Myenteric Plexus-longitudinal Muscle Preparation Research Articles

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, 1 H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (ODQ), concentration-dependently enhanced both basal release (−log EC 50: 6.8) and electrically (10 Hz) -evoked release (−log EC 50: 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 μM) 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 μM) 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 EC 50: 6.0) and longitudinal muscle contractions (−log EC 50: 5.7). ODQ (10 μM) 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.

Inhibition by cannabinoid receptor agonists of acetylcholine release from the guinea-pig myenteric plexus.

1. The dose-related inhibition of the twitch responses of the myenteric plexus-longitudinal muscle preparation of the guinea-pig small intestine by cannabinoid (CB) agonists, (+)-WIN 55212 and CP 55940 during stimulation at 0.1 Hz with supramaximal voltage was confirmed. These agonists inhibited acetylcholine (ACh) release in the presence of physostigmine (7.7 microM) thus indicating a prejunctional site of action. 2. Inhibition of twitch responses and ACh release by CB agonists was reversed by the CB1-selective cannabinoid receptor antagonist, SR141716A. Dose-response curves to (+)-WIN 55212 and CP 55940 were shifted to the right, with no reduction of maximal response, by pretreatment with SR141716A (31.6-1000 nM), but not its vehicle, Tween 80 (1 microM). However, at very high concentrations (25-400 microM), Tween 80 itself caused a dose-related inhibition of the twitch response which was significantly reduced in the presence of SR141716A (1 microM). The opioid receptor antagonist, naloxone (1 microM) had no significant effect on the inhibition by CP 55940 of the twitch response. 3. (+)-WIN 55212, CP 55940 and Tween 80 (50 microM) had no effect on responses to exogenous ACh, confirming that their actions were prejunctional. SR141716A (1 microM) did not increase the sensitivity of the longitudinal muscle to either ACh or histamine, but inhibited the responses to high doses of ACh. 4. The (-)-enantiomer of WIN 55212, was approximately 300 times less active than the (+) enantiomer in inhibiting the twitch response, had no CB1 antagonist activity against the active isomer and did not inhibit the release of ACh in the presence of physostigmine. 5. The dissociation constant (KD) values for SR 141716A against the inhibitory effect of (+)-WIN 55212 and CP 55940 on the twitch response were 12.07 nM (95% confidence intervals 8.55 and 20.83) and 6.44 nM (95% confidence intervals 4.70 and 10.24), respectively. In experiments in which the release of ACh was inhibited by (+)-WIN 55212, the KD values were 9.21 nM and 10.53 nM at SR141716A concentrations of 31.6 nM and 100 nM, respectively. The KD values for the antagonism by naloxone of the inhibition of the twitch responses and the inhibition of ACh release by normorphine in this preparation were found to be 2.38 +/- 0.69 nM and 2.00 +/- 0.9 nM, respectively. 6. During maximal inhibition of ACh release by (+)-WIN 55212, the addition of normorphine (400 nM) caused a further significant decrease in ACh output. 7. SR141716A alone produced a significant increase in ACh release in both the absence and presence of exogenous cannabinoid drugs, hence we conclude that it has a presynaptic site of action. We also conclude that SR141716A acts either by antagonizing the effect of an endogenous CB1 receptor agonist or by having an inverse agonist effect at these receptors.

Evidence that methyl arachidonyl fluorophosphonate is an irreversible cannabinoid receptor antagonist.

1. Methyl arachidonyl fluorophosphonate (MAFP) (1 microM) significantly attenuated the ability of WIN 55,212-2, CP 55,940, (-)-delta 9-tetrahydrocannabinol (THC), nabilone and (R)-(+)-arachidonoyl-1'-hydroxy-2'-propylamide (methanandamide) to inhibit electrically-evoked isometric contractions of the myenteric plexus-longitudinal muscle preparation of guinea-pig small intestine. 2. The sizes of the maximal responses to WIN 55,212-2 and CP 55,940 decreased significantly in the presence of 1 microM MAFP. 3. MAFP (1 microM) essentially abolished the inhibitory effects on the twitch response of the highest concentration of methanandamide used (3.162 microM). The dextral shift it induced in the log concentration-response curve of nabilone was non-parallel. In contrast, the dextral shift in the log concentration-response curve of THC produced by MAFP did not deviate significantly from parallelism and was relatively small with a mean value of 3.45 and 95% confidence limits of 1.19 and 13.08. 4. MAFP (1 microM) did not attenuate the effects of normorphine or clonidine on the twitch response of the myenteric plexus-longitudinal muscle preparation or affect the contractile response of this preparation to acetylcholine. 5. When administered by itself at concentrations of 1 to 1000 nM, MAFP had no detectable effect on the twitch response of the myenteric plexus-longitudinal muscle preparation. 6. These results support the hypothesis that MAFP is an irreversible cannabinoid CB1 receptor antagonist that possesses some degree of selectivity.

