Receptor-dependent formation of endogenous cannabinoids in cortical neurons

Abstract

We investigated the transduction mechanisms mediating formation of the endogenous cannabinoid (endocannabinoid) lipids, anandamide (arachidonylethanolamide) and 2-arachidonylglycerol, in primary cultures of rat cortical neurons. Unstimulated neurons contained 0.3±0.1 pmol of anandamide and 16.5±3.3 pmol of 2-arachidonylglycerol per mg of protein, as determined by gas chromatography/mass spectrometry. Ca²⁺ entry into the neurons via activated glutamate N-methyl-d-aspartate (NMDA) receptors increased 2-arachidonylglycerol levels approximately three times, but had no effect on anandamide levels. By contrast, anandamide formation was stimulated five times by simultaneous activation of NMDA and acetylcholine receptors. Alone, acetylcholine receptor activation had no effect on anandamide or 2-arachidonylglycerol levels. The formation of fatty acid ethanolamides that do not activate cannabinoid receptors, including palmitylethanolamide and oleylethanolamide, was stimulated by coactivation of NMDA and acetylcholine receptors. Pharmacological experiments suggest that the cholinergic contribution to anandamide formation was mediated by α7 nicotinic receptors (antagonized by methyllycaconitine), whereas the contribution to palmitylethanolamide and oleylethanolamide formation was mediated by muscarinic receptors (antagonized by atropine). These findings indicate that cortical neurons produce anandamide and 2-arachidonylglycerol in a receptor-dependent manner, and that brain neurons may generate different endocannabinoid lipids depending on their complement of neurotransmitter receptors.

Introduction

Cannabinoid receptors, the molecular target for the psychoactive constituent of Cannabis Δ⁹-tetrahydrocannabinol (Piomelli et al., 2000), are activated by a family of lipid molecules that primarily include anandamide (arachidonylethanolamide) Devane et al., 1992, Di Marzo et al., 1994 and 2-arachidonylglycerol Mechoulam et al., 1995, Sugiura et al., 1995, Stella et al., 1997. Both anandamide and 2-arachidonylglycerol are produced by brain neurons in an activity-dependent manner Di Marzo et al., 1994, Stella et al., 1997, Giuffrida et al., 1999, bind to and activate cannabinoid CB₁ receptors Felder et al., 1993, Vogel et al., 1993, Mechoulam et al., 1995, Sugiura et al., 1996, and undergo rapid biological inactivation Di Marzo et al., 1994, Beltramo et al., 1997, suggesting that they may act as endogenous modulatory substances (Piomelli et al., 2000).

Unlike classical neurotransmitters, the endogenous cannabinoids (endocannabinoids) may be produced upon demand by enzymatic cleavage of membrane lipid precursors and immediately extruded from neurons without an intermediate step of vesicle storage Piomelli et al., 2000, Schmid, 2000. This feature—unusual in a brain chemical transmitter, but reminiscent of other lipid-derived mediators—prompted the two questions addressed in the present study. The first is how formation of anandamide and 2-arachidonylglycerol is initiated: is it stimulated by action potentials invading the synaptic nerve endings or by receptor-activated mechanisms? Previous studies have shown that anandamide and 2-arachidonylglycerol can be produced during neural activity in vitro and in vivo, but they have not investigated the cellular mechanisms of this response Di Marzo et al., 1994, Stella et al., 1997, Giuffrida et al., 1999. The second question is whether, regardless of the mechanism involved, anandamide and 2-arachidonylglycerol can be generated independently of each other. An affirmative answer to this question would imply that these compounds may be released under different circumstances and, possibly, serve distinct functional roles in neuronal signaling. To begin to address these questions, we have investigated the mechanisms of anandamide and 2-arachidonylglycerol formation in primary cultures of rat brain cortical neurons. The results of our experiments show that anandamide and 2-arachidonylglycerol are produced in a receptor-dependent manner, and that segregated molecular mechanisms may underlie the formation of each of these compounds.