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

Abstract

At nanomolar concentrations, SR141716 and AM251 act as specific and selective antagonists of the cannabinoid CB1 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 CB1 receptors in native tissue. We show here, using [35S]GTPgammaS 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 A1 receptor signaling is not eliminated. Unlike lipophilic A1 receptor antagonists (potency order DPCPX>>N-0840 approximately cirsimarin>caffeine), adenosine deaminase (ADA) was not fully capable in eliminating basal A1 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 A1 receptors are not constitutively active, but that an ADA-resistant adenosine pool is responsible for tonic A1 receptor activity in brain membranes. SR141716 and AM251, at concentrations fully effective in reversing CB1-mediated responses (10-6 m), did not reduce basal G-protein activity, indicating that CB1 receptors are not constitutively active in these preparations.4 At higher concentrations (1-2.5 x 10-5 m), both antagonists reduced basal G-protein activity in control and ADA-treated membranes, but had no effect when A1 receptor signaling was blocked with DPCPX. Moreover, the CB1 antagonists right-shifted A1 agonist dose-response curves without affecting maximal responses, suggesting competitive mode of antagonist action. The CB1 antagonists did not affect muscarinic acetylcholine or GABAB receptor signaling. 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 CB1 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.

Figure 1

Low micromolar concentrations of the CB₁ receptor antagonists SR141716 and AM251 inhibit basal G-protein activity in untreated and ADA-treated rat cerebellar membranes, but have no effect in the presence of the adenosine A₁ receptor selective antagonist DPCPX. Following a 30-min preincubation in the absence (control and DPCPX conditions) or presence of ADA (0.5 U ml⁻¹), [³⁵S]GTPγS-binding assay was conducted, as described in methods, in control conditions or in the presence of ADA (0.5 U ml⁻¹) or DPCPX (10⁻⁶ M). Control incubations contained the vehicle for DPCPX (0.5% DMSO vol vol⁻¹). The vehicle for ADA (0.06% glycerol) did not affect basal binding (data not shown). The data represent the mean of [³⁵S]GTPγS binding over basal±s.e.m. from three independent experiments performed in duplicate. An asterisk (^*) denotes a statistically significant decrease (P<0.05).

Figure 2

At micromolar concentrations, the CB₁ receptor antagonists inhibit adenosine A₁ receptor signaling. Rat cerebellar membranes were preincubated for 30 min in the presence of ADA (0.5 U ml⁻¹), and then incubated with increasing concentrations of the adenosine receptor agonist 2-chloroadenosine (2ClAdo) in the absence (control) or presence of the CB₁ antagonists SR141716 or AM251 (both at 10⁻⁵ M), as indicated. Vehicle for all conditions was 0.5% DMSO (vol vol⁻¹). The data represent the mean±s.e.m. from three independent experiments performed in duplicate. When not visible, error bars fell within the size of the symbol.

Figure 3

Micromolar concentrations of the CB₁ receptor antagonists do not affect mACh- or GABA_B receptor-dependent G-protein activity. Rat cerebellar membranes were incubated with the muscarinic agonist CCh or the GABA_B agonist baclofen at concentrations, producing near half-maximal and maximal stimulation (10⁻⁶ M and 10⁻⁵ M, respectively). The CB₁ antagonists were present at 10⁻⁵ M concentration and agonist responses were determined in the presence of 1 μM DPCPX, to block basal A₁ receptor-mediated G-protein activity. The data represent the mean±s.e.m. from three independent experiments performed in duplicate.

Figure 4

Lipophilic adenosine receptor antagonists decrease basal G-protein activity in rat cerebellar membranes to the same extent and with the potency expected at A₁ receptors (a). Membranes were incubated in the presence of ADA (0.5 U ml⁻¹) and the indicated concentrations of the antagonists, as described in Methods (b). Inhibitory responses to the inverse A₁ receptor agonist DPCPX (10⁻⁶ M) cannot be reversed by the neutral A₁ antagonist N-0840 (5 × 10⁻⁵ M) or other A₁ antagonists, cirsimarin (5 × 10⁻⁵ M) and caffeine (5 × 10⁻⁴ M). The vehicle for all conditions was 0.5% DMSO (vol vol⁻¹). The data represent the mean±s.e.m. from at least three independent experiments performed in duplicate.

Figure 5

Pretreatment of rat cerebellar membranes with MAFP concomitantly potentiates 2-AG-stimulated G-protein activity (a) and prevents enzymatic degradation of 2-AG to AA more efficiently than PMSF (b). Membranes were pretreated with MAFP (10⁻⁵ M), PMSF (10⁻³ M) or the vehicle (DMSO) as a control for 30 min at +25°C in the presence of 0.5% BSA. In (a), membranes were used for [³⁵S]GTPγS-binding assay to determine G-protein activation in response to 2-AG concentrations, producing near half-maximal (10⁻⁶ M) or maximal stimulation (10⁻⁴ M). In (b), pretreated membranes were used for HPLC to assess enzymatic hydrolysis of 2-AG (5 × 10⁻⁵ M) under incubation conditions closely mimicking G-protein activation assay. By HPLC analysis, initial purity of 2-AG was 98% with the rest of the material (2%) representing 1(3)-AG. For (a), the data represent the mean±s.e.m. of [³⁵S]GTPγS binding from basal and, for (b), the mean±s.e.m. of relative (%) peak areas, each from at least three independent experiments performed in duplicate. n.d.: not detectable. An asterisk (^*) denotes the statistically significant (P<0.05) difference from the respective control; # indicates statistically the significant (P<0.05) difference between MAFP and PMSF treatment.

Figure 6

MAFP has no antagonist activity towards cannabinoid CB₁ receptor-dependent G-protein activity in rat cerebellar membranes. Membranes were pretreated with MAFP (10⁻⁵ M) or solvent (DMSO) as control for 30 min at +25°C in the presence of 0.5% BSA. The cannabinoid agonists were tested near to their EC₅₀ and E_max concentrations to reveal possible antagonism. The data represent the mean±s.e.m. from at least three independent experiments performed in duplicate.

Figure 7

Dose–response curves to various cannabinoid agonists in optimized assay conditions where tonic adenosine A₁ receptor activity and enzymatic degradation of 2-AG is fully eliminated. Rat cerebellar membranes were pretreated with 10⁻⁵ M MAFP in the presence of 0.5% BSA, as described in Methods. [³⁵S]GTPγS-binding assay was conducted for 90 min at 25°C in the presence of 10⁻⁶ M DPCPX. The data represent the mean±s.e.m. from three independent experiments performed in duplicate. When not visible, error bars fell within the size of the symbol. AEA, arachidonoyl ethanolamide; 2-AG, 2-arachidonoylglycerol; 2-AGE, 2-arachidonoylglyceryl ether; CP55,940, (−)-3-[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]-4-[3-hydroxypropyl]cyclohexan-1-ol.