Role of nitric oxide in norepinephrine release from myenteric plexus in vitro and in Trichinella spiralis-infected rats.

Nitric oxide (NO) has been implicated as modulator of neural function and inflammatory mediator. Previously, we have demonstrated suppression of norepinephrine (NE) release from myenteric nerves following Trichinella spiralis infection implicating interleukin-1 beta (IL-1 beta) as a mediator of these changes. In the present study, we have examined the role of NO in NE release from the myenteric plexus and in the suppression of NE release induced by IL-1 beta in vitro, and we have determined whether NO is involved in the suppression of NE release from the myenteric plexus observed in T. spiralis-infected rats. Electrically evoked NE release from jejunal longitudinal muscle-myenteric plexus preparations (LMMP) was measured following (a) in vitro exposure of the tissue to the NO donor 3-morpholinosydnonimine (SIN-1), L-arginine, or IL-1 beta, in the presence or absence of NOS inhibitors, and (b) in vivo treatment of control or T. spiralis-infected rats with N6-nitro-L-arginine-methyl-ester (L-NAME), NG-nitro-D-arginine-methyl-ester (D-NAME) or vehicle for 6 days. In vitro inhibition of NO synthesis had no effect on NE release from the myenteric plexus. Treatment with SIN-1 or L-arginine suppressed NE release in a manner similar to that observed with IL-1 beta. Moreover, the effect of IL-1 beta was attenuated by L-NAME. In contrast, treatment of T. spiralis-infected rats with L-NAME had no effect on the suppression of NE release. These results indicate that in the absence of inflammation, the myenteric plexus can generate sufficient NO to inhibit NE release and that NO mediates the action of IL-1 beta on NE release in vitro. However, we have no evidence for the involvement of NO in the suppression of NE release in nematode-infected rats.

Depressant effects of hypoxia and hypoglycaemia on neuro-effector transmission of guinea-pig intestine studied in vitro with a pharmacological model.

1. Since intermittent ischaemia may play an important role in the aetiology of Inflammatory Bowel Disease, particularly Crohn's Disease, a pharmacological model of neuronal ischaemia was applied to guinea-pig isolated intestinal preparations to mimic the acute effects of reduced blood flow on intestinal motility. 2. Neuro-effector transmission and smooth muscle performance were examined in myenteric plexus-longitudinal muscle preparations of guinea-pig ileum exposed to sodium cyanide (NaCN), in order to inhibit oxidative phosphorylation, or to iodoacetic acid (IAA), to block glycolysis. Comparisons were made with the effects due to simple deprivation of oxygen or glucose. 3. Depression of cholinergic neuro-effector transmission induced by hypoxia or NaCN (effective concentration range 0.1-3 mM), given as separate treatments, singly or repetitively over 60-90 min, were apparent within 30 s and were reversible. The maximum inhibition was 90% and the IC50 for NaCN was 0.3 mM. A conspicuous component of these inhibitions was prejunctional. 4. Non-cholinergic neuro-effector contractions were inhibited by up to 90% by anoxia or NaCN but recovery was incomplete and slower than with cholinergic contractions. 5. Glucose-free solutions also caused a reversible failure of cholinergic neuro-effector transmission but of slower onset. In contrast, IAA (0.06-1 mM) abolished contractions irreversibly, apparently by a direct depressant effect on smooth muscle contraction. Unlike NaCN, IAA caused an initial potentiation of electrically-induced contractions, partly by a prejunctional potentiation of cholinergic neuro-effector transmission. 6. It is concluded that a disruption of intestinal activity in pathological conditions associated with intestinal ischaemia may result from disturbances in the function of enteric neurones.

Inhibition of acetylcholine induced intestinal motility by interleukin 1 beta in the rat.

