Amphetamine effects on acetylcholine release and mammalian neuromuscular transmission

The mechanisms by which (+)-amphetamine biphasically modifies neuromuscular transmission were studied in the rat phrenic nervediaphragm preparation. Low to moderate amphetamine concentrations (30–300 μM) enhanced twitch height and potentiated the nerve stimulated release of acetylcholine (ACh) by up to 4.8-fold from the phrenic nerve. Higher amphetamine concentrations depressed muscle twitch and ACh release. Using a cannulated diaphragm preparation, amphetamine enhanced the twitch response to nerve stimulation but markedly depressed the contractions elicited by a pulsed injection of ACh. Amphetamine-induced enhancement of ACh release was prevented by pretreatment of animals with α-methyl-p-tyrosine, suggesting that amphetamine may be acting indirectly by releasing catecholamines. These results support the hypothesis that amphetamine enhancement results from a presynaptic increase in ACh release and the blocking actions are mediated by a postsynaptic inhibitory effect.     

Pre- and postsynaptic adrenergic activation by norepinephrine reuptake inhibitors in the field-stimulated rat vas deferens

Desipramine (DMI), protriptyline, chlorpromazine, amitriptyline and cocaine, alone or in the presence of prazosin, produced a dose-related inhibition of contractions induced by field stimulation of the rat vas deferens. The inhibition of contractions was readily reversed by yohimbine. In contrast, when yohimbine was first added to the bath, all agents, except chlorpromazine, produced a dose-related enhancement of contractions which were readily reversed by prazosin. The potencies of these agents for induction of contractile inhibition, after prazosin, and contractile enhancement, after yohimbine, were similar. Both of the latter contractile responses of DMI were markedly attenuated or absent in tissues taken from rats pretreated with reserpine and alpha-methyl-para-tyrosine. The data indicate that, in the rat vas deferens, inhibition of norepinephrine reuptake results primarily in presynaptic (α₂) receptor activation. Postsynaptic (α₁) adrenergic activation by inhibition of norepinephrine reuptake can be demonstrated in this tissue only after presynaptic (α₂) receptor blockade. The possible implications of the present studies to the delayed clinical onset of action of tricyclic antidepressants is discussed.     

Modulation of transmitter release from the terminals of the locust wing stretch receptor neuron by muscarinic antagonists

The forewing stretch receptor (SR) neuron makes monosynaptic connections with wing depressor motoneruons; in this article the pharmacology of its output onto the first baslar motoneuron (BA1) has been investigated. The SR, like other insect afferents that have been studied so far, appears to be cholinergic; transmission was suppressed reversibly by the nicotinic antagonist gallamine (10^?4M) and irreversibly by α-bungarotoxin (10^?6 M). The choline reuptake blocker hemicholinium-3 (10^?4 M) also caused a reversible reduction in the amplitude of SR excitatory postsynaptic potentials (EPSPs) recorded in BA1. The receptor subtype nonselective muscarinic antagonists atropine (10^?4 M), scopolamine (10^?4 M), and quinuclidinyl benzilate (10^?5 M), unlike nicotinic antagonists, caused an augmentation in EPSP amplitude. This effect does not appear to be caused by an increase in sensitivity of the motoneuron to acetylcholine (ACh), since atropine produced a marked reduction rather than an increase in the amplitude of responses to ACh pressure applied to the soma of BA1. Scopolamine only caused a modest reduction in the amplitude of ACh somatic responses. The simplest explanation for these observations is that muscarinic antagonists bring about an increase in EPSP amplitude by blockade of presynaptic autoreceptors that normally down-regulate the release of ACh from SR terminals. The effects of muscarinic receptor subtype-selective antagonists indicate that presynaptic receptors in this preparation may have a pharmacological profile more similar to that of vertebrate M₂ receptors than to that of M₁ or M₂ subtypes. The functional significance of autoreceptors in this preparation are discussed. © 1995 John Wiley & Sons, Inc.     

