-dendritic part of the cholinergic neuron. The DMPP-induced release of ACh was dose-dependently inhibited by oxotremorine and increased by atropine. The ACh output evoked by either 45 or 108 mM potassium was enhanced by atropine. Oxotremorine did not affect the ACh release by high potassium but prevented the facilitatory effect of atropine. It is concluded that the inhibitory muscarinic mechanism modulates similarly the ACh release evoked by DMPP or high potassium and the release caused by electrical stimulation. From the experiments with high potassium it is concluded that the inhibitory muscarine receptors are localized at the site of ACh release.

[3H]acetylcholine release from the phrenic nerve is increased or decreased by activation or desensitization of nicotine receptors.

Nicotinic agonists (nicotine, 1,1-dimethyl-4-phenylpiperazinium (DMPP) increased [3H]acetylcholine release when the pre-exposure time was short (20 s). This effect was antagonized by pretreatment with d-tubocurarine (d-TC), which indicates a receptor-mediated effect. After a longer pre-exposure time (3 min) 10 microM nicotine significantly decreased the evoked [3H]acetylcholine release, probably because desensitization of the nicotine autoreceptors had abolished the nicotinic autofacilitation of transmitter release.

GABA receptors are involved in the modulation of the release of 5-hydroxytryptamine from the vascularly perfused small intestine of the guinea-pig.

Isolated small intestinal segments of the guinea-pig were perfused arterially and the release of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) into the portal venous effluent was determined by HPLC with electrochemical detection. Test substances were applied intraarterially. Muscimol (1 microM) time dependently first increased then decreased the release of 5-HT and 5-HIAA. The stimulatory effect was prevented by tetrodotoxin (TTx) or scopolamine, indicating that it was mediated by the release of acetylcholine. Bicuculline concentration dependently decreased (1 microM) or increased (10, 50 microM) the release of 5-HT and 5-HIAA, indicating that endogenous GABA also activates stimulatory and inhibitory GABAA receptors. Bicuculline antagonized the stimulatory and inhibitory effect of muscimol. (-)-Baclofen, but not its (+) enantiomer, inhibited the release of 5-HT in the absence and presence of TTx. It was concluded that the release of 5-HT from enterochromaffin cells is directly inhibited by GABAA and GABAB receptors. In addition, acetylcholine released after activation of GABAA receptors stimulates 5-HT release.

Organ selectivity of hexahydrosiladifenidol in blocking pre- and postjunctional muscarinic receptors studied in guinea-pig ileum and rat heart.

Pre- and postjunctional pA2 values of the muscarinic antagonist hexahydrosiladifenidol were determined with guinea-pig ileum and rat heart. Hexahydrosiladifenidol did not discriminate between pre- and postjunctional receptors within the same organ but was more potent on the ileum (20-80 times) than on the heart. It is concluded that pre- and postjunctional muscarinic receptors in the heart may differ from those in the ileum.