The fact that raised interleukin 1 beta (IL 1 beta) concentrations have been found in the colonic mucosa of rats with experimentally induced colitis and of patients with inflammatory bowel disease indicates that this cytokine may participate in the disturbed intestinal motility seen during inflammatory bowel disease. This study investigated whether IL 1 beta could change the contractility of (a) a longitudinal muscle-myenteric plexus preparation from rat jejunum, ileum, and colon and (b) isolated jejunal smooth muscle cells. Isometric mechanical activity of intestinal segments was recorded using a force transducer. Moreover, smooth muscle cell length was measured by image analysis. Although IL 1 beta did not affect jejunal, ileal, and colonic basal contractility, it significantly reduced contractile response to acetylcholine (ACh). This significant inhibition was seen only after 90 or 150 minutes of incubation with IL 1 beta. Pretreatment with cycloheximide blocked IL 1 beta induced inhibition of ACh stimulated jejunal contraction, suggesting that a newly synthesised protein was involved in the effect. NW-nitro-L-arginine (a nitric oxide synthase inhibitor) did not prevent the inhibition induced by IL 1 beta. Blocking neural transmission with tetrodotoxin abolished the IL 1 beta effect on jejunal contractile activity, whereas IL 1 beta had no effect on isolated and dispersed smooth muscle cells. IL 1 beta inhibits ACh induced intestinal contraction and this inhibitory effect involves protein synthesis but is independent of nitric oxide synthesis. This effect does not involve a myogenic mechanism but is mediated through the myenteric plexus.

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.

Further evidence for the presence of cannabinoid CB1 receptors in guinea-pig small intestine.

1. CP 50,556, CP 55,940, nabilone, CP 56,667, delta 9 -tetrahydrocannabinol (THC) and cannabinol each inhibited electrically-evoked contractions of the myenteric plexus-longitudinal muscle preparation of guinea-pig small intestine in a concentration-related manner. The IC50 values of these cannabinoids, respectively 3.45, 3.46, 30.61, 162.94, 214.63, and 3913.5 nM, correlate well with previously obtained potency values for displacement of [3H]-CP 55,940 from cannabinoid binding sites. 2. Electrically-evoked contractions of the myenteric plexus-longitudinal muscle preparation were also inhibited by AM 630 (6-iodo-pravadoline) and by WIN 55,212-2 (IC50 = 1923.0 and 5.54 nM, respectively). The present finding that AM 630 is an agonist, contrasts with a previous observation that it behaves as a cannabinoid receptor antagonist in the mouse isolated vas deferens. 3. SR141716A produced dose-related parallel rightward shifts in the log concentration-response curves of CP 55,940, WIN 55,212-2, THC and AM 630 for inhibition of electrically-evoked contractions of the myenteric plexus-longitudinal muscle preparation. SR141716A (1 microM) did not reverse the inhibitory effects of normorphine and clonidine on electrically-evoked contractions or potentiate the contractile response to acetylcholine. 4. Doses of naloxone and yohimbine that reversed the inhibitory effects of normorphine or clonidine on electrically-evoked contractions of the myenteric plexus-longitudinal muscle preparation did not affect the inhibitory response to WIN 55,212-2. 5. Electrically-evoked release of acetylcholine from strips of myenteric plexus-longitudinal muscle was decreased by 200 nM CP 55,940 and this inhibitory effect was almost completely reversed by 1 microM SR141716A. Acetylcholine-induced contractions were not affected by 200 nM CP 55,940. 6. These results support the hypothesis that guinea-pig small intestine contains prejunctional cannabinoid CB1 receptors through which cannabinoids act to inhibit electrically-evoked contractions by reducing release of the contractile transmitter, acetylcholine. 7. THC was found to be more susceptible to antagonism by SR141716A than CP 55,940 or AM 630, raising the possibility that guinea-pig small intestine contains more than one type of cannabinoid receptor. 8. At concentrations of 10 nM and above, SR141716A produced small but significant increases in the amplitude of electrically-evoked contractions of the myenteric plexus-longitudinal muscle preparation suggesting that this tissue may release an endogenous cannabinoid receptor agonist or that some cannabinoid receptors in this tissue are precoupled and that SR141716A can reduce the number of receptors in this state.

Localization of endothelin ETB receptors on the myenteric plexus of guinea-pig ileum and the receptor-mediated release of acetylcholine.