Nicotinic Autoreceptors Mediating Enhancement of Acetylcholine Release Become Operative in Conditions of "Impaired" Cholinergic Presynaptic Function

Abstract: The existence in the mammalian CNS of release-inhibiting muscarinic autoreceptors is well established. In contrast, few reports have focused on nicotinic autoreceptors mediating enhancement of acetylcholine (ACh) release. Moreover, it is unclear under what conditions the function of one type of autoreceptor prevails over that of the other. Rat cerebrocortex slices, prelabeled with [³H]choline, were stimulated electrically at 3 or 0.1 Hz. The release of [³H]ACh evoked at both frequencies was inhibited by oxotremorine, a muscarinic receptor agonist, and stimulated by atropine, a muscarinic antagonist. Nicotine, ineffective at 3 Hz, enhanced [³H]ACh release at 0.1 Hz; mecamylamine, a nicotinic antagonist, had no effect at 3 Hz but inhibited [³H]ACh release at 0.1 Hz. The cholinesterase inhibitor neostigmine decreased [³H]ACh release at 3 Hz but not at 0.1 Hz; in the presence of atropine, neostigmine potentiated [³H]ACh release, an effect blocked by mecamylamine. In synaptosomes depolarized with 15 mM KCI, ACh inhibited [³H]ACh release; this inhibition was reversed to an enhancement when the external [Ca²⁺] was lowered. The same occurred when, at 1.2 mM Ca²⁺, external [K⁺] was decreased. Oxotremorine still inhibited [³H]ACh release at 0.1 mM Ca²⁺. When muscarinic receptors were inactivated with atropine, the K⁺ (15 mM)-evoked release of [³H]ACh (at 0.1 mM Ca²⁺) was potently enhanced by ACh acting at nicotinic receptors (EC₅₀? 0.6 µM). In conclusion, synaptic ACh concentration does not seem to determine whether muscarinic or nicotinic autoreceptors are activated. Although muscarinic autoreceptors prevail under normal conditions, nicotinic autoreceptors appear to become responsive to endogenous ACh and to exogenous nicotinic agents under conditions mimicking impairment of ACh release. Our data may explain in part the reported efficacy of cholinesterase inhibitors (and nicotinic agonists) in Alzheimer's disease.     

Effects of reserpine and adenosine agonist on α2-adrenergic receptor of rat vas deferens examined with [3H]yohimbine and [3H]clonidine

The changes of [³H]yohimbine and [³H]clonidine binding sites in rat vas deferens on treatments with adenosine receptor agonists (2-chloroadenosine, adenosine) or reserpine were examined. Treatment with adenosine agonist in vitro increased [³H]clonidine binding sites but had no influence on affinity and number of binding sites of α₂-antagonist, [³H]yohimbine. Amount of [³H]yohimbine binding sites was found to be higher than that of [³H]clonidine with or without the treatment. Inhibition curves of α₂-agonists, clonidine and norepinephrine, on [³H]yohimbine binding were less than unity though α₂-antagonist inhibited with about 1.0 of n_H. The treatment with adenosine agonist reduced the IC₅₀ value of agonists on the [³H]yohimbine binding but had no influence on the inhibitory effect of antagonist. These effect of adenosine agonists was completely blocked by theophylline. Accordingly it was considered that activation of adenosine receptor caused configurational change in α₂-adrenergic receptor from low affinity state for agonist to the high affinity state, though both states had same affinity for antagonist.On the other hand, treatment with reserpine in vivo increased the affinity of clonidine for α₂-adrenergic receptors and also increased the amount of the α₂-receptors.     

