The specific activity of [3H]-CP55

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. separate individual data points (i.e. the number of assay tubes in binding and cAMP experiments or number of brain slices in superfusion experiments, respectively). To assess the significance of differences between two means, Student’s being the specific binding in pmol (mg protein)?1 (dependent variable) and lg[CP]=log10 of the concentration (M) of [3H]-CP55.940/CP55.940 (independent variable). The parameters to be estimated were served to decide whether a bimolecular reaction between the CP55.940 and its binding site occurred (Feuerstein & Limberger, 1999). Thus, an estimate of near unity with a sufficiently narrow CI95 allowed assumption of a bimolecular reaction and a single served to decide whether a bimolecular reaction between CB1 receptor and cannabinoids occurred. In this case, that is, being near unity, another evaluation could take place, which yielded an estimate of the dissociation constant between receptor and inhibitor, (i.e. Hill coefficients) were near unity (1.230.03, respectively, 1.200.04), a single binding site in both brain regions was assumed. Open in a separate window 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 around unity, suggesting a single binding site in both brain areas. In the presence of PMSF, the apparent affinity of anandamide was significantly enhanced, providing evidence for amidase activity in synaptosomal preparations of frozen human neocortical and amygdala tissue. Open in JDTic a separate window Figure 2 Inhibition of specific [3H]-CP55.940 binding (20 pM) by CP55.940, AM251, 9-THC, WIN55212-2 (a, c) and anandamidePMSF (50 (nM)(nM)was around unity (0.910.33). The inhibitory effect of CP55.940 was fully reversed in the presence of AM251 (Figure 5, inset), which, given bioassays. First, it was necessary to demonstrate the existence of cannabinoid CB1 receptor-binding sites in the human neocortex, as commonly demonstrated in rat neocortical preparations by using, for example, radioligand-binding assays (Devane was only 0.62, a single site of action can nevertheless be assumed, since (i) the JDTic CI95 of largely overlapped unity, (ii) may be influenced by an endocannabinoid tone (see below), and since (iii) of the saturation-binding curve was near unity. The inhibitory effect of CP55.940 was probably mediated by the CB1 receptor, as the antagonist AM251 fully reversed the observed effect and the functional IC50 value (6.76 nM) obtained from the concentrationCresponse curve was in the same order of magnitude of the would have any effect on DA release. AM251 concentration-dependently increased DA release in the human neocortex, whereas it had no effect in the rat. The increasing effect was MTC1 diminished by CP55.940, suggesting a CB1 receptor-mediated action. The enhanced release may be explained by the blockade of constitutively active CB1 receptors (Bouaboula CB1 receptors, since the antagonist AM251 completely abolished the inhibitory effect. The IC50 values of CP55.940 for inhibiting electrically evoked [3H]-DA release and for inhibiting forskolin-stimulated cAMP accumulation (6.76 and 20.9 nM) were rather similar. One may speculate that, among other mechanisms (e.g. blockade of N-type Ca2+ channels), inhibition of adenylyl cyclase participates in the CB1 receptor-mediated inhibition of DA release in the human neocortex. A similar degree of inhibition of the JDTic cAMP accumulation by cannabinoids was recently detected in neocortical membranes of post-mortem human brain. These results differ from the more pronounced inhibition in cells transfected with the human CB1 receptor (Felder led to a stimulatory response although the percentage effect was.