EFS-induced Acetylcholine Release Research Articles

Cystic fibrosis transmembrane conductance regulator modulates enteric cholinergic activities and is abnormally expressed in the enteric ganglia of patients with slow transit constipation.

Cystic fibrosis transmembrane conductance regulator (CFTR) was recently found in the enteric nervous system, where its role is unclear. We aimed to identify which enteric neuronal structures express CFTR, whether CFTR modulates enteric neurotransmission and if altered CFTR expression is associated with slow transit constipation (STC). Immunofluorescence double labeling was performed to localize CFTR with various neuronal and glial cell markers in the human colon. The immunoreactivity (IR) of CFTR and choline acetyltransferase (ChAT) on myenteric plexus of control and STC colon was quantitatively analyzed. In control colonic muscle strips, electrical field stimulation (EFS) evoked contractile responses and the release of acetylcholine (ACh) was measured in the presence of the CFTR channel inhibitor, CFTR(inh)-172. CFTR-IR was densely localized to myenteric ganglia, where it was co-localized with neuronal markers HuC/D and β-tubulin, and glial marker S-100 but little with glial fibrillary acidic protein. Vesicular ACh transport was almost exclusively co-localized with CFTR, but neurons expressing nitric oxide synthase were CFTR negative. Significant reductions of CFTR-IR (P < 0.01) and ChAT-IR (P < 0.05) were observed on myenteric ganglia of STC compared to control. Pre-treatment of colonic muscle strips with CFTR(inh)-172 (10µM) significantly inhibited EFS-evoked contractile responses (P < 0.01) and ACh release (P < 0.05). Co-localization of CFTR-IR with cholinergic markers, inhibition of EFS-induced colonic muscle contractility and ACh release by CFTR(inh)-172 suggest that CFTR modulates enteric cholinergic neurotransmission. The downregulation of CFTR and ChAT in myenteric ganglia of STC correlated with the impaired contractile responses to EFS.

Na+/Ca2+ exchanger 2‐heterozygote knockout mice display decreased acetylcholine release and altered colonic motility in vivo

The Na(+) /Ca(2+) exchanger (NCX) is a plasma membrane transporter involved in regulating intracellular Ca(2+) concentrations. NCX is critical for Ca(2+) regulation in cardiac muscle, vascular smooth muscle, and nerve fibers. However, little is known about the physiological role of NCX in the myenteric neurons and smooth muscles of the gastrointestinal tract. To determine the role of NCX1 and NCX2 in gastrointestinal tissues, we examined electric field stimulation (EFS)-induced responses in the longitudinal smooth muscle of the distal colon in NCX1- and NCX2-heterozygote knockout mice. We found that the amplitudes of EFS-induced relaxation that persisted during EFS were greater in NCX2 heterozygous mice (HET) than in wild-type mice (WT). Under the nonadrenergic, noncholinergic (NANC) condition, EFS-induced relaxation in NCX2 HET was similar in amplitude to that of WT. In addition, an NCX inhibitor, YM-244769 enhanced EFS-induced relaxation but did not affect EFS-induced relaxation under the NANC condition, as in NCX2 HET. Unlike NCX2 HET, NCX1 HET displayed no marked changes in colonic motility. These results indicate that cholinergic function in the colon is altered in NCX2 HET. The magnitude of acetylcholine (ACh)-induced contraction in NCX2 HET was similar to that in WT. In contrast, EFS-induced ACh release was reduced in NCX2 HET compared with that in WT. In this study, we demonstrate that NCX2 regulates colonic motility by altering ACh release onto the myenteric neurons of the distal colon.