Nonadrenergic modulation by clonidine of the cosecretion of catecholamines and enkephalins in adrenal chromaffin cells

Cultured bovine chromaffin cells cosecrete catecholamines and enkephalins following cholinergic nicotinic stimulation. Initial reports on the inhibitory effect of clonidine on catecholamine secretion raised the possibility of a modulation of chromaffin cell function through a presynaptic adrenergic mechanism. The purpose of this work was to investigate the pharmacological characteristics of this inhibitory effect of clonidine on the cosecretion of catecholamines and enkephalins in 4-day-old cultured chromaffin cells. We observed that clonidine completely inhibits nicotine-stimulated secretion of both leucine-enkephalin and catecholamines with an IC50 of 34 microM. Treatment of chromaffin cells for 3 days with 100 nM reserpine leads to a 67% increase in nicotine-stimulated secretion of leucine-enkephalin without any effect on the IC50 of clonidine. In reserpine-treated chromaffin cells, norepinephrine (100 microM) inhibits only by 27% nicotine-stimulated secretion of leucine-enkephalin with an IC50 of 50 microM. Neither the alpha 2-adrenergic antagonist yohimbine nor the alpha 1-adrenergic antagonist prazosin could fully reverse the inhibitory effect of clonidine on leucine-enkephalin secretion at 10 nM. These results tend to rule out the role of alpha-adrenergic receptors in the mediation of clonidine inhibition of cosecretion in chromaffin cells.     

Modulation of transmitter release from the locust forewing stretch receptor neuron by GABAergic interneurons activated via muscarinic receptors

The role of muscarinic receptors in the down‐regulation of acetylcholine (ACh) release from the locust forewing stretch receptor neuron (fSR) terminals has been investigated. Electrical stimulation of the fSR evokes monosynaptic excitatory postsynaptic potentials (EPSPs) in the first basalar motoneuron (BA1), produced mainly by the activation of postsynaptic nicotinic cholinergic receptors. The general muscarinic antagonists scopolamine (10⁻⁶ M) and atropine (10⁻⁸ to 10⁻⁶ M) caused a reversible increase in the amplitude of electrically evoked EPSPs. However, scopolamine (10⁻⁶ M) caused a slight depression in the amplitude of responses to ACh pressure‐applied to the soma of BA1. These observations indicate that the EPSP amplitude enhancement is due to the blockade of muscarinic receptors on neurons presynaptic to BA1. The muscarinic receptors may be located on the fSR itself and act as autoreceptors, and/or they may be located on GABAergic interneurons which inhibit ACh release from the fSR. Electron microscopical immunocytochemistry has revealed that GABA‐immunoreactive neurons make presynaptic inputs to the fSR. The GABA antagonist picrotoxin (10⁻⁶ M) caused a reversible increase in the EPSP amplitude, which does not appear to be due to an increase in sensitivity of BA1 to ACh, as picrotoxin (10⁻⁶ M) slightly decreased ACh responses recorded from BA1. Application of scopolamine (10⁻⁶ M) to a preparation preincubated with picrotoxin did not cause the EPSP amplitude enhancement normally seen in control experiments; in fact, it caused a slight depression. This indicates that at least some of the presynaptic muscarinic receptors are located on GABAergic interneurons that modulate transmission at the fSR/BA1 synapse. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 420–431, 1999     

Cholinergic modulation of the synaptic transmission in the frog spinal cord

It was found during experiments on isolated frog spinal cord involving extracellular recording from the dorsal roots (sucrose bridging) and intracellular recording from motoneurons by microelectrodes that 10 mM of the M-cholinomimetic arecoline produces motoneuronal depolarization which is matched by depolarizing electronic ventral root potentials and a rise in motoneuronal input resistance. Arecoline changes synaptic transmission by increasing the amplitude of postsynaptic potentials during intracellular recording and that of motoneuronal reflex discharges in the ventral roots but reduces the duration of dorsal root potentials. In the presence of arecoline, L-glutamate-induced motoneuronal response increases. Facilitation of synaptic transmission produced by arecoline in the spinal cord is bound up with cholinergic M₂- activation, since it is suppressed by atropine but not by low concentrations of pirenzipine; it is also coupled with a reduction in adenylcyclase activity. When motoneuronal postsynaptic response has been suppressed, as in the case of surplus calcium or theophylline, arecoline produces an inhibitory effect on the amplitude of motoneuronal monosynaptic reflex discharges which is suppressed by pirenzipine at a concentration of 1×10^–7 M. This would indicate the presence at the primary afferent terminals of presynaptic cholinergic M₁ receptors which mediate its inhibition of impulses of transmitter release. This effect is independent of changes in cyclic nucleotide concentration.A. M. Gorkii Medical Institute, Donetsk. Translated from Neirofiziologiya, Vol. 19, No. 3, pp. 399–405, May–June, 1987.     

