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3-Azidophenyl- and 3-isothiocyanatophenyl-and 2-(5'-azidopentyl)- and 2-(5'-isothiocyanatopentyl)pyrazoles were synthesized to determine whether these compounds could behave as covalently binding ligands for the CB1 cannabinoid receptor in rat brain membranes. Heterologous displacement of [3H]CP55940 indicated that the apparent affinity of these compounds for the CB1 receptor was similar to that of the parent compound, SR141716A, with the exception of the 3-isothiocyanato derivatives, which showed a 10-fold loss of affinity. The 3-azidophenyl and 3-isothiocyanatophenyl compounds behaved as antagonists against the cannabinoid agonist desacetyllevonantradol in activation of G proteins [guanosine 5'-O-(y-[35S]thio)triphosphate ([35S]GTPgammaS) binding] and regulation of adenylyl cyclase. The 2-(5'-azidopentyl)- and 2-(5'-isothiocyanatopentyl)pyrazoles were poor antagonists for [35S]GTPgammaS binding, and both compounds failed to antagonize the cannabinoid regulation of adenylyl cyclase. After incubation with the isothiocyanato analogues or UV irradiation of the azido analogues, the 3-substituted aryl pyrazoles formed covalent bonds with the CB1 receptor as evidenced by the loss of specific binding of [3H]CP55940. In the case of the isothiocyanato analogues, the log concentration-response curve for cannabinoid-stimulated [35S]GTPgammaS binding was shifted to the right, indicating that loss of receptors compromised signal transduction capability. These irreversibly binding antagonists might be useful tools for the investigation of tolerance and receptor down-regulation in both in vitro and in vivo studies.

Under reducing conditions of SDS-polyacrylamide gel electrophoresis, the CB(1) receptor exists in its monomeric form as well as in an SDS-resistant high molecular weight form that appears to be devoid of G proteins. The CB(1) cannabinoid receptor was immunoprecipitated from 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate-solubilized rat brain membranes using an antibody against the CB(1) receptor N terminus. The CB(1) receptor was coimmunoprecipitated with its associated G proteins, specifically those of the Galpha(i/o) family, but not Galpha(s), Galpha(q), or Galpha(z). The CB(1) receptor-Galpha(i/o) complex existed in the absence of exogenous agonists, and the cannabinoid receptor agonist desacetyllevonantradol failed to alter the stoichiometry of the receptor-Galpha(i/o) interaction. Guanosine-5'-O-(3-thio)triphosphate could disrupt the interaction. A peptide derived from the CB(1) receptor juxtamembrane C-terminal domain, peptide CB(1)401-417, autonomously activates G(i/o) proteins. Peptide CB(1)401-417 competitively disrupted the CB(1) receptor association with Galpha(o) and Galpha(i3) but not Galpha(i1) or Galpha(i2). This G protein specificity was also observed in detergent extracts from membranes of the frontal cortex, striatum, and cerebellum. Alternative peptides, including peptides from the CB(1) receptor third intracellular loop and the G protein activating peptide mastoparan-7, failed to promote uncoupling from Galpha(o). A CB(2) receptor juxtamembrane C-terminal peptide failed to disrupt the CB(1) receptor-Galpha(o) complex. These studies illustrate that the CB(1) receptor can exist as an SDS-resistant multimer. In 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate detergent, the CB(1) receptor exists in a complex with G proteins of the G(i/o) family in the absence of exogenous agonists. Furthermore, this study provides the first description of domain specificity for interaction with a selective set of G proteins.

Multiple affinity states are revealed by agonist competition for radioantagonist [3H]SR141716A binding to rat brain CB1 cannabinoid receptors. Desacetyllevonantradol (DALN), a tricyclic cannabinoid, and WIN55212-2, an aminoalkylindole, both bound in two discrete affinity states (30% high affinity), but the ratios of the IC50 revealed distinct differences. Other affinity-state differences between the agonists were: Na+ reduced the affinity for the membrane-bound receptor by 10-fold for DALN but minimally for WIN55212-2; a nonhydrolysable GTP analogue decreased the fraction of high-affinity WIN55212-2 binding but not that of DALN unless Na+ was also present. Detergent solubilisation increased the fraction of high-affinity binding for both agonists but eliminated any effect of Na+ on the agonist affinities. In detergent solution, the GTP analogue reduced the WIN55212-2 high-affinity fraction but not that of DALN, even though the IC50 values increased for both DALN and WIN55212-2. The differential modulation of CB1 receptor-G-protein coupling by Na+ and guanine nucleotides is dependent upon the cannabimimetic agonist bound.

