Calcium transduces and amplifies external signals in order to produce cellular responses such as growth or differentiation, contraction, chemotaxis, secretion, or changes in metabolism and transport. The balance between Ca influx via channels and efflux through the plasma membrane determines total cell Ca. Mammalian cells have two mechanism for expelling Ca against a ten thousand fold concentration gradient: the Ca ATPase and the Na-Ca exchanger (NCX). The ATPase is ubiquitous in mammalian cells, whereas NCX is selectively expressed in certain cell types. NCX is a reality reversible carrier, whereas the ATPase is an essentially irreversible pump. The direction of net Ca flux via NCX depends on the electrochemical Na and Ca gradients. Cardiac NCX was recently shown to consist of a single polypeptide of 938 amino acids, 520 of which from a large, apparently cytoplasmic loop that is flanked by five 5' and six 3' putative transmembrane domains. The long term goal of this project is to elucidate biochemical mechanisms of NCX regulation in vascular smooth muscle. NCX is plentiful in arterial myocytes, but latent because it makes no detectable contribution to basal free Ca regulation in aortic myocytes. NCX makes a major contribution to net Ca efflux evoked by stimulating with a hormone that releases Ca from internal stores. In contrast to the activation of Ca efflux via NCX, raising cytosolic free Ca feedback inhibits the Ca influx via NCX. The Ca dependence of the inhibition of the Ca Influx via NCX will be determined. Calcium influx via NCX correlates closely with cellular ATP levels in the myocytes and renal epithelial cells. The biochemical basis for the ATP dependence of exchange activity will be investigated. Aortic myocytes that stably express cGMP-dependent protein kinase will be used to determine whether the kinase modulates NCX activity. The hypothesis that chronic inhibition of NCX increase the amount of hormonally releasable Ca in the sarcoplasmic reticulum will be tested. Biochemical mechanisms of regulation of NCX expression and activity by intracellular messengers will be elucidated. The 5'-flanking segment of the smooth muscle NCX gene (a few thousand bp) will be cloned and sequenced. Cis-acting elements in the 5'-flanking region of the NCX gene that account for the regulation of transcription by glucocorticoids and other stimuli such as growth factors will be identified by expression of reporter constructs with nested deletions in aortic myocytes.
