Regulation of Adenylyl Cyclase Type V/VI in Smooth Muscle: Interplay of Inhibitory G Protein and Ca Influx

The characteristics of inhibitory regulation of adenylyl cyclase V/VI by Ca and G proteins were examined in dispersed gastric smooth muscle cells. The mechanisms were evoked separately, sequentially, or concurrently using ligand-gated and G protein-coupled receptor agonists and receptor-independent probes (e.g, thapsigargin). During the initial phase of agonist stimulation, a,b-methylene-ATP, UTP, and ATP inhibited forskolin-stimulated cAMP formation in a concentration-dependent fashion. Inhibition by a,b-methylene-ATP, which activates ligand-gated P2X receptors, was abolished by zero Ca , whereas inhibition by UTP, which activates P2Y2 receptors coupled to Gq/11 and Gi3, was not affected by zero Ca 21 but was abolished by pertussis toxin (PTX). Inhibition by ATP, which activates both P2X and P2Y2 receptors, was not affected by zero Ca alone; but after inhibition mediated by Gai3 was blocked with PTX, inhibition by Ca influx was unmasked and was abolished by zero Ca. Inhibition by cholecystokinin-8 was observed only during the phase of capacitative Ca influx and was blocked by zero Ca. Inhibition by UTP during this phase was not affected by zero Ca alone; but after inhibition mediated by Gai3 was blocked with PTX, inhibition by Ca 21 influx was unmasked and was abolished by zero Ca. Inhibition of adenylyl cyclase V/VI activity in smooth muscle can be mediated independently by inhibitory G proteins and Ca influx but is exclusively mediated by inhibitory G proteins when both mechanisms are triggered. Functional regulation of the 10 cloned isoforms of adenylyl cyclase is diverse, with no two isoforms displaying identical regulation (Cooper et al., 1995; Sunahara et al., 1996). Nevertheless, three broad categories can be distinguished comprising (1) types I and VIII, predominantly expressed in neurons and stimulated by submicromolar concentrations of Ca and calmodulin and a more widely expressed type III, stimulated by low micromolar concentrations of Ca (Choi et al., 1992a, 1992b; Xia et al., 1992; Cali et al., 1994), (2) types II, IV, and IX, which are not affected by Ca or inhibited (in the case of types II and IV) by the GTP-binding proteins Gi and Go (Tang and Gilman, 1992; Taussig et al., 1993, 1994; Premont et al., 1996), and (3) types V and VI, which are inhibited by Gi and Go and by submicromolar concentrations of Ca elicited by Ca influx but not by Ca release from intracellular stores (Boyajian et al., 1991; Yoshimura and Cooper, 1992; Chiono et al., 1995; Taussig and Gilman, 1995). We have shown recently that adenylyl cyclase types V and VI, but not types II, III, or IV, are expressed in gastrointestinal smooth muscle (Murthy and Makhlouf, 1997). The cyclases are inhibited, depending on the agonist, by Gi1, Gi2, Gi3, and Go. Inhibition via somatostatin sst3 receptors is mediated by Gi1 and Go (Murthy et al., 1996), whereas inhibition via opioid m, d, or k receptors is mediated by Gi2 and Go (Murthy and Makhlouf, 1996). Inhibition via adenosine A1 receptors, muscarinic m2 receptors, and P2Y2 receptors is mediated by Gi3 (Murthy and Makhlouf, 1995a, 1997, 1998). Stimulation of adenylyl cyclase via muscarinic m3 receptors is mediated by the bg subunit of Gq/11; the stimulation is masked by the predominant inhibition mediated via m2 receptors by Gi3 (Murthy and Makhlouf, 1997). The coexistence of receptor subtypes coupled to distinct signaling pathways is likely to elicit various patterns of regulation of adenylyl cyclase V/VI in smooth muscle. For example, stimulation of adenylyl cyclase activity by adenosine A2 receptors coupled to Gs is attenuated by A1 receptors coupled to inhibition of adenylyl cyclase via the a subunit of Gi3 and to activation of PLC-b3 and IP3-dependent Ca 21 release via the a and bg subunits of Gi3 (Murthy and Makhlouf, 1995a): inhibitory regulation could be mediated by Ca influx, inhibitory G protein, or both. Whether these inhibitory mechanisms operate in concert or are mutually exclusive has not