Pharmacological Effects of Solifenacin on Human Isolated Urinary Bladder

To investigate the effects of solifenacin on human detrusor smooth muscles, we evaluate the effects of solifenacin on the contractions induced by carbachol, KCl, CaCl₂ and electrical field stimulation (EFS), and the EFS-induced acetylcholine release from detrusor smooth muscle strips by using the muscle bath and microdialysis technique. The effects of solifenacin were also compared with effects of other antimuscarinic agents (atropine, oxybutynin and propiverine). Pretreatment with various antimuscarinic agents caused parallel shifts to the right of the concentration-response curves to carbachol. The pA₂ value of the Schild plots for solifenacin was similar to that for oxybutynin. Atropine did not inhibit the KCl- and CaCl₂-induced contractions, while solifenacin, oxybutynin and propiverine significantly inhibited these contractions. EFS-induced contractions were inhibited by various antimuscarinic drugs in a concentration-dependent manner. In the presence of atropine, solifenacin tended to inhibit the residual atropine-resistant contractions induced by EFS, but it was not significant. However, oxybutynin and propiverine inhibited them under the same conditions. Although pretreatment with atropine and propiverine did not cause significant changes in EFS-induced acetylcholine release, solifenacin and oxybutynin caused significant decreases in acetylcholine release. The present results suggest that solifenacin inhibits contractions of human detrusor smooth muscles mainly by the antimuscarinic action and that the high concentration of solifenacin has Ca²⁺ channel antagonist action. Moreover, solifenacin may block not only postjunctional receptors, but also prejunctional receptors to modulate acetylcholine releases in cholinergic nerve endings in human detrusor smooth muscles. The findings support that muscarinic-receptor-inhibitory actions in human bladder mainly contribute to the usefulness of solifenacin as a therapeutic drug for overactive bladder.

Change in acetylcholine release from rat bladder with partial outlet obstruction

To investigate changes in acetylcholine release from the bladder of rats with partial bladder outlet obstruction (BOO), as partial BOO leads to hypertrophy and an alteration in the contractions of the detrusor smooth muscle, and acetylcholine plays an important role in urinary bladder contractions but there is little available information on acetylcholine release after BOO. Partial BOO was induced in adult female rats by ligating the proximal urethra over a 1 mm angiocatheter; sham-operated rats served as controls. The rats were killed 2 weeks, 3 and 6 months after induction of BOO. We investigated the contractions induced by carbachol, KCl (80 mm), ATP and electrical-field stimulation (EFS, 2.5-40 Hz), and collected the dialysate obtained from a microdialysis probe inserted into the muscle strips during EFS, and measured the amount of acetylcholine in the dialysate fraction by high-performance liquid chromatography with electro-chemical detection. S-100 immunohistochemical staining of the bladder preparations was used for histological examination in BOO and control rats. The bladder weight gradually increased after BOO. There were no significant changes in KCl-induced contractions throughout the experimental period in either group. There were no significant changes in carbachol-induced contractions until 3 months after BOO but there was a significant reduction at 6 months. ATP-induced contractions were significantly increased 2 weeks and 3 months after BOO. EFS-induced contractions were gradually reduced after BOO. Acetylcholine release from the bladder strips was not significantly different between the groups until 2 weeks after BOO. However, acetylcholine release in BOO rats was significantly decreased 3-6 months after BOO, being significantly lower than that of the control rats. In the histological study, the number of nerve fibres in the BOO rats was significantly lower than in the control rats. We suggest that the prolonged BOO caused a decrease in EFS-induced acetylcholine release and the number of nerves in the rat urinary bladder, which might contribute to bladder underactivity in BOO.

Age-Related Alterations in the Biochemical and Functional Properties of the Bladder in Type 2 Diabetic GK Rats