Cholinergic Receptors in the Human vas Deferens

Abstract

This study represents the first investigation demonstrating the contractile response to exogenous acetylcholine (ACh) in the isolated human vas deferens. Pharmacological characterization of cholinergic receptors was achieved using selective antagonists to define receptor subtypes. In the HVD the effect of exogenous ACh is revealed as a dose-dependent sudden increase in the basal tension of the vasa. The ACh receptors of the HVD were competitively antagonized by atropine (ATR) with a high pA₂ value (8.78). The main finding of this study is the presence of cholinergic receptors of the pharmacologically defined M₂-ACh subtype in the isolated HVD, according to the pA₂ values obtained with pirenzepine (PRZ) 7.39, AF-DX 116 (AF) 5.92 and 4-DAMP 5.65, M₁-ACh, _M2-ACh and M₃-ACh selective antagonists, respectively. Prazosin (PZ), a selective α₁-adrenergic antagonist, displayed a similar competitive antagonism for the contractile response evoked both by ACh (pA₂ = 8.69) and NE (pA₂ = 8.58) in the HVD. The antagonism exerted by PZ on the ACh-induced contractile response of the HVD, suggests that ACh probably acts at a presynaptic level stimulating the release of NE from an adrenergic neuron. According to these findings, the receptor involved in this action, located in the proximity of the nerve terminals, seems to be of the M₂-ACh subtype.     

Role of alpha 1 adrenoceptors in the turnover of phosphatidylinositol and of alpha 2 adrenoceptors in the regulation of cyclic AMP accumulation in hamster adipocytes

The incorporation of radioactive phosphate into phosphatidylinositol was stimulated by epinephrine in hamster fat cells. This action was inhibited by alpha-adrenergic antagonists in the potency order: Prazosin?phentolamine>yohimbine. Methoxamine, but not clonidine, was able to mimic the effect of epinephrine. These data indicate that the phosphatidylinositol effect in fat cells is due to activation of alpha₁ adrenoceptors. On the other hand, the accumulation of cyclic AMP due to epinephrine was potentiated by alpha-adrenergic antagonists in the potency order phentolamine>yohimbine ?prazosin, in hamster fat cells. Clonidine significantly decreased the accumulation of cyclic AMP due to isoproterenol or ACTH in hamster fat cells, suggesting that the alpha-adrenergic modulation of cyclic AMP levels in hamster fat cells is mediated by alpha₂ adrenoceptors. Radioligand binding studies with plasma membranes from hamster adipocytes demonstrated the presence of both alpha₁ and alpha₂ adrenoceptors but about 90% of the binding sites were alpha₂. These data support the hypothesis that alpha₂ effects of catecholamines are due to inhibition of adenylate cyclase while the increases in phosphatidylinositol turnover that seem to be involved in the mobilization of calcium are linked exclusively to alpha₁ adrenoceptor activation.     

Selective alpha2-adrenergic properties of dexmedetomidine over clonidine in the human forearm.

