Actions of gamma-aminobutyric acid on neurones of guinea-pig myenteric plexus.

The effects of gamma-aminobutyric acid (GABA) applied by ionophoresis, pressure ejection and superfusion to myenteric neurones of the guinea-pig ileum were investigated by intracellular recording techniques. Ionophoretic or pressure application of GABA (10 pC-30 nC) caused membrane depolarizations of AH neurones but not S neurones. This depolarization was associated with a conductance increase. It reversed polarity at a membrane potential of -18 mV when intracellular electrodes contained KCl, and -39 mV when electrodes contained K acetate, citrate or sulphate. The ionophoretic depolarization was antagonized by bicuculline (1-30 microM) in an apparently competitive manner. During prolonged or repeated ionophoretic application of GABA, both the depolarization and conductance increase desensitized. Superfusion of GABA (1-100 microM) caused a membrane depolarization in AH neurones, associated with an increase in membrane conductance. The increase in conductance was always smaller than that evoked by ionophoresis of GABA. Bicuculline only partially depressed the depolarization induced by superfusion of GABA, particularly slowing its rising phase. beta-p-Chlorophenyl GABA (baclofen) (10 microM) caused a depolarization similar to that observed with GABA in the presence of bicuculline. The depolarization induced by baclofen and GABA (in presence of bicuculline) superfusion did not decline during prolonged applications; superfusion of GABA but not baclofen reversibly reduced or eliminated the effects of GABA ionophoresis. It is concluded that GABA has two effects on the membrane of myenteric neurones. The first is a bicuculline-sensitive, rapidly desensitizing chloride activation: the second is a bicuculline-insensitive, non-desensitizing depolarization.     

The calcium-dependent slow after-hyperpolarization in myenteric plexus neurones with tetrodotoxin-resistant action potentials

Intracellular recordings were made from neurones of the myenteric plexus of the guinea-pig ileum. The slow after-hyperpolarization which followed an action potential in some neurones was abolished by Mn(++), La(+++) and by solutions which contained no Ca(++). In these neurones, the action potential and the slow after-hyperpolarization persisted in Na(+)-free solutions or in the presence of tetrodotoxin (2 muM). The findings suggest that an inward Ca(++) current during the action potential is essential for the slow after-hyperpolarization.     

Extracellular recording from the guinea-pig myenteric plexus and the action of morphine

Extracellular recordings were made with glass suction electrodes from neurons in isolated ganglia of the myenteric plexus of the guinea-pig ileum. The activity recorded was considered to arise in neuronal somata as a consequence of the mechanical contact with the recording electrode. The spike activity was depressed by normorphine (10 nM—1 μM) and this action was prevented by low concentrations of naloxone. Morphine remained effective in solutions which contained 1 or 12 mM K⁺. The findings support other evidence that morphine acts by hyperpolarizing the soma membrane of myenteric neurons, but do not elucidate the ionic mechanism of the effect.     

Actions of bradykinin on electrical and synaptic behavior of neurones in the myenteric plexus of guinea-pig small intestine

1. Electrophysiologic methods were used to study actions of bradykinin (BK) in neurones of the myenteric plexus of guinea-pig small intestine in vitro. Exposure to BK depolarized the membrane potential and elevated excitability in AH- and S-type neurones. Neuronal input resistance associated with the depolarizing responses was either decreased or unchanged in S-type and increased in AH-type neurones. 2. The selective B(2) BK receptor antagonist HOE-140, but not the selective B(1) receptor antagonist des-arg(10)-HOE-140, suppressed the BK-evoked responses. RT-PCR confirmed the expression of B(2) receptor mRNA, but not B(1) receptor mRNA. 3. Binding of fluorescently- labeled HOE-140 (HOE741) was localized to ganglion cells in whole-mount preparations. BK B(2) receptors were coexpressed with immunoreactivity for calbindin or nitric oxide synthase. 4. Exposure to BK suppressed the amplitude of both fast and slow excitatory postsynaptic potentials. Depolarizing responses evoked by application of serotonin or substance P and nicotinic responses to acetylcholine were not reduced by BK. This suggested that BK action on neurotransmission was presynaptic suppression of neurotransmitter release. Presence of HOE-140 in the bathing solution suppressed or abolished the presynaptic inhibitory action of BK. 5. The cyclooxygenase inhibitor, piroxicam, suppressed both the direct excitatory action of BK and its presynaptic inhibitory action. Application of prostaglandin E(2), D(2), F(2alpha) or I(2) mimicked the BK-evoked responses. 6. The results suggest that BK acts at B(2) BK receptors on myenteric neurones to stimulate the formation of prostaglandins. Once formed and released, the prostaglandins act to elevate the excitability of ganglion cells in the myenteric plexus and to suppress the synaptic release of neurotransmitters.     