Previous studies have demonstrated that experimental type 1 diabetes induced by streptozotocin causes alterations in the biochemical and functional properties of several receptor systems in the rat bladder. However, the exact mechanism involved in the pathophysiology of voiding dysfunction in type 2 diabetic patients is unknown. Because the GK rat is a widely accepted genetically determined rodent model for human type 2 diabetes, we investigated diabetes-induced changes in the bladder smooth muscle of the GK rats at several time points. Male GK rats and age-matched Wistar rats, as controls, were maintained for 4, 8, 16, and 32 weeks. Contractile responses to KCl, carbachol, ATP, and electrical field stimulation (EFS) were measured by using the isolated muscle bath techniques. Acetylcholine (ACh) release induced by EFS from bladder muscle strips was measured by using high-performance liquid chromatography coupled with a microdialysis procedure. Maximum contractile responses to carbachol and ATP, the release of ACh, and tissue sorbitol levels were similar in bladders from GK and control rats until 8 weeks of age. At 16 weeks of age, however, the contractile responses to carbachol and ATP, and tissue sorbitol levels were increased, and the EFS-induced ACh release was decreased in GK rats compared with controls. Although the maximum contractile responses to EFS were unchanged until 16 weeks of age, they were decreased in 32-week-old GK rats, compared with controls. Our data indicate the presence of age-related alterations in the biochemical and functional properties of the bladder in type 2 diabetic GK rats.

Roles of M2 and M4 Muscarinic Receptors in Regulating Acetylcholine Release From Myenteric Neurons of Mouse Ileum

We investigated the subtype of presynaptic muscarinic receptors associated with inhibition of acetylcholine (ACh) release in the mouse small intestine. We measured endogenous ACh released from longitudinal muscle with myenteric plexus (LMMP) preparations obtained from M1-M5 receptor knockout (KO) mice. Electrical field stimulation (EFS) increased ACh release in all LMMP preparations obtained from M1-M5 receptor single KO mice. The amounts of ACh released in all preparations were equal to that in the wild-type mice. Atropine further increased EFS-induced ACh release in the wild-type mice. Unexpectedly, atropine also increased, to a similar extent, EFS-induced ACh release to the wild-type mice in all M1-M5 receptor single KO mice. In M2 and M4 receptor double KO mice, the amount of EFS-induced ACh release was equivalent to an atropine-evoked level in the wild-type mouse, and further addition of atropine had no effect. M2 receptor immunoreactivity was located in both smooth muscle cells and enteric neurons. M4 receptor immunoreactivity was located in the enteric neurons, being in co-localization with M2 receptor immunoreactivity. These results indicate that both M2 and M4 receptors mediate the muscarinic autoinhibition in ACh release in the LMMP preparation of the mouse ileum, and loss of one of these subtypes can be compensated functionally by a receptor that remained. M1, M3, and M5 receptors do not seem to be involved in this mechanism.

Acetylcholine Release from Urinary Bladder Smooth Muscles of Non-Insulin-Dependent Diabetic Rats

Introduction: To investigate the mechanism of voiding dysfunction in non-insulin-dependent diabetes mellitus, we attempted to measure the acetylcholine (ACh) release using an in vivo microdialysis technique and measuring the detrusor pressure after electrical field stimulation (EFS) of the pelvic nerve. Materials and Methods: Eight- and 32-week-old female Goto-Kakizaki (GK) rats (non-insulin-dependent diabetes mellitus model) and age-matched female Wistar rats (controls) were used in this study. The pelvic nerve was exposed on a bipolar platinum electrode to EFS, and a cannula was inserted into the bladder to measure the detrusor pressure. The microdialysis probe was inserted into the bladder wall and was connected to a microinfusion syringe pump. Dialysate was constantly perfused, collected in a microtube, and then injected into the ACh assay system. Histological examinations were performed by staining with hematoxylin and eosin and S-100 immunohistochemical staining in bladder preparations of both GK and control rats. Results: In 8-week-old rats, both detrusor pressures and amounts of ACh release of GK rats were not significantly different from those of control rats. In 32-week-old rats, both detrusor pressures and ACh releases were only significantly increased at 5 and 10 Hz of EFS. In the histological study, the number of nerve fibers or bundles of 32-week-old GK rats was significantly decreased as compared with control rats. Conclusion: The present data suggest that the decrease in EFS-induced ACh release in GK rats, which may be caused by the decreased number of nerve fibers, may contribute to the decrease in bladder contractions.

