INHIBITION OF NITRIC-OXIDE SYNTHASE ACTIVITY AND NITRIC OXIDE-DEPENDENT CALCIUM INFLUX IN RENAL EPITHELIAL-CELLS BY CYCLIC ADENOSINE-MONOPHOSPHATE - IMPLICATIONS FOR CELL INJURY

Cell injury frequently occurs in the setting of tissue destruction and inflammation and is associated with a rise in intracellular calcium ( Ca-i) and increased NO production. The mechanisms that trigger rises i n Ca-i and NO during cell injury are not fully defined, but they may i nvolve activation of G protein-coupled receptors for substances such a s bradykinin, Ang II, thromboxane, and thrombin. These receptors act t hrough G proteins from different families that have distinct functions . Receptors for bradykinin and Ang II act through members of the G alp ha(i) and G alpha(q) families, whereas receptors for thrombin and thro mboxane act through members of the G alpha(i), G alpha(q), and G alpha (12/13) families. These G proteins cooperate to regulate Ca-i and NO i n epithelial cells through distinct mechanisms. In a number of experim ental settings, activators of the adenylyl cyclase system reduce the s everity of cell injury. To understand the mechanisms by which G protei n-dependent signaling systems may contribute to cell injury and to def ine the role of adenylyl cyclase in ameliorating cell injury, the effe cts of adenylyl cyclase on bradykinin-stimulated Ca influx and NO in c ultured renal epithelial cells that stably overexpress G alpha(q) and G alpha(13) were studied. This system allowed for the separation of di fferent components of the signals initiated by receptors for thromboxa ne and thrombin. G alpha(13) increased bradykinin-stimulated Ca influx by a mechanism that depends on NO and cGMP. The increased Ca influx w as blocked by inhibitors of NO synthase and guanylyl cyclase and by ac tivation of adenylyl cyclase. NO production was inhibited by activator s of cAMP-dependent protein kinase, which indicated that cAMP blocks C a influx by inhibiting NO production. Expression of G alpha(q), the G protein that regulates phospholipase C, also increased bradykinin-stim ulated Ca influx, but by an NO, cGMP-independent mechanism that was in sensitive to inhibition by adenylyl cyclase. The authors conclude that Ca influx is modulated by NO-dependent and independent mechanisms, an d that to the extent that increased NO production contributes to incre ased Ca influx and cell injury, cell injury may be reduced by agents t hat activate adenylyl cyclase.