1. The type of endothelin (ET) receptor located on the myenteric neurones of guinea-pig ileum was determined by receptor autoradiography and function of the receptor was examined by release experiments of acetylcholine (ACh) from the longitudinal muscle myenteric plexus (LM-MP) preparations. 2. Specific [125I]-ET-1 binding sites were distributed in muscle layers, myenteric and submucous plexuses, and mucosa layers. High-grain densities were detected in both myenteric and submucous plexuses. 3. Binding in the myenteric plexus was abolished by incubation with either IRL 1620 (endothelin ETB receptor agonist) or BQ 788 (endothelin ETB receptor antagonist), but not with BQ 123 (endothelin ETA receptor antagonist). The [125I]-IRL 1620 binding sites were evident in the myenteric plexus. Thus, the endothelin receptor located on the myenteric neurones is of the ETB type. 4. ET-1 (10(-10)-3 x 10(-8) M) and ET-3 (10(-10)-3 x 10(-8) M) evoked 3H outflow from LM-MP preparations of ileum preloaded with [3H]-choline, in a concentration-dependent manner. There was no significant difference between maximum amounts of ET-1-evoked and ET-3-evoked 3H outflow. 5. ET-1 and ET-3 evoked outflow of 3H was BQ 788-sensitive, but BQ 123-insensitive. Both evoked outflows of 3H were Ca(2+)-dependent and tetrodotoxin-sensitive. 6. These results indicate that the endothelin ETB receptor is located on the enteric cholinergic neurones and that stimulation evokes the release of ACh.

Modulation of kappa-opioid receptor mediated tolerance in the guinea-pig ileum by chronic co-administration of dihydropyridines.

The influence of the L-type Ca2+ channel modulators nimodipine (a Ca2+ blocker) and BAY K 8644 (a Ca2+ activator) on the expression of tolerance to the inhibitory effects of kappa- and mu-opioid agonists in the guinea-pig ileum from guinea-pigs rendered tolerant to the kappa-opioid receptor agonist U-50,488H was investigated. Tolerance to U-50,488H was induced by its administration (15 mg/kg twice a day) for 4 days. Control groups received saline at the same time schedule. Chronic infusion of guinea-pigs with nimodipine (2 micrograms/microliters/h for 7 days) or BAY K 8644 (0.5 microgram/microliters/h for 7 days), did not cause any modification of the height of contractions induced by electrical stimulation of the myenteric plexus-longitudinal muscle (MPLM) preparation from naïve guinea-pigs. The Ca2+ antagonist nimodipine increases the potency of U-50,488H (selective kappa agonist) to reduce the amplitude of neurogenic contractions of the MPLM strip in naïve animals, whereas the Ca2+ activator BAY K 8644 induced the opposite effect. However, the effect of DAMGO (selective mu agonist) was not modified in guinea-pigs infused with nimodipine or BAY K 8644. Tolerance to the inhibitory effects of both U-50,488H and DAMGO was observed following administration of U-50,488H for 4 days and was revealed as a rightward shift of the concentration-response curves for the two agonists. Chronic infusion of guinea-pigs with nimodipine concurrently with chronic U-50,488H, markedly attenuated the expression of selective tolerance to U-50,488H as well as the cross-tolerance between U-50,488H and DAMGO. By the contrary, the magnitude of tolerance to U-50,488H and to DAMGO was enhanced by concomitant infusion of BAY K 8644. The results suggest that, in the GPI, kappa-opioid receptor may be functionally linked to the dihydropyridine sensitive Ca2+ channel: The blockade of the channel increased whereas its activation reduced the potency of U-50,488H. In chronic experiments, nimodipine prevented the expression of tolerance to U-50,488H and the cross-tolerance between U-50,488H and DAMGO, whereas BAY K 8644 produced the opposite effect. These results suggest that, in the GPI, selective tolerance to kappa-agonist as well as cross-tolerance between kappa- and mu-opioid agonists would involve activation of L-type Ca2+ channels, which could indicate that intracellular Ca2+ may be the final common pathway through which myenteric neurons adapt to the chronic opioid exposure.

ATP and nitric oxide: inhibitory NANC neurotransmitters in the longitudinal muscle-myenteric plexus preparation of the rat ileum.