We tested the hypothesis that dexmedetomidine (Dex) has greater alpha(2)- vs. alpha(1) selectivity than clonidine and causes more alpha(2)-selective vasoconstriction in the human forearm. After local beta-adrenergic blockade with propranolol, forearm blood flow (plethysmography) responses to brachial artery administration of Dex, clonidine, and phenylephrine (alpha(1)-agonist) were determined in healthy young adults before and after alpha(2)-blockade with yohimbine (n = 10) or alpha(1)-blockade with prazosin (n = 9). Yohimbine had no effect on phenylephrine-mediated vasoconstriction but blunted Dex-mediated vasoconstriction (mean +/- SE: -41 +/- 5 vs. -11 +/- 2%; before vs. after yohimbine) more than clonidine-mediated vasoconstriction (-39 +/- 5 vs. -28 +/- 4%; before vs. after yohimbine) (P < 0.02). Prazosin blunted phenylephrine-mediated vasoconstriction (-39 +/- 4 vs. -8 +/- 2%; before vs. after prazosin) but had similar effects on both Dex- (-30 +/- 4 vs. -39 +/- 6%; before vs. after prazosin) and clonidine-mediated vasoconstriction (-29 +/- 3 vs. -41 +/- 7%; before vs. after prazosin) (P > 0.7). Both Dex and clonidine reduced deep forearm venous norepinephrine concentrations to a similar extent (-59 +/- 12 vs. -55 +/- 10 pg/ml; Dex vs. clonidine, P > 0.6); this effect was abolished by yohimbine and blunted by prazosin. These results suggest that Dex causes more alpha(2)-selective vasoconstriction in the forearm than clonidine. The similar vasoconstrictor responses to both drugs after prazosin might be explained by the presynaptic effects on norepinephrine release.     

Dopaminergic Regulation of Striatal Acetylcholine Release: The Critical Role of Acetylcholinesterase Inhibition

Abstract: This study examined the effects of different levels of acetylcholinesterase (AChE) inhibition on dopaminergic regulation of striatal acetylcholine (ACh) release as estimated by in vivo brain microdialysis. Systemic administration of d-amphetamine (2 or 10 mg/kg) increased the striatal output of ACh when the AChE inhibitor neostigmine (0.1 µM) was present in the perfusion fluid. In contrast, when the same experiments were conducted at 0.01 µM neostigmine, d-amphetamine failed to affect (2 mg/kg) or significantly decreased (10 mg/kg) striatal ACh output. The inhibitory action of the D₂ receptor agonist quinpirole (0.2 mg/kg) was significantly greater at 0.01 µM than at 0.1 µM neostigmine. Similarly, there was a nonsignificant trend for the D₂ antagonist raclopride (1 mg/kg) to stimulate ACh release to a greater extent at the low neostigmine concentration. In contrast, the stimulant effects of systemic administration of the D₁ agonist A-77636 (1.46 mg/kg) on striatal ACh release were the same at the two neostigmine concentrations. These results demonstrate that the concentration of an AChE inhibitor in the perfusion solution can quantitatively and even qualitatively influence the manner in which dopaminergic agents regulate ACh overflow in the striatum. On comparing the present results with earlier reports concerning the effects of d-amphetamine on tissue concentrations of ACh, it is tentatively concluded that a low neostigmine concentration is the more physiologically relevant condition. Under such conditions, at moderate doses d-amphetamine does not appear to alter striatal ACh release, with this likely being due to the opposing actions of D₁ and D₂ receptors. Nevertheless, until the endogenous interstitial concentrations of striatal ACh can be measured by other methods, the physiological relevance of ACh microdialysis studies in the striatum will remain uncertain.     

Development of clonidine-tolerance in the rat vas deferens: Cross tolerance to other presynaptic inhibitory agents

Possibility of the development of clonidine-tolerance in the peripheral nervous tissue was examined using vas deferens isolated from rats chronically treated with clonidine. Rats were treated with clonidine for 10 days by adding the drug to drinking water (10 μg/ml). For the control rats, drug-free tap water was provided. Electrically evoked twitch response of vas deferens was suppressed by adenosine, β-endorphine and α₂-adrenergic agonists, such as clonidine and B-HT 933, both in control and clonidine-treated groups. Vas deferens isolated from clonidine-treated rats showed significantly lower responsiveness to the inhibitory effects of clonidine and B-HT 933 compared to those from control rats. Vas deferens from clonidine-treated rats also was less responsive to adenosine and β-endorphin, both of which interact with presynaptic inhibitory receptors other than α₂-adrenergic and muscarinic cholinergic stimulation responsiveness of the postsynaptic smooth muscle to both α-adrenergic and muscarinic cholinergic stimulation did not change after 10 days of treatment with clonidine. These results suggest that clonidinetolerance can be induced in the peripheral nervous system by chronic treatment of this drug and that the tolerance is not specific to α₂-adrenergic agonists. Some common pathway in the inhibitory mechanisms of various agents or possible interactions between the different types of presynaptic inhibitory receptors may be involved in this phenomenon.     