Effect of 1DMe, a Neuropeptide FF Analog, on Acetylcholine Release From Myenteric Plexus of Guinea Pig Ileum

Since neuropeptide FF (NPFF) is a putative neurotransmitter to exert anti-opioid activity, we examined the effects of [D-Tyr', (NMe)Phe3]neuropeptide FF (IDMe), a stable NPFF analog, on acetylcholine (ACh) release from a longitudinal muscle-myenteric plexus (LMMP) preparation of guinea pig ileum in which opioids were known to inhibit ACh release when muscarinic autoinhibition was not fully activated. In the presence of atropine, 1DMe increased spontaneous and electrical field stimulation (EFS)-evoked ACh release in a concentration-dependent manner. Naloxone also increased ACh release. The stimulatory effects of 1DMe and naloxone were not additive. In the absence of atropine, 1DMe did not affect ACh release. Morphine decreased spontaneous and EFS-evoked ACh release in the presence of 1 microM atropine. 1DMe as well as naloxone counteracted the inhibitory effects of morphine on EFS-evoked ACh release. The combination of 1DMe and naloxone was not more inhibitory than either drug alone. 1DMe had no appreciable effect on norepinephrine-induced inhibition of spontaneous and EFS-evoked ACh release. These results first demonstrated the effects of a NPFF analog on neurotransmitter release: 1DMe had a stimulatory effect on spontaneous and EFS-induced ACh release from the LMMP preparation of guinea pig ileum, probably by counteracting the inhibitory effect of endogenous opioids on ACh release.

Direct measurement of acetylcholine release in detrusor smooth muscles isolated from rabbits.

In the present study, we measured acetylcholine (ACh) released from rabbit detrusor smooth muscle strips induced by electrical field stimulation (EFS) using high-performance liquid chromatography coupled with microdialysis procedure. There were frequency- and duration-dependent increases in contractile response and ACh release. There was a significant, but not simple correlation between EFS-induced contraction and ACh release. Atropine caused a decrease and increase in the contractile response and ACh release, respectively. Pretreatment with propranolol increased ACh release, but pretreatment with phentolamine had no significant effect. These results demonstrate that this method is applicable to direct measurement of ACh release by EFS, and that neurotransmitters other than ACh may relate to EFS-induced contraction. In addition, it is suggested that there are prejunctional inhibitory muscarinic receptors and beta-adrenoceptors, which contribute to ACh release induced by EFS in the rabbit detrusor smooth muscles.

Measurement of acetylcholine released from rabbit detrusor smooth muscle using HPLC with electro-chemical detection coupled with microdialysis procedure

We measured the amount of acetylcholine (ACh) released from rabbit detrusor smooth muscles induced by electrical field stimulation (EFS) using microdialysis procedure. The dialysis probe was inserted through the detrusor muscle strip and was continuously perfused with a Ringer solution containing physostigmine sulfate, at a rate of 2 μl/min. The strip was suspended in an organ bath filled with the modified Krebs-Henseleit solution and then EFS was delivered. The isometric force was recorded and monitored in each muscle preparation. The dialysates were collected every 10 min. Ach was determined by a high performance liquid chromatography with electro-chemical detection. The contraction of the muscle strip and ACh release induced by EFS were increased in a frequency and duration dependent manner. There were some differences between frequency response curves of contraction and frequency dependent ACh release. In the contractile response, the maximum contractions were observed at lower frequencies, while ACh releases reached the maximum at higher frequencies. There was a significant, but not simple correlation between EFS-induced contraction and ACh release. The results suggest that this new method is useful to investigate the ACh release from rabbit detrusor smooth muscles, and that other neurotransmitters than ACh possibly contribute to EFS-induced contraction.

Abnormal modulation of cholinergic neurotransmission by endogenous nitric oxide in the bronchus of rats with hyperresponsiveness induced by allergen challenge.

