An optimized approach to study endocannabinoid signaling: evidence against constitutive activity of rat brain adenosine A₁ and cannabinoid CB₁ receptors

Abstract

1 At nanomolar concentrations, SR141716 and AM251 act as specific and selective antagonists of the cannabinoid CB 1 receptor. In the micromolar range, these compounds were shown to inhibit basal G-protein activity, and this is often interpreted to implicate constitutive activity of the CB 1 receptors in native tissue. We show here, using [ 35 S]GTPgS binding techniques, that micromolar concentrations of SR141716 and AM251 inhibit basal G-protein activity in rat cerebellar membranes, but only in conditions where tonic adenosine A 1 receptor signaling is not eliminated. 2 Unlike lipophilic A 1 receptor antagonists (potency order DPCPXbN-0840 Ecirsimarin4caf-feine), adenosine deaminase (ADA) was not fully capable in eliminating basal A 1 receptor-dependent G-protein activity. Importantly, all antagonists reduced basal signal to the same extent (20%), and the response evoked by the inverse agonist DPCPX was not reversed by the neutral antagonist N-0840. These data indicate that rat brain A 1 receptors are not constitutively active, but that an ADA-resistant adenosine pool is responsible for tonic A 1 receptor activity in brain membranes. 3 SR141716 and AM251, at concentrations fully effective in reversing CB 1 -mediated responses (10 À6 M), did not reduce basal G-protein activity, indicating that CB 1 receptors are not constitutively active in these preparations. 4 At higher concentrations (1 -2.5 Â 10 À5 M), both antagonists reduced basal G-protein activity in control and ADA-treated membranes, but had no effect when A 1 receptor signaling was blocked with DPCPX. Moreover, the CB 1 antagonists right-shifted A 1 agonist dose -response curves without affecting maximal responses, suggesting competitive mode of antagonist action. The CB 1 antagonists did not affect muscarinic acetylcholine or GABA B receptor signaling. 5 When further optimizing G-protein activation assay for the labile endocannabinoid 2-arachidonoylglycerol (2-AG), we show, by using HPLC, that pretreatment of cerebellar membranes with methyl arachidonoyl fluorophosphonate (MAFP) fully prevented enzymatic degradation of 2-AG and concomitantly enhanced the potency of 2-AG. In contrast to previous claims, MAFP exhibited no antagonist activity at the CB 1 receptor. 6 The findings establish an optimized method with improved signal-to-noise ratio to assess endocannabinoid-dependent G-protein activity in brain membranes, under assay conditions where basal adenosinergic tone and enzymatic degradation of 2-AG are fully eliminated.

Presynaptic Monoacylglycerol Lipase Activity Determines Basal Endocannabinoid Tone and Terminates Retrograde Endocannabinoid Signaling in the Hippocampus

“…MAFP and ATFMK might depress IPSC/EPSC by acting directly on CB1 receptors. This possibility is, however, inconsistent with our finding that the effects of MAFP/ATFMK on basal IPSCs were primarily suppressed by THL that inhibits 2-AG synthesis and also with the biochemical study showing that MAFP has neither agonist nor antagonist activity at CB1 receptors (Savinainen et al, 2003). Thus, it is reasonable to conclude that the effects of MAFP and ATFMK presented here result mostly from the inhibition of MGL activity, although contribution of some unknown nonspecific effects of MAFP/ATFMK cannot be excluded completely.…”

“…Alternatively, if URB754 does not inhibit MGL, as demonstrated recently by Saario et al (2006), the results in Figure 1 are inconclusive and a possibility remains that MGL contributes to DSI termination. To test this possibility, we examined effects of the classical MGL inhibitors MAFP (Goparaju et al, 1999;Dinh et al, 2002Dinh et al, , 2004Savinainen et al, 2003;Saario et al, 2004;Walter et al, 2004) and ATFMK (Dinh et al, 2002;Walter et al, 2004) on DSI and 2AG-induced suppression of IPSCs.…”

Signal transduction of the CB1 cannabinoid receptor

“…Some other data suggest that the constitutive activity is not present in all CB 1 R expressing cells, or it can be inhibited by Ca 2C chelation (Katona et al 1999, Hoffman & Lupica 2000, Wilson & Nicoll 2001, Chevaleyre & Castillo 2003, Savinainen et al 2003, Breivogel et al 2004, Hentges et al 2005, Zhu & Lovinger 2005, Neu et al 2006 or by inhibition of DAGL (Turu et al 2007). There is also an observation that SR141716A, the CB 1 R-inverse agonist, can inhibit other receptors as well (Savinainen et al 2003, Lauckner et al 2008, and this can complicate the interpretation of its inverse agonist effects.…”

The pharmacology of cannabinoid receptors and their ligands: an overview

“…For example, at concentrations above those at which it is capable of blocking the CB 1 receptor, SR141716A behaves as an antagonist of the CB 2 receptor, the putative abnormal-cannabidiol receptor, the adenosine A 1 receptor, the TRPV1 receptor and possibly also the TRPV1-like receptor. 18,20 Finally, evidence is now emerging for the presence of an allosteric site on the CB 1 receptor. 29 1 antagonists instead of competitive CB 1 receptor antagonists for the clinic (e.g.…”