Nicotinic acetylcholine receptors on a cholinergic nerve terminal in the cockroach,Periplaneta americana

Summary Intracellular microelectrode recording and ionophoretic application of carbamylcholine (CCh) were used to compare the cholinergic sensitivity of postsynaptic dendrites of an identified neurone with that of an identified presynaptic cholinergic axon.The axon of the lateral filiform hair sensory neurone (LFHSN) in the first-instar cockroachPeriplaneta americana was found to be as sensitive to CCh as the dendritic regions of giant interneurone 3 (GI 3). The CCh response of both neurones was unaffected by replacing Ca²⁺ with Mg²⁺, confirming that the ACh receptors are present on the neurones under test. The CCh response of both neurones was mimicked by ionophoretic application of nicotine. The responses were blocked by 10^–5 M mecamylamine and 10^–6 M d-tubocurarine and were not affected by muscarinic antagonists, suggesting that the ACh receptors present on GI 3 and LFHSN are predominantly nicotinic.The muscarinic agonist oxotremorine and the antagonists atropine and quinuclidinyl benzilate had no modulatory effect on LFHSN-GI 3 synaptic transmission.The latency of the LFHSN response to CCh was consistent with the hypothesis that ACh receptors are situated on the main axon/terminal within the neuropil of the ganglion. It has previously been shown that this region of the axon does not form output synapses (Blagburn et al. 1985a). This indirect evidence indicates that presynaptic or extrasynaptic ACh receptors are present in the membrane of a cholinergic axon.LFHSN was depolarized by synaptically-released ACh after normal or evoked spike bursts, suggesting that the nicotinic ACh receptors act as autoreceptors. However, it was not possible to obtain direct evidence to support the hypothesis that these receptors modulate ACh release.Abbreviations CCh carbamylcholine - GI giant interneurone - FHSN filiform hair sensory neurone - LFHSN lateral filiform hair sensory neurone - R _in input resistance - V depolarization - V _m resting potential     

Pharmacological identification of acetylcholine receptor subtypes in echinoderm smooth muscle (Sclerodactyla briareus)

Contractions of an echinoderm (sp. Sclerodactyla briareus) smooth muscle, the longitudinal muscle of the body wall (LMBW), were evoked by acetylcholine (ACh) and agonists: epibatidine, muscarine and nicotine (in order of force generation: ACh>muscarine=epibatidine>nicotine). ACh-induced contractions were blocked by atropine by 50%, and methoctramine, by 30%. ACh responses were also blocked by 25% by methyllycaconitine (MLA) but not by d-tubocurarine (dTC). Muscarine initiated large contractions that were completely blocked by atropine. To elucidate possible muscarinic ACh receptor (mAChR) subtypes, muscarinic agonists (oxotremorine, pilocarpine) and antagonists (methoctramine, pirenzepine) were tested. Oxotremorine, pilocarpine, and pirenzepine each enhanced resting tonus and potentiated ACh-induced contractions (order of potency: pilocarpine>oxotremorine=pirenzepine). Muscarine, oxotremorine or pirenzepine generated phasic, rhythmic contractions. Nicotine-induced contractions were almost completely blocked by dTC but were not altered by atropine. Large contractions evoked by epibatidine were potentiated by dTC whereas atropine had no effect on them. MLA blocked spontaneous rhythmicity. Cholinesterase inhibitors, neostigmine or physostigmine, caused marked potentiation of ACh-induced contractions and initiated rhythmic slow wave contractions in previously quiescent muscles. The present pharmacological evidence points to the co-existence of excitatory nicotinic ACh receptor (nAChRs) and mAChRs where nAChRs possibly modulate tone, and the mAChRs initiate and enhance rhythmicity.     