The action of neurotensin on single myenteric neurones.

The action of neurotensin was studied on single myenteric neurones within ganglia of the myenteric plexus isolated from the guinea-pig ileum. Drugs were applied by adding them to the perfusing Krebs solution. Extracellular recording with glass suction electrodes indicated that neurotensin (100 pM-300 nM) caused a dose-dependent excitation of about 50% of myenteric neurones; the remaining neurones were unaffected. This effect persisted in calcium-free solutions. Intracellular recording showed that a similar proportion of Type 1 myenteric neurones were depolarized by neurotensin: this was associated with an increase in membrane resistance. Type 2 cells were either depolarized or hyperpolarized by neurotensin. The depolarization persisted in calcium-free solutions. The hyperpolarization disappeared in calcium-free solutions, suggesting either that the potential change itself is calcium-dependent or that it was due to release by neurotensin of a hyperpolarizing substance.     

Effects of cannabinoid receptor ligands on electrophysiological properties of myenteric neurones of the guinea-pig ileum

1. The effect of cannabinoid receptor agonists was studied in guinea-pig myenteric neurones in vitro by use of conventional intracellular recording techniques.
2. Exposure of myenteric neurones of the S-cell type to the cannabinoid receptor agonists WIN 55,212-2 (100 nM) and CP 55,940 (100 nM) reversibly and significantly depressed the amplitude of fast excitatory synaptic potentials (fast e.p.s.ps) by 46% and 37%, respectively.
3. The depressant effect of WIN 55,212-2 and CP 55,940 on fast e.p.s.p. amplitude (expressed as the area above the amplitude-time curve (mVs)) was significantly greater than that of the vehicle, Tween 80, which had no detectable effect.
4. The inhibitory effect of WIN 55,212-2 appeared to be concentration-dependent over the range 1–100 nM. WIN 55,212-3, its (−)-enantiomer (100 nM), was inactive.
5. The cannabinoid CB₁ receptor antagonist, SR141716A (1 μM), reversed the inhibitory effects of WIN 55,212-2 on fast e.p.s.ps in 38% of neurones tested (3/8) and acetylcholine (ACh)-induced depolarizations in 42% of neurones tested (5/12).
6. When tested on its own, SR141716A (1 μM) caused a 40–50% reduction in the amplitude of fast e.p.s.ps (n=9).
7. WIN 55,212-2 reversibly depressed the amplitude of the slow e.p.s.p. and, in 2 out of 7 neurones, this effect was reversed by SR141716A (1 μM).
8. It is concluded that cannabinoid-induced inhibition of fast cholinergic synaptic transmission occurred by reversible activation of both presynaptic and postsynaptic CB₁ receptors and that slow excitatory synaptic transmission can also be reversibly depressed by cannabinoids. Furthermore, it would seem that subpopulations of myenteric S-neurones and their synapsing cholinergic and non-cholinergic, non-adrenergic terminals are not endowed with cannabinoid receptors.

     

Effects of morphine on electrical activity of single myenteric neurons in cat small bowel

Quantitative parameters of the discharge patterns of burst-type units and single-spike units were increased, decreased or unchanged after application of morphine. The results were the same when morphine was added in the presence of naloxone. The results suggest that morphine had no consistent effect on the spike discharge of continuously active neurons in Auerbach's plexus of cat small intestine.     