The involvement of endogenous nitric oxide (NO) in bronchial cholinergic neurotransmission was compared between normal rats and airway hyperresponsive (AHR) rats. Male Wistar rats were sensitized and repeatedly challenged with dinitrophenylated (DNP)-Ascaris antigen. Twenty-four hours after the last antigenic challenge, enhancements of both the electrical field stimulation (EFS)-induced bronchoconstriction and acetylcholine (ACh) release were observed. NG-Monomethyl-L-arginine (L-NMMA; NO synthase inhibitor, 0.1 mM) augmented the EFS-induced bronchoconstriction and ACh release without affecting exogenously applied ACh-induced bronchoconstriction in normal rats. Interestingly, the augmentative effects of L-NMMA seen in normal rats were not manifested in AHR rats. Sodium nitroprusside inhibited the EFS-induced bronchoconstriction in a concentration-dependent manner; the inhibition was much larger than that of exogenously applied ACh-induced constriction in both normal and AHR rats. Furthermore, dibutyryl cGMP (3 mM) inhibited the EFS-induced bronchoconstriction with no effect on the ACh-induced bronchoconstriction in both normal and AHR rats. These findings suggest that endogenous NO may have a modulatory role in bronchial cholinergic neurotransmission in normal rats and that the augmented ACh release in the AHR rats may result from the defect of endogenous NO-induced modulation of cholinergic nerve transmission.

Abnormal Modulation of Cholinergic Neurotransmission by Endogenous Nitric Oxide in the Bronchus of Rats with Hyperresponsiveness Induced by Allergen Challenge

The involvement of endogenous nitric oxide (NO) in bronchial cholinergic neurotransmission was compared between normal rats and airway hyperresponsive (AHR) rats. Male Wistar rats were sensitized and repeatedly challenged with dinitrophenylated (DNP)-Ascaris antigen. Twenty-four hours after the last antigenic challenge, enhancements of both the electrical field stimulation (EFS)-induced bronchoconstriction and acetylcholine (ACh) release were observed. NG-Monomethyl-L-arginine (L-NMMA; NO synthase inhibitor, 0.1 mM) augmented the EFS-induced bronchoconstriction and ACh release without affecting exogenously applied ACh-induced bronchoconstriction in normal rats. Interestingly, the augmentative effects of L-NMMA seen in normal rats were not manifested in AHR rats. Sodium nitroprusside inhibited the EFS-induced bronchoconstriction in a concentration-dependent manner; the inhibition was much larger than that of exogenously applied ACh-induced constriction in both normal and AHR rats. Furthermore, dibutyryl cGMP (3 mM) inhibited the EFS-induced bronchoconstriction with no effect on the ACh-induced bronchoconstriction in both normal and AHR rats. These findings suggest that endogenous NO may have a modulatory role in bronchial cholinergic neurotransmission in normal rats and that the augmented ACh release in the AHR rats may result from the defect of endogenous NO-induced modulation of cholinergic nerve transmission.Keywords: Airway hyperresponsiveness, Nitric oxide, Cholinergic neurotransmission, Nitrergic dysfunction, Dinitrophenylated (ONP)-Ascaris

Incubation with IgE increases cholinergic neurotransmission in human airways in vitro.

Airway cholinergic hyperresponsiveness is frequently observed in asthmatic patients. Recent reports suggest the possible involvement of IgE in hyperresponsiveness, although the exact mechanism is still uncertain. In this study, we examined whether incubation with IgE could facilitate the cholinergic function in human airways. Bronchi were obtained from 20 patients undergoing lung resection. Cholinergic contractile responses were induced by electrical field stimulation (EFS) or exogenous acetylcholine (ACh), and they were assessed by isometric tension measurement. EFS-induced ACh release from cholinergic nerves was also measured by high performance liquid chromatography. Incubation with IgE significantly enhanced EFS-induced bronchial contraction and ACh release as compared with the values of the bronchi incubated with heat inactivated IgE (control) (p < 0.05, respectively), but it did not alter the contractile responses induced by exogenous ACh. Pretreatment with the muscarinic M2-receptor agonist pilocarpine reduced the EFS-induced ACh release in the control tissues (p < 0.05), but not in the tissues incubated with IgE. The M2-receptor antagonist methoctramine significantly enhanced the EFS-induced contraction in control bronchi (p < 0.05), but this augmentation was not observed in the tissues incubated with IgE. These results suggest that IgE itself can enhance cholinergic bronchial contraction via facilitation of ACh release from cholinergic nerves and that this augmentation is related to autoreceptor M2 dysfunction at nerve endings.