Effect of clonidine on neuromuscular transmission and the nicotinic acetylcholine receptor

The effects of clonidine on neuromuscular transmission were investigated in the mouse phrenic nerve-diaphragms and chicken biventer cervicis. Clonidine inhibited the indirect twitch response dose-dependently and reversibly without an effect on the direct response of the muscles to electrical stimulation and KCl. This effect was antagonized effectively by diaminopyridine but not by yohimbine, phentolamine or physostigmine. The quantal content was not affected although the amplitudes of end-plate potential (epp) and spontaneous miniature epp (mepp) were markedly depressed. Clonidine also decreased the slope of the ACh dose-response curve and maximal response in denervated mouse diaphragms as well as the carbachol response in the chinck muscle. In the latter, ACh response was not depressed by clonidine probably because of its inherent anticholinesterase activity. Clonidine facilitated the fading of ACh-contracture either in mouse or chick muscle. It is concluded that clonidine impairs the neuromuscular transmission by a noncompetitive blockade of ACh receptors, most likely affecting the ACh channel but not the recognition site of the ACh receptor. Its inhibitory effect is not mediated by alpha 2-adrenoceptor, suggesting that there is no alpha 2-adrenoceptor on the motor nerve terminal to modulate the transmitter release.     

Dysfunctional Presynaptic M2 Receptors in the Presence of Chronically High Acetylcholine Levels: Data from the PRiMA Knockout Mouse

The muscarinic M₂ receptor (M2R) acts as a negative feedback regulator in central cholinergic systems. Activation of the M₂ receptor limits acetylcholine (ACh) release, especially when ACh levels are increased because acetylcholinesterase (AChE) activity is acutely inhibited. Chronically high ACh levels in the extracellular space, however, were reported to down-regulate M2R to various degrees. In the present study, we used the PRiMA knockout mouse which develops severely reduced AChE activity postnatally to investigate ACh release, and we used microdialysis to investigate whether the function of M2R to reduce ACh release in vivo was impaired in adult PRiMA knockout mice. We first show that striatal and hippocampal ACh levels, while strongly increased, still respond to AChE inhibitors. Infusion or injection of oxotremorine, a muscarinic M₂ agonist, reduced ACh levels in wild-type mice but did not significantly affect ACh levels in PRiMA knockout mice or in wild-type mice in which ACh levels were artificially increased by infusion of neostigmine. Scopolamine, a muscarinic antagonist, increased ACh levels in wild-type mice receiving neostigmine, but not in wild-type mice or in PRiMA knockout mice. These results demonstrate that M2R are dysfunctional and do not affect ACh levels in PRiMA knockout mice, likely because of down-regulation and/or loss of receptor-effector coupling. Remarkably, this loss of function does not affect cognitive functions in PRiMA knockout mice. Our results are discussed in the context of AChE inhibitor therapy as used in dementia.     

Presynaptic alpha 2 -adrenergic stimulation leads to growth hormone release in the dog

In previous studies we have shown that the alpha 2 -adrenergic receptor agonist clonidine (CLON) releases growth hormone (GH) in conscious dogs, an effect abolished by the selective alpha 2-receptor antagonist yohimbine (YOH) and by reserpine, but not by the alpha 1-receptor antagonist prazosin (1). In the present work intravenous (iv) administration of CLON in conscious dogs evoked a dose-related rise in plasma GH at doses of 2-8 /micrograms/Kg, but not at 16 and 32 /micrograms/Kg. Acute pretreatment with the selective inhibitor of norepinephrine (NE) synthesis, DU-18288, or with a potent antagonist of presynaptic alpha 2-receptors, mianserin abolished the GH rise induced by CLON (4 /micrograms/Kg iv). In contrast, a 10-day-pretreatment with YOH greatly enhanced the GH-releasing effect of CLON (2 /micrograms/Kg iv). In all these data indicate that in the dog: 1) CLON induces GH release via activation of alpha 2-adrenergic receptors; 2) these receptors are likely located on presynaptic sites [experiments with reserpine (1), DU-18288, mianserin, dose-response curve with CLON 2-32/micrograms/kg iv]; 3) the adrenergic receptors involved in GH release exhibit supersensitivity upon (YOH-induced) chronic pharmacologic denervation. In view of the inhibitory action of presynaptic alpha 2-adrenergic receptors (autoreceptors) on NE function, it may be envisioned that in the dog noradrenergic activation is inhibitory and not stimulatory to GH release.     