猫贲门失弛缓症模型肌间神经丛的超微结构研究

目的应用苯基二甲基十四烷氯化铵(BAC)建立猫贲门失弛缓症模型,研究其下食管括约肌(LES)肌间神经丛的微观结构。方法10只猫随机分为两组,模型组胃镜下LES处环形注射BAC,对照组注射生理盐水,8周后检测食管动力学变化,然后应用电子显微镜观察LES肌间神经丛的形态学变化。结果模型组LES压力较对照组明显升高(P<0.01),两组食管收缩幅度无显著性差异;神经终末内有多种不同类型的突触小泡,两组神经末梢内清亮小泡的数量无显著差异(P>0.05),模型组突触小泡的数量较对照组明显减少(P<0.05)。结论肌间神经丛的突触小泡的减少可能是贲门失弛缓症的发病原因之一。     

An inhibitory action of tetracyclines on guinea-pig myenteric plexus

In plexus containing preparations of the longitudinal muscle of the guinea-pig ileum, an inhibitory action of tetracyclines on twitch-responses to electrical field stimulation was found. Tetracycline, chlortetracycline, minocycline and doxycycline, but not oxytetracycline (0.02 to 1.6 mmol/l) caused a concentration-dependent presynaptic inhibition of acetylcholine release. The inhibitory effect of the tetracyclines was also obtained after ganglion block by hexamethonium (30 mumol/l). The inhibitory effect of the tetracyclines was not antagonized by piperoxan (2 mumol/l) or yohimbine (1 mumol/l) and was partly reduced by the presence of naloxone (1 to 50 nmol/l). After exposing the preparation the peptidase inhibitors, i.e., to the combination of bestatin (10 mumol/l), captopril (10 mumol/l) and thiorphan (0.3 mumol/l), the inhibitory effect of tetracyclines was significantly increased. From these results it would appear that twitch-inhibition caused by tetracycline, chlortetracycline, minocycline and doxycycline is mainly mediated via the release of endogenous opioids from the myenteric plexus.     

Effects of crotoxin on autonomic neuromuscular transmission in the guinea-pig myenteric plexus and vas deferens

A. Anadön and M. R. Martinez-Larran?aga. Effects of crotoxin on autonomic neuromuscular transmission in the guinea-pig myenteric plexus and vas deferens. Toxicon23, 963–972, 1985. — The effects of crotoxin, the neurotoxic complex from the venom of the South American rattlesnake Crotalus durissus terrificus on mammalian autonomic neuromuscular transmission, have been investigated. In the longitudinal muscle of the guinea-pig ileum, crotoxin induced a dose-dependent contraction which was followed by relaxation, in spite of the continued presence of the toxin. The contractile response was inhibited by indomethacin, tetrodotoxin, verapamil or nifedipine, but was unaffected by atropine, propranolol, mepyraroine or methysergide. In addition, crotoxin caused a presynaptic inhibition of the electrically-evoked twitch of the longitudinal muscle of the guinea-pig ileum. In the guinea-pig vas deferens crotoxin also caused an inhibition of the response to field stimulation. The inhibition was reversible after washing and the preparation remained insensitive to further doses of the toxin. The inhibitory effects of crotoxin were not mediated by noradrenaline and were not due to a non-specific smooth muscle depression, because it was not associated with any reduction in motor responses to acetylcholine, ATP, bradykinin or substance P. Pre-incubation of the guinea-pig vas deferens with indomethacin blocked the inhibitory effects of the toxin. This suggests that the presynaptic activity of crotoxin in the vas deferens might be mediated by prostaglandins.     

Clonidine activates membrane potassium conductance in myenteric neurones

1 Intracellular recordings were made from neurones in the myenteric plexus of the ileum removed from guinea-pigs. The effects of clonidine and adrenaline on membrane potential and resistance were observed.