Excitatory prejunctional β 2-adrenoceptor distribution within equine airway cholinergic nerves

We examined the effect of activation of β 2-adrenoceptor (AR) by isoproterenol (ISO) on acetylcholine (ACh) release evoked by electrical field stimulation (EFS: 20 V, 0.5 Hz, 0.5 msec) from cholinergic nerves in five regions of equine airways. We also tested if the effect of ISO was dependent on epithelium or prostanoids by examining the effect of ISO on ACh release in the presence and absence of epithelium or cyclooxygenase inhibition. Trachealis strips or bronchial rings were preincubated for 60 min with 10 −7 M atropine, 10 −6 M neostigmine, and 10 −5 M guanethidine. The ACh amount was measured by high-performance liquid chromatography with electrochemical detection. Isoproterenol (10 −8−10 −6 M) augmented ACh release throughout the whole airway in a concentration-dependent manner. Rubbing off the epithelium potentiated EFS-induced ACh release but neither epithelium removal nor cyclooxygenase inhibition affected the magnitude of ISO-induced augmentation of ACh release. These results indicate that in equine airway parasympathetic nerves: (1) excitatory prejunctional β 2-adrenoceptors are distributed throughout the tracheobronchial tree; (2) the function of this excitatory β 2-adrenoceptor is independent of endogenous prostanoids and epithelium; and (3) there is an epithelium-derived factor that inhibits ACh release.

Potentiation of acetylcholine release from tracheal parasympathetic nerves by cAMP.

We tested the hypothesis that increasing intracellular levels of adenosine 3', 5'-cyclic monophosphate (cAMP) increases acetylcholine (ACh) release from airway parasympathetic nerves. Muscle strips from equine trachea were preincubated for 60 min with 10(-7)M atropine, 10(-6)M neostigmine, and 10(-5) M guanethidine. The ACh release was evoked by electrical field stimulation (EFS, 20 V, 0.5 ms, 0.5 Hz) and measured by high-performance liquid chromatography with electrochemical detection. Agents known to increase cAMP, i.e., forskolin (10(-6) - 10(-4) M), 8-bromoadenosine 3', 5'-cyclic monophosphate (8-BrcAMP; 10(-5)-10(-3) M), and 3-isobutyl-1-methylxanthine (IBMX ; 10(-5)-10(-3)M) was potentiated by IBMX but not mimicked by 1,9 dideoxyforskolin. To determine if the augmentation of Ach release facilitated EFS-induced ACh release in a concentration-dependent manner. Forskolin-induced augmentation of ACh release induced by activation of beta 2-adrenoceptors is mediated via cAMP-dependent pathways, we also examined the additive effects of 8-BrcAMP, forskolin, and IBMX with 10(-6)M isoproterenol (ISO), the concentration that maximally augments ACh release. Neither forskolin nor 8-BrcAMP potentiated the maximal augmentation produced by ISO, but inhibition of phosphodiesterase with IBMX (10(-4) and 10(-3)M) augmented the maximal effect of ISO. These observations indicate that neuronal cAMP is a physiological modulator of ACh release from airway parasympathetic nerves and mediates ISO-induced augmentation of ACh release. Bronchodilators that increase cAMP may therefore paradoxically augment ACh release while relaxing smooth muscle.

