Elevation of cytosolic free Ca2+ inhibits the type VI adenylyl cyclase that predominates in C6-2B cells. However, it is not known whether there is any selective requirement for Ca2+ entry or release for inhibition of cAMP accumulation to occur. In the present study, the effectiveness of intracellular Ca2+ release evoked by three independent methods (thapsigargin, ionomycin, and UTP) was compared with the capacitative Ca2+ entry that was triggered by these treatments. In each situation, only Ca2+ entry could inhibit cAMP accumulation (La3+ ions blocked the effect); Ca2+ release, which was substantial in some cases, was without effect. A moderate inhibition, as was elicited by a modest degree of Ca2+ entry, could be rendered substantial in the absence of phosphodiesterase inhibitors. Such conditions more closely mimic the physiological situation of normal cells. These results are particularly significant, in demonstrating not only that Ca2+ entry mediates the inhibitory effects of Ca2+ on cAMP accumulation, but also that diffuse elevations in [Ca2+]i are ineffective in modulating cAMP synthesis. This property suggests that, as with certain Ca(2+)-sensitive ion channels, Ca(2+)-sensitive adenylyl cyclases may be functionally colocalized with Ca2+ entry channels.

A number of the currently described adenylyl cyclase species can be regulated by Ca2+ in the submicromolar concentration range in in vitro assays. The regulatory significance of these observations hinges on whether a physiological elevation in intracellular Ca2+ can regulate these cyclase activities in intact cells. However, achieving a physiological elevation in cytosolic Ca2+ is complicated by the fact that hormonal increases in cytosolic Ca2+ can be accompanied by additional effects, such as liberation of beta gamma-subunits of G-proteins and activation of protein kinase C, which can have disparate type-specific effects on cyclase activities. Therefore we have devised a strategy based on capacitative Ca2+ entry to show that, when types I and VI adenylyl cyclase are expressed in human embryonic kidney 293 cells, they are stimulated and inhibited respectively by Ca2+ entry. Blockade of Ca2+ entry by La3+ ions blocks the effects of Ca2+ entry on cyclic AMP synthesis. These studies establish that adenylyl cyclases deemed to be sensitive to Ca2+ in in vitro assays can be regulated by physiological Ca2+ entry, and therefore, such cyclases are poised to respond to changes in intracellular Ca2+ in tissues in which they are expressed.