2 Clonidine (100 pM—30 nM) caused a concentration-dependent membrane hyperpolarization associated with a fall in neurone input resistance.

3 The amplitude of the clonidine hyperpolarization, but not the conductance increase, was greater in cells with lower resting potentials and smaller in more polarized neurones. In a given cell, membrane hyperpolarization decreased and membrane depolarization increased the clonidine effect.

4 Low potassium solutions enhanced and high potassium solutions reduced the hyperpolarizing action of clonidine but did not significantly change the conductance increase caused by clonidine.

5 The concentration-effect curve for clonidine was displaced to the left when the extracellular calcium concentration was reduced. Conversely, clonidine was almost ineffective in elevated calcium concentrations. This was true for both the hyperpolarization and the conductance increase.

6 It is suggested that clonidine activates a potassium conductance by causing an elevation in the free intracellular calcium concentration.

7 Clonidine reversibly depressed the amplitude of the nicotinic fast excitatory postsynaptic potential and the noncholinergic slow excitatory postsynaptic potential.

8 All the effects of clonidine were shared by adrenaline and the actions of both were reversed or prevented by phentolamine (100 nM—1 μM).

     

Dependence and cross-dependence in the guinea-pig myenteric plexus

Summary Chronic activation of opioid receptors results in the development of tolerance and dependence. Tolerance may be confined to a single receptor type and thus has been termed selective tolerance. The present investigation reveals that prolonged activation of an inhibitory acting receptor type not only results in dependence associated with this receptor but also brings about cross-dependence. Cross-dependence involves both opioid receptors as well as non-opioid receptors, e. g. adrenoceptors. The experimental design employed did not permit conclusions to be drawn about whether those receptors exhibiting cross-dependence also developed tolerance. Regardless of the receptors and their specific subsequent transduction systems, all the receptors which showed dependence and cross-dependence proved sensitive to pertussis toxin, suggesting a critical function of GTP-binding proteins for the development of not only opioid dependence but also for drug dependence in general. Since multiple transmitter receptors may converge on the same ion channel, the concept of convergent dependences may be linked to GTP-binding proteins. However, no conclusions can be drawn with regard to the precise biochemical mechanisms underlying dependence. Send offprint requests to R. Schulz at the above address     

Morphine augments calcium-dependent potassium conductance in guinea-pig myenteric neurones

Intracellular recordings were made from myenteric neurones removed from guinea-pig ileum and maintained in vitro. Action potentials were elicited by passing brief depolarizing currents through the recording electrode. In AH cells they were followed by afterhyperpolarizations resulting from an increase in potassium conductance (GK,Ca). Morphine (1 nM - 1 microM), applied by superfusion, increased the duration of the afterhyperpolarization (and the underlying GK,Ca) which followed from 1 to 30 action potentials. Morphine did not change the peak amplitude of the afterhyperpolarization. This action of morphine occurred both in cells which showed no change in resting membrane potential or resistance and in cells which were hyperpolarized. It was prevented by naloxone (10 nM - 1 microM). The possibility is proposed that morphine inhibits one of the mechanisms by which myenteric neurones control their free intracellular calcium concentration close to the plasma membrane.     

Cross-tolerance and convergent dependence between morphine and cannabimimetic agent WIN 55,212-2 in the guinea-pig ileum myenteric plexus.