1. The nature of neurotransmitter(s) involved in non-adrenergic non-cholinergic (NANC) relaxations induced by electrical stimulation (10 s trains, 1-8 Hz) was investigated in the precontracted longitudinal muscle-myenteric plexus preparation of the rat ileum. 2. Electrical stimulation of the tissue induced complex responses, consisting of a primary contraction, a primary relaxation, an off-relaxation and a rebound contraction, which were all tetrodotoxin(TTX)-sensitive. 3. Vasoactive intestinal polypeptide (VIP) and carbon monoxide (CO) did not induce relaxations. alpha-Chymotrypsin did not reduce the relaxations induced by electrical stimulation, while zinc protoporphyrin IX had non-specific effects. 4. Nitric oxide (NO) induced concentration-dependent relaxations. NG-nitro-L-arginine methylester (L-NAME) abolished the primary contractions and off-relaxations, while it partially reduced the primary relaxations. 5. ATP induced relaxations and ATP-desensitization of the tissues partially reduced the primary relaxations. Suramin and reactive blue 2 did not consistently influence the primary relaxations. 6. The ATP-induced relaxations were not influenced by L-NAME or TTX. The inhibitory effect of ATP-desensitization and L-NAME did not summate. 7. The cyclic AMP content of the tissue did not increase upon electrical stimulation or after addition of NO or ATP. The cyclic GMP content of the tissue increased upon electrical stimulation and addition of NO, but not after addition of ATP. 8. It is concluded that the relaxation induced by electrical stimulation consists of two types of responses. The off-relaxation is completely nitrergic, while the primary relaxation is mediated by NO, ATP and an as yet unknown transmitter which is not VIP or CO.

Influence of age on cholinergic and inhibitory nonadrenergic noncholinergic responses in the rat ileum

In view of the previously reported marked reduction in the number of neurons in the rat small intestine, the influence of age on the responses to electrical stimulation of the longitudinal muscle-myenteric plexus preparation of the ileum of young (3–4 months), adult (12–13 months) and old (24–25 months) rats was investigated. The differences in responses between the three age groups were varied. At the basal tone level, electrical train stimulation induced complex responses consisting of a primary contraction, which was partly cholinergic, a secondary contraction, which was completely cholinergic and a rebound contraction, which was partly cholinergic. These responses did not differ between young, adult and old rats. The inhibiting effects of atropine and potentiating effects of physostigmine on these responses did not change with age. The acetylcholine-induced concentration-response curve did not differ between the three age groups. Electrical train stimulation of the precontracted longitudinal muscle-myenteric plexus preparation under nonadrenergic noncholinergic (NANC) conditions induced multiphasic responses consisting of a primary contraction, a primary relaxation, a postrelaxation and a rebound contraction. The primary contraction and the postrelaxation, which were nitrergic in nature, were not influenced by age. The primary relaxation was slightly more pronounced in young rats. In young rats, this relaxation was partly inhibited by N G-nitro- l-arginine methyl ester (L-NAME) and ATP desensitization, while in adult and old rats these primary relaxations were only influenced by ATP desensitization, but not by L-NAME. Nitric oxide-induced relaxations were similar in the three age groups. The cAMP content was not increased by electrical stimulation, while the cGMP content increased with electrical stimulation, but no differences due to age were observed. These results suggest that cholinergic responses in the rat small intestine are well-maintained with age while the nitrergic contribution to NANC relaxation decreases with age.

Azabicyclic indole esters as potent 5-HT 4 receptor antagonists

The synthesis of a series of azabicyclic indole esters is described and their potency reported as 5-HT 4 receptor antagonists. Optimization of the most potent compound ( 19) by preparing the corresponding oxazino[3,2- a]indole ester afforded 34, which had a pIC 50 of 9.5 in the guinea pig distal colon longitudinal muscle myenteric plexus preparation.

Comparative effects of K + channel modulating agents on contractions of rat intestinal smooth muscle

The effects of six K + channel openers were investigated on contractions of the rat ileum longitudinal muscle-myenteric plexus preparation elicited by electrical field stimulation and by K +. Levcromakalim, pinacidil, RP 49356 ( N-methyl-2-(3 pyridyl)-tetrahydro-thiopyran-2-carbothioamide-1-oxide) and SDZ PCO 400 ((3 S,4 R)-3,4-dihydro-3-hydroxy-2,2-dimethyl-4-[(3-oxo-1-cyclopenten-1-yl)oxy]-2 H-1-benzopyran-6-carbonitrile) completely abolished contractions elicited by electrical stimulation and caused complete relaxation of contractions elicited by K + with comparable IC 50 values. Minoxidil sulphate was much less potent and diazoxide was without effect in either protocol. The relaxant effects of these agents were antagonized by glibenclamide, tetraethylammonium and yohimbine in a manner which was not surmountable. The present study indicates that the relaxant effect of these compounds in intestinal smooth muscle is mediated through glibenclamide-sensitive ATP-dependent K + channels. These compounds did not preferentially inhibit either direct smooth muscle- or nerve-mediated responses. The present data may point to differences in the channels or their regulatory sites, in intestinal, compared with vascular, smooth muscle.

