No abstract

While acute activation of inhibitory G i/o -coupled receptors leads to inhibition of adenylyl cyclase, chronic activation of such receptors leads to an increase in cAMP accumulation. This phenomenon, observed in many cell types, has been referred to as adenylyl cyclase superactivation. At this stage, the mechanism leading to adenylyl cyclase superactivation and the nature of the isozyme(s) responsible for this phenomenon are largely unknown. Here we show that transfection of adenylyl cyclase isozymes into COS-7 cells results in an isozymespecific increase in AC activity upon stimulation (e.g. with forskolin, ionomycin, or stimulatory receptor ligands). However, independently of the method used to activate specific adenylyl cyclase isozymes, acute activation of the -opioid receptor inhibited the activity of adenylyl cyclases I, V, VI, and VIII, while types II, IV, and VII were stimulated and type III was not affected. Chronic -opioid receptor activation followed by removal of the agonist was previously shown, in transfected COS-7 cells, to induce superactivation of adenylyl cyclase type V. Here we show that it also leads to superactivation of adenylyl cyclase types I, VI, and VIII, but not of type II, III, IV, or VII, demonstrating that the superactivation is isozyme-specific. Not only were isozymes II, IV, and VII not superactivated, but a reduction in the activities of these isozymes was actually observed upon chronic opiate exposure. These results suggest that the phenomena of tolerance and withdrawal involve specific adenylyl cyclase isozymes. The synthesis of cAMP by adenylyl cyclase (AC)1 is modulated by hormones and neurotransmitters acting via receptors that activate GTP-binding proteins (G proteins). To date, mRNAs encoding nine distinct isozymes of AC have been identified (1-11). Sequence and functional similarities allow the categorization of these ACs into six classes: (a) AC type I (AC-I) is stimulated by Ca 2ϩ /calmodulin, possibly independently of G ␣s stimulation, and is inhibited by G ␤␥ subunits; (b) AC-VIII is stimulated by Ca 2ϩ /calmodulin; (c) AC-V and AC-VI are inhibited by low levels of Ca 2ϩ but are unaffected by G ␤␥ subunits; (d) AC-II, AC-IV, and AC-VII comprise a subfamily, where AC-II and AC-IV are highly activated by G ␤␥ subunits in the presence of activated G ␣s , while AC-II and AC-VII are stimulated by activation of protein kinase C; (e) AC-III is stimulated by a high concentration of Ca 2ϩ /calmodulin in the presence of G ␣s but is unaffected by G ␤␥ subunits; (f) AC-IX, which has only recently been cloned, has thus far only been found to be affected by G ␣s . The activities of all AC isozymes seem to be stimulated by G ␣s , but to different extents (5, 9, 10, 12-15).Stimulation of seven-transmembrane domain inhibitory receptors (e.g. -, ␦-, and -opioid receptors and m 2 -and m 4 -muscarinic receptors) activates G i/o proteins, as a result of which these G proteins dissociate into G ␣ and G ␤␥ dimers (16 -18). The G ␣i subunit interacts with AC, leading to its acute inhibition ...

Interaction of certain cytokines with their corresponding cell-surface receptors induces programmed cell death. Interferon-gamma induces in HeLa cells a type of cell death with features characteristic of programmed cell death. Here, we report the isolation of a novel gene, DAP3 (death-associated protein-3), involved in mediating interferon-gamma-induced cell death. The rescue of this gene was performed by a functional selection approach of gene cloning that is based on transfection with an antisense cDNA expression library. The antisense RNA-mediated inactivation of the DAP3 gene protected the cells from interferon-gamma-induced cell death. This property endowed the cells expressing it with a growth advantage in an environment restrictive due to the continuous presence of interferon-gamma and thus provided the basis of its selection. The gene is transcribed into a single 1.7-kilobase mRNA, which is ubiquitously expressed in different tissues and codes for a 46-kDa protein carrying a potential P-loop motif. Ectopic expression of DAP3 in HeLa cells was not compatible with cell growth, resulting in a 16-fold reduction in the number of drug-resistant stable clones. The data presented suggest that DAP3 is a positive mediator of cell death induced by interferon-gamma.

Several derivatives of cannabinol and the 1,1-dimethylheptyl homolog (DMH) of cannabinol were prepared and assayed for binding to the brain and the peripheral cannabinoid receptors (CB1 and CB2), as well as for activation of CB1- and CB2-mediated inhibition of adenylylcyclase. The DMH derivatives were much more potent than the pentyl (i.e., cannabinol) derivatives. 11-Hydroxycannabinol (4a) was found to bind potently to both CB1 and CB2 (Ki values of 38.0 +/- 7.2 and 26.6 +/- 5.5 nM, respectively) and to inhibit CB1-mediated adenylylcyclase with an EC50 of 58.1 +/- 6.2 nM but to cause only 20% inhibition of CB2-mediated adenylylcyclase at 10 microM. It behaves as a specific, though not potent, CB2 antagonist. 11-Hydroxycannabinol-DMH (4b) is a very potent agonist for both CB1 and CB2 (Ki values of 100 +/- 50 and 200 +/- 40 pM; EC50 of adenylylcyclase inhibition 56.2 +/- 4.2 and 207.5 +/- 27.8 pM, respectively).

Two cannabinoid receptors, designated neuronal (or CB1) and peripheral (or CB2), have recently been cloned. Activation of CB1 receptors leads to inhibition of adenylate cyclase and N-type voltage-dependent Ca 2+ channels. Here we show, using a CB2 transfected Chinese hamster ovary cell line, that this receptor binds a variety of tricyclic cannabinoid ligands as well as the endogenous ligand anandamide. Activation of the CB2 receptor by various tricydic cannabinoids inhibits adenylate cyclase activity and this inhibition is pertussis toxin sensitive indicating that this receptor is coupled to the GilGo GTP-binding proteins. Interestingly, contrary to results with CB1, anandamide did not inhibit the CB2 coupled adenylate cyclase activity and A9-tetrahydrocannabinol had only marginal effects. These results characterize the CB2 receptor as a functional and distinctive member of the cannabinoid receptor family.