Cannabinoid CB1 receptor-mediated modulation of evoked dopamine release and of adenylyl cyclase activity in the human neocortex

Abstract

1. The present study investigated the binding characteristics of various ligands to cannabinoid CB(1) receptors in human neocortex and amygdala. In addition, the functionality of CB(1) receptors in the human neocortex was assessed by examining the effects of CB(1) receptor ligands on evoked [(3)H]-dopamine (DA) release in superfused brain slices and on synaptosomal cAMP accumulation. 2. Saturation-binding assays in human neocortical and amygdala synaptosomes using a radiolabelled cannabinoid receptor agonist ([(3)H]-CP55.940) revealed pK(d) values of 8.96 and 8.63, respectively. The numbers of binding sites (B(max)) were 3.99 and 2.67 pmol (mg protein)(-1), respectively. 3. Various cannabinoid receptor ligands inhibited [(3)H]-CP55.940 binding with rank order potencies corresponding to those of previous studies in animal tissues. 4. Electrically evoked [(3)H]-DA release from human neocortical slices was inhibited by CP55.940 (IC(50) 6.76 nm, I(max) 65%) and strongly enhanced by the cannabinoid receptor antagonist AM251. However, [(3)H]-DA release was not influenced in rat neocortex. In human tissue, the estimated endocannabinoid concentration in the biophase of the release-modulating CB(1) receptors was 1.07 nm, expressed in CP55.940 units. 5. K(+)-evoked [(3)H]-DA release in the presence of tetrodotoxin (TTX) was strongly inhibited by CP55.940 in humans, but not in rats. 6. In human tissue, CP55.940 inhibited forskolin-stimulated cAMP accumulation (IC(50) 20.89 nm, I(max) 35%). AM251 blocked this effect and per se increased forskolin-stimulated cAMP accumulation by approximately 20%. 7. In conclusion, cannabinoids modulate [(3)H]-DA release and adenylyl cyclase activity in the human neocortex. CB(1) receptors are located on dopaminergic nerve terminals and seem to be tonically activated by endocannabinoids.

Figure 1

Saturation characteristics of CP55.940 binding in human neocortical and amygdala synaptosomes. The specific activity of [³H]-CP55.940 (158 Ci mmol⁻¹) was diluted with unlabelled CP55.940 to obtain final assay concentrations from 0.032 to 10 nM. Synaptosomes (∼100 μg protein) were incubated for 60 min at 30°C. Nonspecific binding was determined by using 10 μM CP55.940. (a, c) show saturation curves and parameter estimates of specific CP55.940 binding evaluated by nonlinear regression analysis. (b, d) show the corresponding Scatchard analyses of specific CP55.940 binding. Data points of each curve are means±s.e.m. of three experiments (i.e. tissue of 4–5 different patients), each performed in quadruplicate.

Figure 2

Inhibition of specific [³H]-CP55.940 binding (20 pM) by CP55.940, AM251, Δ⁹-THC, WIN55212-2 (a, c) and anandamide±PMSF (50 μM; b, d) in human neocortical (upper panel) and amygdala (lower panel) synaptosomes. Synaptosomes (∼100 μg protein) were incubated for 60 min at 30°C with radiolabelled CP55.940 and various concentrations of drugs under study. Nonspecific binding was determined by using 10 μM CP55.940. The data points of each curve are means±s.e.m. of four experiments (i.e. tissue of 4–7 different patients), each performed in quadruplicate.

Figure 3

Effect of CP55.940 on electrically evoked [³H]-DA release from human neocortical slices. Following incubation with [³H]-DA, slices were superfused and stimulated electrically (S₁–S₃; 90 pulses, 40 mA, 3 Hz). (+)-Oxaprotiline and fluvoxamine (each 1 μM) were present in the incubation- and superfusion medium. CP55.940 was added in increasing concentrations 70 min before S₂ and S₃. The mean S_X/S₁ values (±s.e.m.) represent evoked tritium overflow, normalized to controls. The solid curve was obtained by using function (4), the dotted curve by using function (2). Data were obtained from a total of 12 patients.

Figure 4

Effect of AM251 on electrically evoked [³H]-DA release from human or rat neocortical slices. Following incubation with [³H]-DA, slices were superfused and stimulated electrically (S₁–S₃; 90 pulses, 40 mA, 3 Hz). (+)-Oxaprotiline and fluvoxamine (1 μM each) were present in the incubation and superfusion medium. AM251 was either given alone at increasing concentrations 70 min before S₂ and S₃ or, when tested in combination with 0.1 μM CP55.940, present from 70 min before S₂ until the end of superfusion (in this case, CP55.940 was given 70 min before S₃). Inset: Effect of 0.1 μM CP55.940 in the absence or presence of AM251 in the human neocortex (taken from data points presented in Figures 3, 4). Effects are given as mean normalized S_X/S₁ ratios±s.e.m., obtained from the neocortical tissue of four different patients or two different rats each resulting in 4–15 single data points. ^*P<0.05, ^**P<0.01, compared to the corresponding controls. ^•P<0.05, compared to the effect of 0.1 μM CP55.940 in the absence of 0.1 μM AM251.

Figure 5

Effect of CP55.940 on forskolin (1 μM)-induced cAMP accumulation in human neocortical synaptosomes. Drug effects are expressed as a fraction of forskolin-treated controls. Inset: Effect of 1 μM CP55.940 in the absence and presence of 1 μM AM251. Data represent means±s.e.m. of four experiments (i.e. tissue of four different patients) each performed in duplicate. ^**P<0.01, compared to the corresponding controls.

Figure 6

Effect of AM251 (0.1–10 μM) on forskolin-stimulated cAMP accumulation in human neocortical synaptosomes. Drug effects are expressed as a fraction of forskolin-treated controls. Data represent means±s.e.m. of four experiments (i.e. tissue of four different patients) each performed in duplicate. ^*P<0.05, compared to the corresponding controls.