Examination of the effect of the cannabinoid receptor agonist, CP 55,940, on electrically evoked transmitter release from rat brain slices

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Abstract

In the present study we examined the effect of the cannabinoid receptor agonist, {[1a,2-(R)-5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-phenol; CP 55,940} on [14C]acetylcholine and [3H]norepinephrine release from hippocampal slices and on [14C]acetylcholine release from striatal slices. CP 55,940 potently inhibited electrically evoked [14C]acetylcholine release from hippocampal slices, with an EC50 of 0.02 μM and a maximal inhibition of 61% at 1 μM. The inhibition of acetylcholine release by CP 55,940 was partially antagonized (60%) by the cannabinoid receptor antagonist, {[N-piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride; SR 141716A}. Alone, SR 141716A significantly enhanced stimulated [14C]acetylcholine release. In contrast to the effects of CP 55,940 on [14C]acetylcholine release, electrically evoked [3H]norepinephrine release from hippocampal slices and [14C]acetylcholine release from striatal slices were both unaffected by this compound. Similarly, hippocampal [3H]norepinephrine release and striatal [14C]acetylcholine release were not affected by SR 141716A. In conclusion, the results of this study extend our previous data indicating that cannabinoid receptors modulate acetylcholine release in the hippocampus. The effects of cannabinoid receptor activation on [3H]acetylcholine release in the hippocampus does not appear to extend to [3H]norepinephrine release from this region or to acetylcholine release from the striatum. © 1997 Elsevier Science B.V. All rights reserved.

Introduction

Cannabinoid receptors are G-protein-coupled receptors that are linked to the inhibition of adenylate cyclase (Howlett et al., 1988). Two types of cannabinoid receptor, designated the cannabinoid CB1 and CB2 receptors, have been identified and cloned (Matsuda et al., 1990; Munro et al., 1993). The cannabinoid CB1 receptor is located both in the central nervous system (Herkenham et al., 1990) and in some peripheral tissues such as the testis (Pacheco et al., 1991), ileum (Pertwee et al., 1992) and uterus (Das et al., 1995). The cannabinoid CB2 receptor has been identified in macrophages and in the spleen (Munro et al., 1993).

The screening of brain extracts for binding activity at the cannabinoid receptor has identified an arachidonic acid derivative, termed anandamide, which binds with moderately high affinity to these receptors (Devane et al., 1992). This compound appears to be a partial agonist (Mackie et al., 1993), at cannabinoid CB1 receptors. Anandamide, along with other related compounds (Priller et al., 1995), has been proposed to be an endogenous transmitter for cannabinoid receptors.

A number of synthetic cannabinoid receptor agonists have been developed with an affinity for the cannabinoid receptors greater than that of Δ-9-tetrahydrocannabinol (Devane et al., 1988; Howlett et al., 1988). The most potent of these has been {[1a,2-(R)-5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxy-propyl)cyclohexyl]-phenol; CP 55,940} (Herkenham et al., 1990). In addition to the synthetic cannabinoid receptor agonists, an unrelated class of compounds that are agonists at the cannabinoid receptor, are aminoalkyindoles such as {R-(+)-(2,3-dihydro-5-methyl-3-[{4-morpholinyl}methyl]pyrol[1,2,3-de]-1,4-benzozoxazin-6-yl)(naphthalenyl)methanone monomethanesulfate; WIN 55212-2} (Compton et al., 1992). These compounds were based on the structure of pravadoline, which was first developed as a cyclooxygenase inhibitor for use as an antinociceptive agent, but was also found to have an action on cannabinoid receptors (Compton et al., 1992; D'Ambra et al., 1992). More recently, a synthetic cannabinoid receptor antagonist, {[N-piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride; SR 141716A}, has been developed (Rinaldi-Carmona et al., 1994, Rinaldi-Carmona et al., 1995). This compound is structurally distinct from both the synthetic cannabinoids and the aminoalkylindole cannabinoid compounds.