Beta 2-adrenoceptor activation augments acetylcholine release from tracheal parasympathetic nerves

We determined the effects of isoproterenol (Iso) on parasympathetic neurotransmission in isolated equine trachealis strips by comparing the effects of Iso on the contractile response to electrical field stimulation (EFS) and acetylcholine (ACh), as well as by measuring EFS-induced ACh release. The interaction of Iso with muscarinic receptors and endogenous nitric oxide was also investigated. ACh release was measured by high-performance liquid chromatography with electrochemical detection. Iso (10(-7) M or greater) caused significantly more inhibition of EFS- than of ACh-induced contraction, an observation usually interpreted as evidence of prejunctional inhibition of ACh release. However, the latter conclusion was not supported by measurement of ACh release. Iso concentration dependently augmented ACh release, which was reversed by the beta 2-adrenoceptor antagonist ICI-118,551 but not by the beta 1-adrenoceptor antagonist CGP-20,712A. Our results indicate that activation of beta 2-adrenoceptors augments ACh release. Moreover, the comparison of inhibitory effects on EFS- and ACh-induced contraction does not provide correct information about the prejunctional actions of beta-agonists. ACh release was increased more by atropine (10(-7) M) than by Iso (10(-6) M), indicating the predominance of prejunctional inhibitory muscarinic autoreceptors over excitatory beta 2-adrenoceptors. Additionally, we found that inhibition of nitric oxide synthase by NG-nitro-L-arginine did not affect either the cholinergic contractile response or ACh release in both the absence and presence of Iso.

ACh release from horse airway cholinergic nerves: effects of stimulation intensity and muscle preload.

This study was conducted to determine the effects of stimulation parameters and muscle preload on acetylcholine (ACh) release induced by electrical field stimulation (EFS) of horse airway cholinergic nerves. Trachealis strip bundles were prepared and suspended in 2-ml tissue baths. The tissues were stimulated three to five times for 30 min each. Increasing frequency (0.5-16 Hz) and voltage (5-20 V) increased ACh release; increasing pulse duration (0.5-3 ms) had only a minor effect. Alterations in muscle preload (2-20 g) had no effect on ACh release. ACh release was fairly constant for up to five repeated stimulation periods with the same EFS parameters. Stimulation of the tissues for 15 min released the same amount of ACh as 30 min if the amount was expressed as picomoles per gram per minute, suggesting that ACh release rate was constant during the 30-min period of stimulation. Atropine (10(-6) M) potentiated the release of ACh four- to fivefold, presumably by removing the autoinhibitory effect of ACh on the cholinergic nerves. Tetrodotoxin (10(-6) M) abolished the EFS-induced ACh release.

Effects of Adenylate Cyclase Inhibitors, Phosphodiesterase Inhibitors, and Dibutyryl Cyclic AMP on Spontaneous and Various Stimuli-lnduced Acetylcholine Release from Guinea Pig Ileum Myenteric Plexus

In order to examine a possible contribution of cyclic AMP to acetylcholine (ACh) release from guinea pig ileum myenteric plexus, effects of adenylate cyclase inhibitors, phosphodiesterase (PDE) inhibitors and dibutyryl cyclic AMP on the spontaneous and the various stimuli-induced ACh release were investigated. A PDE inhibitor, theophylline (1 mM) increased the ACh release induced by nicotine (6.16 microM) significantly. Another PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 1 mM) and dibutyryl cyclic AMP (4 mM) had no effect. The adenylate cyclase inhibitors dithiobisnitrobenzoic acid (DTNB, 1 mM) and alloxan (4 mM) both decreased the nicotine-induced ACh release remarkably. PDE inhibitors increased and adenylate cyclase inhibitors decreased the high-K+-induced ACh release. Dibutyryl cyclic AMP brought about a slight but significant increase of the high-K+-induced ACh release. All the drugs failed to alter the ACh release induced by electrical field stimulation (EFS) at 10 Hz. Effects of all drugs except dibutyryl cyclic AMP on the spontaneous ACh release were the same as those on the nicotine-induced one. Dibutyryl cyclic AMP decreased it significantly. These results suggest that the cyclic AMP system is involved in the spontaneous, the nicotine-induced and the high-K+-induced ACh release and that the EFS-induced ACh release is independent of cyclic AMP.