Involvement of eicosanoids and macrophage-like cells in cytokine-mediated changes in rat myenteric nerves

Background & Aims: Proinflammatory cytokines alter function in enteric nerves, but little is known about underlying mechanisms. This study was designed to investigate the roles of prostanoids and of macrophage-like cells in cytokine-induced suppression of [ 3H]norepinephrine release from rat myenteric plexus. Methods: The release of 3H from jejunal longitudinal muscle-myenteric plexus preparations that had been loaded with [ 3H]norepinephrine was measured. Measurements of 3H release as well as concentrations of prostaglandin E 2 and leukotriene were made in preparations exposed to interleukin 1β plus interleukin 6 and in the presence or absence of piroxicam, 5-lipoxygenase inhibitor MK886, cycloheximide, or cyclosporin A. An ultrastructural analysis was also performed to investigate the presence of macrophage-like cells in the myenteric plexus. Results: Interleukin 1β plus interleukin 6 suppressed 3H release and caused an increase in tissue prostaglandin E 2 butnot leukotriene E 4. Piroxicam and cycloheximide but not MK886 attenuated the cytokine-induced increase in prostaglandin E 2 and the suppression of [ 3H]norepinephrine release. Ultrastructural analysis showed macrophage-like cells in the plexus, and the cytokine effects were inhibited by cyclosporin A. Conclusions: Prostanoids but not leukotrienes mediate the cytokine-induced suppression of norepinephrine release, and the results of this study suggest that macrophage-like cells are also involved.

Stimulation-induced 3H-L-citrulline accumulation in isolated longitudinal muscle myenteric plexus preparations of rat small intestine: a measure to characterize nitrergic neurons.

Nitric oxide (NO) synthase activity in rat isolated longitudinal muscle myenteric plexus preparations of the small intestine was determine by measuring the accumulation of 3H-L-citrulline during 30 min incubation with 3H-L-arginine. In untreated preparations a significant amount of 3H-L-citrulline accumulated in the tissue, about 2000 dpm/30 mg per 30 min, accounting for about 1.7% of the tissue radioactivity. Intermittent electrical field stimulation (15 Hz, 10 s trains with 10 s intervals for total of 20 min) caused a threefold increase in 3H-L-citrulline accumulation. The NO synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) reduced the spontaneous accumulation of 3H-L-citrulline by 65% and prevented the electrically evoked increase. Removal of extracellular calcium or addition of tetrodotoxin blocked the electrically evoked increase in 3H-L-citrulline accumulation without affecting spontaneous accumulation. Application of the calcium ionophore A 23187 (10 mumol/l) or 45 mmol/l) or 45 mmol/l potassium caused a twofold increase in the accumulation of 3H-L-citrulline. The muscarine receptor agonist oxotremorine (1 mumol/l) had no effect on spontaneous accumulation of 3H-L-citrulline, but inhibited the electrically evoked increase by about 50%, and this effect was blocked by scopolamine. A substantial amount of 3H-L-citrulline (15000 dpm) accumulated also in the incubation media, and this was increased 1.7-fold by the presence of A 23187 and 2.7-fold by electrical stimulation. However, electrically evoked increase in 3H-L-citrulline was not prevented by tetrodotoxin, in contrast to observation on tissue levels. In conclusion, during incubation with 3H-L-arginine tissue levels of 3H-L-citrulline in rat isolated longitudinal muscle myenteric plexus preparations, but not accumulation in incubation media may be used as a biochemical marker of the activity of nitrergic intestinal neurons which appear to be inhibited via muscarine receptors.