Two insulin mediators, inositol phosphoglycans, were isolated from bovine liver by methods previously developed for rat liver, i.e. chromatography on an AG 1 x 8 ion exchange column and selective elution with HCl at pH 2.0 and 1.3. The pH 2.0 mediator containing D-chiroinositol stimulated pyruvate dehydrogenase phosphatase, whereas the pH 1.3 mediator containing myo-inositol inhibited cAMP-dependent protein kinase. Each mediator was further purified by thin layer and Bio-Gel P4 column chromatography and injected ip into normal fed rats together with [U-14C]glucose. After 2.5 h, diaphragms were removed, and glycogen isolated. Insulin mediators, like insulin, stimulated [U-14C]glucose incorporation into glycogen by 150-160% in a dose-dependent manner in the nanomolar range. Mediators injected iv in the nanomolar range into low dose streptozotocin-diabetic rats decreased plasma glucose 30-45% in 30-60 min, with a return to basal concentrations after 150-180 min. These in vivo insulin-like effects of mediator were observed without changes in serum insulin concentrations. The pH 2.0 mediator was 50-100 times more active (per nmol organic phosphate) than the pH 1.3 mediator in the ip diaphragm glycogenesis assay. Mediator effects on diaphragm were completely blocked by preincubation with an immunopurified inositol phosphoglycan antibody. Both mediators were equally active iv in lowering plasma glucose (per nmol inositol) at concentrations comparable to those of insulin.

The present investigation was undertaken to characterize cannabinoid receptor binding in the absence of the membrane environment, inasmuch as cannabinoid drugs have been noted to influence the behavior of integral membrane proteins. The zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) was able to solubilize the cannabinoid receptor from rat brain membranes, with the greatest yield and specific activity being obtained at a detergent/protein ratio of 0.5:1. [3H]CP-55940 bound to a single class of binding sites in the CHAPS extract, which exhibited a Kd of 0.94 nM as determined by nonlinear regression analysis of equilibrium binding data. The order of potency for cannabinoid agonists in heterologous equilibrium binding studies was CP-55244 > or = desacetyllevonantradol > delta 9-tetrahydrocannabinol > cannabinol >> cannabidiol, consistent with the relative affinities for these agonists in brain membrane preparations. CP-55243, the biologically inactive enantiomer of CP-55244, competed for binding of [3H]CP-55940 by < 50% at 1 microM, similar to its poor affinity for the receptor in membranes. The CHAPS-solubilized cannabinoid receptor exhibited functional interactions with guanine nucleotide-binding proteins (G proteins). GTP and nonhydrolyzable analogs decreased [3H]CP-55940 binding by 75%. The concentration-effect curves for guanine nucleotides exhibited a potency order similar to that observed for other G protein-linked receptors. Kinetic analyses indicated that GTP analogs increased the rate of agonist dissociation, decreasing the t1/2 from 60 min at 0-4 degrees to a multiphasic dissociation that exhibited a component having a t1/2 of < 1 min. The cannabinoid agonist desacetyllevonantradol was able to reduce pertussis toxin-catalyzed ADP-ribosylation of G proteins by 50%, demonstrating a receptor effect on G protein functions. These studies demonstrate that the membrane environment is not necessary for agonist binding to the cannabinoid receptor. Furthermore, the cannabinoid receptor maintains its functional interactions with pertussis toxin-sensitive G proteins in detergent solution.

The preservation and maintenance of natural park ecosystems, with modern man's being restricted to generally nonconsumptive uses of the park, represents one end of a spectrum of land use that extends through exploitation of natural ecosystems to the development of simplified agricultural ecosystems. Criteria for management of a park ecosystem must, of necessity, differ from criteria for other uses of land, since park management involves preventing or compensating for the influence of man. The objectives for natural areas appear to be ecologically feasible if it is recognized that these areas have a finite capacity for absorbing man's consumptive and disruptive influences. The interpretation of ecosystems to park visitors provides an opportunity to contribute to an environmental ethic that extends beyond the park environment.