INHIBITORY REGULATION OF ADENYLATE CYCLASE

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A continuing goal of this project is to delineate the role of the inhibition of adenylyl cyclase in signal transduction pathways. It has become apparent in recent years that the various signal transduction pathways in cells interact at a number of levels. As a result, an individual neurotransmitter that directly modifies (e.g. by a GTP regulatory protein) the activity of one signal generating enzyme, may, via the generation of a signal, modulate the activities of a number of other pathways. Such interactions impart fine coordination of cellular activity. Understanding how these interactions modulate overall cellular responsiveness and identifying the molecular aspects of the signal transduction systems that permit such interactions is one of the most challenging and insightful areas of current hormone and neurotransmitter research. The present proposal aims to dissect a novel mode of interaction between the two major signalling systems, i.e. [Ca2+]i and cAMP. In a number of cell types (including pituitary and cardiac tissue, but not, for instance, liver) Ca2+, in submicromolar concentrations (i.e. physiologically elevated intracellular concentrations) exerts a powerful inhibition of plasma membrane adenylyl cyclase. Although preliminary evidence suggests that the inhibition may be exerted at the catalytic unit of adenylyl cyclase, the mechanism of the Ca2+ effect is unknown. Consequently, the present proposal takes three approaches: i) to characterize this inhibition biochemically and to determine whether the effect is mediated directly at the catalytic unit of adenylyl cyclase, or by some other factor that modifies catalytic activity; ii) to determine whether, in intact cells, hormones that elevate [Ca2+]i can inhibit cAMP accumulation by this mechanism; and iii) using probes that are available from currently-cloned adenylyl cyclases, to clone the species of adenylyl cyclase that is expressed in NCB-20 cells, which is potently inhibited by submicromolar [Ca2+], and determine whether expression of the cDNA will display Ca2+-inhibition. The NCB-20 cell line is a convenient model system in which to address many of the issues relating to antagonistic interactions between [Ca2+]i and cAMP, independent of ion channel effects. These non-excitable cells express opiate and alpha2-receptors, which inhibit cAMP production, serotonin and PGE1 receptors that stimulate adenylyl cyclase; and bradykinin and muscarinic cholinergic receptors that stimulate phospholipase C. Such a system should allow a determination of whether direct inhibition of adenylyl cyclase by submicromolar [Ca2+]i contributes to antagonistic interactions between the inositol phosphate pathway and cAMP, and the mechanisms involved.