Experimental colitis alters myenteric nerve function at inflamed and noninflamed sites in the rat

Background & Aims : Studies in inflammatory bowel disease have shown extensive structural abnormalities in the enteric nervous system of inflamed and noninflamed gut; however, functional correlates are lacking. The aim of this study was to determine the effect of colitis on myenteric nerve function at inflamed and noninflamed sites in rat intestine. Methods : Tritiated noradrenaline release was measured from longitudinal muscle myenteric plexus preparations from the distal and transverse colon and terminal ileum of rats with colitis induced by trinitrobenzene sulfonic acid or Trichinella spiralis larvae. Results : As characterized by myeloperoxidase activity and histology, both models induced inflammation restricted to the distal colon. In the distal colon in trinitrobenzene sulfonic acid colitis, KCl- or electrical field stimulation-evoked 3H release was suppressed by 56% and 60%, respectively; in T. spiralis-infected rats, the KCl-evoked release was suppressed by 58%. 3H release was also suppressed by similar magnitudes in noninflamed transverse colon and terminal ileum of each model. Conclusions : Experimental distal colitis alters myenteric nerve function in inflamed distal colon and noninflamed gut regions. These changes are independent of the manner in which colitis is induced and provide a basis for the extensive disruption of physiological function observed in inflammatory bowel disease.

Subclassification of presynaptic ?2-adrenoceptors: ?2D-autoreceptors and ?2D-adrenoceptors modulating release of acetylcholine in guinea-pig ileum

The study was designed to classify in terms of alpha 2A, alpha 2B, alpha 2C and alpha 2D the presynaptic alpha 2-autoreceptors, as well as the alpha 2-receptors modulating the release of acetylcholine, in the myenteric plexus-longitudinal muscle (MPLM) preparation of the guinea-pig ileum. A set of antagonists was chosen that was able to discriminate between the four subtypes. Small pieces of the MPLM preparation were preincubated with 3H-noradrenaline or 3H-choline and then superfused and stimulated electrically. The stimulation periods used (3H-noradrenaline: 3 trains of 20 pulses, 50 Hz, train interval 60 s; 3H-choline: single trains of 30 pulses, 0.2 Hz) did not lead to alpha 2-autoinhibition or inhibition of 3H-acetylcholine release by endogenous noradrenaline. The alpha 2-selective agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) reduced the evoked overflow of tritium in both 3H-noradrenaline and 3H-choline experiments. Most (3H-noradrenaline) or all (3H-choline) of the 10 antagonists shifted the concentration-inhibition curves of UK 14,304 to the right. pKd values of the antagonists were calculated from the shifts. pKd values from 3H-noradrenaline experiments correlated with pKd values from 3H-choline experiments (r = 0.981). It is concluded that alpha 2-autoreceptors and alpha 2-heteroreceptors modulating the release of acetylcholine in the MPLM preparation are of the same subtype. Comparison with antagonist affinities for prototypic native alpha 2 binding sites, binding sites in cells transfected with alpha 2 subtype genes, and previously classified presynaptic alpha 2-adrenoceptors--all taken from the literature--indicates that both are alpha 2D.(ABSTRACT TRUNCATED AT 250 WORDS)

Benzimidazolones and renzapride facilitate acetylcholine release from guinea-pig myenteric plexus via 5-HT4 receptors.

The effects of the 5-HT4 receptor agonists BIMU 8, BIMU 1, renzapride and of the 5-HT1p receptor agonist 5-hydroxyindalpine on basal and electrically evoked outflow of tritium were studied in guinea-pig longitudinal muscle myenteric plexus preparations preincubated with [3H]choline. Muscle contractions were recorded simultaneously. BIMU 8 caused a calcium dependent and tetrodotoxin sensitive increase in basal [3H]outflow that was assumed to represent release of [3H]acetylcholine. In addition, BIMU 8 enhanced the release of [3H]acetylcholine and twitch contractions evoked by submaximal electrical stimulation. Ondansetron (1 mumol/l) did not change the effects of BIMU 8, but DAU 6285 and tropisetron (each 1 mumol/l) competitively antagonized the various facilitatory effects of BIMU 8 with pA2 values of 7.0-7.2 (DAU 6285) and 7.0-7.3 (tropisetron). The phosphodiesterase inhibitors IBMX and rolipram did not increase the effects of BIMU 8. BIMU 1 and renzapride also concentration-dependently increased basal release of acetylcholine, and release and contractions caused by submaximal stimulation. The effects of BIMU 1 and renzapride were competitively antagonized by 1 mumol/l tropisetron (pA2 6.6-7.1). The EC50 values for the increase in the evoked [3H]acetylcholine release and contractions were closely similar. 5-Hydroxyindalpine did not change basal release and slightly inhibited the evoked release of [3H]acetylcholine. Release of acetylcholine and contractions elicited by submaximal stimulation were strongly inhibited by (+)-tubocurarine which indicates that nicotine ganglionic transmission is involved in this kind of release. The results suggest that BIMU 8, BIMU 1 and renzapride stimulate 5-HT4 receptors at cholinergic interneurones and thereby facilitate nicotinic ganglionic transmission in the myenteric plexus.(ABSTRACT TRUNCATED AT 250 WORDS)