Acetylcholine potentiation of field stimulated rat vas deferens: A pre-synaptic muscarinic mechanism?

Acetylcholine (ACh) was found to markedly enhance the nerve stimulation induced twitch response of isolated, field-stimulated rat vas deferens (RVD). The ED₂₀₀ (concentration which enhances the twitch response to 200% of control) for this potentiation was 6 × 10^?6M with the maximum twitch response being increased by more than 3 fold (325 ± 30%). Carbachol (ED₂₀₀ = 8.5 × 10^?7M) showed identical results. With each drug the potentiation was competitively antagonized by atropine (10^?7?10^?5M). Physostigmine 10^?8?10^?6M) both enhanced the basal twitch response (215 ± 8% of control at 10^?5M) and the sensitivity of the RVD to ACh (ED₂₀₀ = 3.3 × 10^?7M) but not to carbachol. Atropine, on the other hand reduced the basal twitch response by 18 ± 3% at 10^?5M. Hemicholinium (10^?4M) also reduced the basal twitch responses by 23 ± 5%. ACh (10^?7M?10^?5M) did not modify the responses of unstimulated RVD to norepinephrine or KCl suggesting a pre-synaptic site of action. Taken together these results are compatible with the presence of a pre-junctional, excitatory muscarinic mechanism in the field stimulated RVD. That this cholinergic system may be of physiological significance is supported by the observations that atropine and hemicholinium depress while physostigmine enhances the twitch response in the absence of exogenous ACh.     

Pre- and postsynaptic mechanisms in the clonidine- and oxymetazoline-induced inhibition of gastric motility in the rat

The inhibitory effect of clonidine (non-selective alpha2-adrenoceptor agonist) and oxymetazoline (alpha2A-adrenoceptor selective agonist) was compared on basal and stimulated gastric motor activity (gastric tone and contractions) using the balloon method in the rat. It was shown that oxymetazoline (0.2-1.7 micromol/kg, i.v.) decreased the basal motility, while clonidine (1.9-3.8 micromol/kg, i.v.) failed to affect it. When motility was stimulated centrally by insulin (5 IU/rat, i.v.), both clonidine (1.9-3.8 micromol/kg, i.v.) and oxymetazoline (0.1-3.4 micromol/kg, i.v.) inhibited the gastric motor activity. However, while the effect of clonidine was antagonized by the non-selective alpha2-adrenoceptor antagonist yohimbine (5 micromol/kg, i.v.) and the alpha2A-adrenoceptor selective antagonist BRL 44408 (3 micromol/kg, i.v.), the effect of oxymetazoline was only partially affected. Prazosin (alpha1- and alpha2B-adrenoceptor antagonist, 0.07-0.28 micromol/kg, i.v.) also failed to reverse the effect of oxymetazoline. Furthermore, when gastric motility was stimulated peripherally by activation of postsynaptic cholinergic muscarinic receptors by the combination of carbachol (0.14 micromol/kg, i.v.) and hexamethonium (37 micromol/kg, i.v.), clonidine (3.8 micromol/kg, i.v.) failed to affect the increased motor activity, however, oxymetazoline (0.8-3.4 micromol/kg, i.v.) exerted a pronounced inhibition. These results suggest that different mechanisms may be involved in the inhibitory effect of clonidine and oxymetazoline; while clonidine reduces the gastric motility by activation of presynaptic alpha2-adrenoceptors, postsynaptic component in the effect of oxymetazoline has also been raised.