The cross-tolerance and convergent dependence between morphine and the cannabimimetic agent R(+)-[2,3-dihydro-5-methyl-3[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-+ ++benzoxazin-yl]-(1-naphthalenyl) methanone mesylate (WIN 55,212-2) were assessed in vitro on guinea-pig ileum. To induce tolerance and dependence the myenteric plexus-longitudinal muscle was incubated at 37 degrees C for 5 h with a fixed concentration representing the IC50 for each compound. Myenteric plexus-longitudinal muscle exposed to WIN 55,212-2 (5 x 10(-8) M) was less sensitive to its inhibitory effect on electrically evoked contractions than naive myenteric plexus-longitudinal muscle. The exposure to cannabinoid induced a parallel rightward shift in the lower part of the concentration-response curve of WIN 55,212-2 and a marked reduction in the maximal inhibitory effect of the drug. Myenteric plexus-longitudinal muscle tolerant to WIN 55,212-2 was subsensitive to the inhibitory effect of morphine on the twitch response. The cross-tolerance between WIN 55,212-2 and morphine was bidirectional. In fact, after 5 h the morphine (10(-7) M)-incubated myenteric plexus-longitudinal muscle was less sensitive to the inhibitory effect of WIN 55,212-2. The tissue tolerant to morphine or WIN 55,212-2 was tested for the presence of physical dependence. Naloxone (10(-5) M) produced a typical withdrawal contracture in morphine-tolerant myenteric plexus-longitudinal muscle which could be reduced by a 15-min pretreatment with WIN 55,212-2 (5 X 10(-8) M). In contrast, SR141716 (10(-6) M) [N-(piperidino)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyr azole-carboxamide], a concentration which fully antagonized the inhibitory effect of WIN 55,212-2 (10(-7) M) in control preparations, did not produce significant contracture in WIN 55,212-2-tolerant myenteric plexus-longitudinal muscle. The mechanisms underlying the cross-tolerance and convergent dependence remain to be ascertained.     

Characterization of somatodendritic neuronal nicotinic receptors located on the myenteric plexus.

The effects of nicotine and dimethylphenylpiperazinium (DMPP) on resting and stimulation-evoked release of [3H]-acetylcholine ([3H]ACh) from cholinergic interneurons and neuro-effector neurons of the ileal longitudinal muscle and the responses of the smooth muscle to nicotinic agonists were studied. (-)-Nicotine was 15 times more effective than (+)-nicotine in releasing ACh. Since tetrodotoxin (1 microM) completely antagonized the effect of nicotinic agonists, the site of action of the nicotinic agonists studied was on the somatodendritic nicotinic receptors. The electrical field stimulation-evoked release was not affected by nicotinic agonists and antagonists, indicating that the axon terminals of cholinergic interneurons are not equipped with nicotinic receptors. This preparation proved to be useful to study the effect of nicotinic agonists on somatodendritic receptors, to determine the affinity constants of nicotinic antagonists, and to characterize these receptors. The rank order of antagonists was d-tubocurarine = mecamylamine greater than pipecuronium greater than pancuronium greater than vecuronium greater than hexamethonium; the apparent affinity constants (KD) were 1.15, 1.55, 3.06, 3.98, 13.59 and 32.88 microM, respectively. alpha-Bungarotoxin had no antagonistic activity at all. This finding indicates that nicotine and the endogenous ligand ACh act via a postsynaptic, somatodendritic nicotinic receptor that is pharmacologically similar to those located on the axon terminals of sympathetic neurons or in ganglions, but is dissimilar to those located at the postsynaptic site of the neuromuscular junction.     

Effect of ranitidine on ileal myenteric plexus preparation and on acetyl- and butyrylcholinesterase

Ranitidine at concentrations from 1 microM to 0.1 mM brought about a dose-dependent potentiation of the twitch responses elicited by electrical stimulation of the ileal myenteric preparation. At higher concentrations (0.3-3 mM) ranitidine also caused irregular slow contractions of the unstimulated ileal preparation which were potentiated by eserine and blocked by atropine and tetrodotoxin. In order to identify the mechanism of these apparently cholinomimetic actions, the effects of ranitidine on AChE and BuChE were studied. Ranitidine showed an instantaneous and promptly reversible inhibitory action at concentrations between 0.5 and 30 microM. Double reciprocal plots were prepared and equilibrium dissociation constants calculated. It appears that ranitidine exerts an inhibition of the "mixed" type on both AChE and BuChE, but the dissociation constants for BuChE were markedly higher than those for AChE. Since AChE inhibition occurs in the same concentration range potentiating the twitch responses on the ileal myenteric preparation, it may explain the cholinomimetic effect of ranitidine.