The interaction of antidepressant drugs with central and peripheral (enteric) 5‐HT3 and 5‐HT4 receptors

1. A combined study of receptor binding in central neuronal cell membranes and functional responses in isolated segments of guinea-pig small intestine allowed characterization of the interaction of four antidepressant drugs with central and peripheral 5-HT3 and 5-HT4 receptors. 2. Clomipramine, paroxetine and fluoxetine inhibited [3H]-DAU 6215 binding to 5-HT3 recognition sites in NG 108-15 cells with IC50 values in the range 1.3-4 microM. Litoxetine had an IC50 of 0.3 microM. The specific binding of [3H]-GR 113808 to 5-HT4 recognition sites in pig striatal membranes was inhibited by all four antidepressants with negligible potency (IC50 values > or = 20 microM). 3. In whole ileal segments, concentration-response curves to 5-HT were biphasic, with the high- and low-potency phases involving 5-HT4 and 5-HT3 receptors, respectively. Curves to 2-methyl-5-hydroxytryptamine (2-methyl-5-HT: a 5-HT3 receptor agonist) and 5-methoxytryptamine (5-MeOT: a 5-HT4 receptor agonist) were monophasic. All antidepressants were used at concentrations lacking anticholinoceptor properties, as demonstrated in both electrically stimulated longitudinal muscle-myenteric plexus preparations (LMMPs) and in unstimulated LMMPs following addition of acetylcholine (100 nM). 4. Fluoxetine (0.1-1 microM) and litoxetine (0.3-3 microM) antagonized both the high- and low-potency phases of the 5-HT curve. Schild analysis for the low-potency phase yielded pA2 estimates of 6.6 +/- 0.3 (Schild slope of 1.1) and of 6.6 +/- 0.1 (Schild slope of 1.1), respectively. At higher concentrations (3 microM), fluoxetine markedly inhibited the 5-HT response maximum. Clomipramine (10-300 nM) inhibited, by a mechanism independent of concentration, both phases of the 5-HT curve with a reduction of the maximum response. Paroxetine (1 microM) was ineffective on the high-potency phase, but caused a rightward shift of the low-potency phase (pKB: 6.1 +/- 0.01). 5. Responses to 2-methyl-5-HT were inhibited by 1 microM fluoxetine (pKB: 5.4 +/- 0.02). Like clomipramine(30 and 100 nM), litoxetine (1 and 3 microM) produced rightward displacements of 2-methyl-5-HT-induced contractions, which were virtually independent of antidepressant concentration (pKB values: 6.0 +/- 0.02 and 5.5 +/- 0.01, respectively). At higher concentrations, fluoxetine (3 microM) and clomipramine (300 nM)markedly reduced the 2-methyl-5-HT response maximum. Paroxetine (1 micro M) was ineffective.6. Responses to 5-MeOT were shifted to the right by fluoxetine (0.1-1 micro M) and litoxetine (1 and 3 microM)in a concentration-dependent manner. At higher concentrations, fluoxetine (3 microM) markedly reduced the 5-MeOT response maximum, an effect also observed with 100 and 300 nM clomipramine. Paroxetine(1 microM) was ineffective.7. In unstimulated LMMPs, the excitatory effects evoked by 5-HT, 2-methyl-5-HT and 5-MeOT and the antagonism produced by 300 nM clomipramine were comparable to those obtained in whole ileal segments. This suggests that 5-HT contained in the mucosa of whole preparations does not interfere with agonist-induced contractile responses and with the inhibitory effect of antidepressant drugs.8. In conclusion, our results show that clomipramine, fluoxetine, paroxetine and litoxetine possess low to moderate potency/affinity at both central and peripheral (enteric) 5-HT3 receptors. In contrast, all four antidepressants are virtually ineffective at central 5-HT4 receptors. Inhibition of 5-HT4 receptor mediated ileal contractions by fluoxetine, litoxetine and clomipramine may result from allostericant agonism or, more likely, from post-receptor blockade of second messenger generation. The interaction of antidepressants with central and peripheral 5-HT3 and 5-HT4 receptors may be relevant for both potential therapeutic action and adverse effects at gastrointestinal level.