It has been known for some time that chronic treatment of neuronal cells and tissues with opioids, contrary to their acute effect, leads to an increase in cAMP accumulation. This phenomenon, defined as adenylyl cyclase superactivation, has been implicated in opiate addiction, yet the mechanism by which it is induced remains unclear. Here, we show that this phenomenon can be reproduced and studied in COS-7 cells cotransfected with adenylyl cyclase type V and mu-opioid receptor cDNAs. These cells display acute opioid inhibition of adenylyl cyclase activity, whereas prolonged exposure to the mu-agonist morphine or [-Ala2, N-methyl-Phe4, Gly-ol5]enkephalin leads to a time-dependent superactivation of adenylyl cyclase. This superactivated state is reversible, because it is gradually lost following agonist withdrawal. Adenylyl cyclase superactivation can be prevented by pertussis toxin pretreatment, indicating the involvement of Gi/o proteins, or by cotransfection with the carboxyl terminus of beta-adrenergic receptor kinase or with alpha-transducin (scavengers of Gbetagamma dimers), indicating a role for the G protein betagamma dimers in adenylyl cyclase superactivation. However, contrary to several other Gbetagamma-dependent signal transduction mechanisms (e.g. the extracellular signal-regulated kinase 2/MAP kinase pathway), adenylyl cyclase superactivation is not affected by the Ras dominant negative mutant N17-Ras.

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 ...

Adenylyl cyclase superactivation, a phenomenon by which chronic activation of inhibitory Gi/o-coupled receptors leads to an increase in cAMP accumulation, is believed to play an important role as a compensatory response of the cAMP signaling system in the cell. However, to date, the mechanism by which adenylyl cyclase activity is regulated by chronic exposure to inhibitory agonists and the nature of the adenylyl cyclase isozymes participating in this process remain largely unknown. Here we show, using COS-7 cells transfected with the various AC isozymes, that acute activation of the D2 dopaminergic and m4 muscarinic receptors inhibited the activity of adenylyl cyclase isozymes I, V, VI, and VIII, whereas types II, IV, and VII were stimulated and type III was not affected. Conversely, chronic receptor activation led to superactivation of adenylyl cyclase types I, V, VI, and VIII and to a reduction in the activities of types II, IV, and VII. The activity of AC-III also was reduced. This pattern of inhibition/stimulation of the various adenylyl cyclase isozymes is similar to that we recently observed on acute and chronic activation of the mu-opioid receptor, suggesting that isozyme-specific adenylyl cyclase superactivation may represent a general means of cellular adaptation to the activation of inhibitory receptors and that the presence/absence and intensity of the adenylyl cyclase response in different brain areas (or cell types) could be explained by the expression of different adenylyl cyclase isozyme types in these areas.

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Many types of cells exhibit increased adenylyl cyclase (AC) activity after chronic agonist treatment of G(i/o)-coupled receptors. This phenomenon, defined as AC superactivation or sensitization, has mostly been studied for the opioid receptors and is implicated in opiate addiction. Here we show that this phenomenon is also observed on chronic activation of the CB(1) cannabinoid receptor. Moreover, using COS-7 cells cotransfected with CB(1) receptor and individual AC isozymes, we could show selective superactivation of AC types I, III, V, VI, and VIII. The level of superactivation was dependent on the concentration of agonist and time of agonist exposure and was not dependent on the AC stimulator used. No superactivation of AC types II, IV, or VII was observed in COS-7 cells cotransfected with CB(1). The superactivation of AC type V was abolished by pretreatment with pertussis toxin and by cotransfection with the carboxy terminus of beta-adrenergic receptor kinase, which serves as a scavenger of G(betagamma) dimers, implying a role for the G(i/o) proteins and especially G(betagamma) dimers in the cannabinoid-induced superactivation of AC.

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The accepted dogma concerning the regulation of adenylyl cyclase (AC) activity by G ␤␥ dimers states that the various isoforms of AC respond differently to the presence of free G ␤␥ . It has been demonstrated that AC I activity is inhibited and AC II activity is stimulated by G ␤␥ subunits. This result does not address the possible differences in modulation that may exist among the different G ␤␥ heterodimers. Six isoforms of G ␤ and 12 isoforms of G ␥ have been cloned to date. We have established a cell transfection system in which G ␤ and G ␥ cDNAs were cotransfected with either AC isoform I or II and the activity of these isoforms was determined. We found that while AC I activity was inhibited by both G ␤1/␥2 and G ␤5/␥2 combinations, AC II responded differentially and was stimulated by G ␤1/␥2 and inhibited by G ␤5/␥2 . This finding demonstrates differential modulatory activity by different combinations of G ␤␥ on the same AC isoform and demonstrates another level of complexity within the AC signaling system.

An intriguing development in the G-protein signaling field has been the finding that not only the Galpha subunit, but also Gbetagamma subunits, affect a number of downstream target molecules. One of the downstream targets of Gbetagamma is adenylyl cyclase, and it has been demonstrated that a number of isoforms of adenylyl cyclase can be either inhibited or stimulated by Gbetagamma subunits. Until now, adenylyl cyclase type I has been the only isoform reported to be inhibited by free Gbetagamma. Here we show by transient cotransfection into COS-7 cells of either adenylyl cyclase V or VI, together with Ggamma2 and various Gbeta subunits, that these two adenylyl cyclase isozymes are markedly inhibited by Gbetagamma. In addition, we show that Gbeta1 and Gbeta5 subunits differ in their activity. Gbeta1 transfected alone markedly inhibited adenylyl cylcase V and VI (probably by recruiting endogenous Ggamma subunits). On the other hand, Gbeta5 produced less inhibition of these isozymes, and its activity was enhanced by the addition of Ggamma2. These results demonstrate that adenylyl cyclase types V and VI are inhibited by Gbetagamma dimers and that Gbeta1 and Gbeta5 subunits differ in their capacity to regulate these adenylyl cyclase isozymes.