Despite their abundance in the central nervous system, the function of these receptors is not clear. Electrophysiological studies on cultured neurons have demonstrated that cannabinoid receptor activation inhibits calcium channels (Mackie and Hille, 1992) and opens potassium channels (Deadwyler et al., 1995; Henry and Chavkin, 1995). If cannabinoid receptors have this same action on calcium and potassium channels on the synaptic terminals then these receptors may function to inhibit neurotransmitter release. This is supported by recent studies reporting an inhibitory effect of cannabinoids on norepinephrine release in the mouse vas deferens (Ishac et al., 1996), acetylcholine release in the guinea-pig small intestine (Pertwee et al., 1996) and glutamate release in cultured hippocampal cells (Shen et al., 1996). We have recently reported that WIN 55212-2 produces a strong inhibition of electrically evoked [14C]acetylcholine release from hippocampal slices (Gifford and Ashby, 1996), an area with a high density of cannabinoid receptors (Herkenham et al., 1991a). In addition, SR 141716A alone produced a substantial enhancement in the evoked [14C]acetylcholine release from this region (Gifford and Ashby, 1996). This latter finding suggests either that cannabinoid receptors are constitutively active and produce a tonic inhibition of acetylcholine release or that SR 141716A is antagonizing the effects of an endogenous cannabinoid agonist that is released along with acetylcholine.

The objectives of the present study were firstly to determine whether our original observations on the effects of WIN 55212-2 on hippocampal [14C]acetylcholine release could be reproduced with the synthetic cannabinoid agonist, CP 55,940, and secondly to determine whether this compound also affects the release of other neurotransmitters. For the latter objective, we examined the effects of CP 55,940 and SR 141716A on [3H]norepinephrine release from hippocampal slices and on [14C]acetylcholine release from striatal slices. The striatum was chosen since, like the hippocampus, it also has a high density of cannabinoid receptors and functional brain slices can be readily prepared from this region.

Section snippets

Materials

CP 55,940 was obtained from Pfizer (Groton, CT, USA) and SR 141716A from Sanofi Recherche (Montpellier, France). [14C]acetylcholine (55 mCi/mmol) and [3H]norepinephrine (15 Ci/mmol) were purchased from Amersham (Amersham, UK) and Dupont NEN (Wilmington, DE, USA), respectively.

Superfusion procedure

Male Sprague-Dawley rats (200–350 g, Taconic, Germantown, NY, USA) were decapitated, their brains quickly removed and the hippocampus or striatum dissected out on an ice-cold aluminum block. Following dissection, 300 μm

Effect of CP 55,940 on hippocampal [14C]acetylcholine release

In the hippocampus, CP 55,940 produced a dose-dependent inhibition of electrically evoked [14C]acetylcholine release (Fig. 1). The maximum inhibition of [14C]acetylcholine release by CP 55,940 was 61% and the EC50 for CP 55,940 in producing half of its maximal effect was 22 nM. Basal [14C]acetylcholine release was unaffected by CP 55,940 (data not shown).

Effect of SR 141716A on hippocampal [14C]acetylcholine release

The inhibition of [14C]acetylcholine release by 0.1 μM CP 55,940 was dose dependently antagonized by SR 141716A (Fig. 2). However, SR 141716A

Effect of CP 55,940 on stimulation-evoked transmitter release.

The inhibition of electrically evoked release of [3H]acetylcholine by CP 55,940 supports our previous observations, obtained using the aminoalkylindole, WIN 55,212-2, suggesting that cannabinoid receptors have a role in controlling the release of this neurotransmitter in this region (Gifford and Ashby, 1996). Lesioning studies in animals and observations in humans on the effects of muscarinic antagonists have shown that the hippocampal cholinergic system is necessary for short-term memory (

Acknowledgements

The authors wish to thank Pfizer Inc. for providing CP 55,940 and Sanofi Recherche for providing SR 141716A and funding part of this work. Additional funding was also provided by MH 52155 to C.R.A. Jr.

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