The mechanisms involved in the """"""""active"""""""" (uphill) extrusion of calcium from muscle cells, and in the regulation of steady Ca balance, are incompletely understood. Many types of cells, including invertebrate muscle and vertebrate smooth and cardiac muscle, employ a Ca transport mechanism in which Na ions exchange for Ca (""""""""Na/Ca exchange""""""""). Thus, the Na electrochemical gradient may provide at least some of the energy for Ca extrusion; moreover, Na/Ca exchange may also mediate Ca entry when Na electrochemical gradient is reduced. ATP also plays a role, in part via an effect on the Na/Ca exchange kinetics, and in part as fuel for a parallel Ca-ATPase driven """"""""Ca pump"""""""". Internally perfused single giant barnacle muscle cells are employed as a model system to study Ca transport and metabolism in muscle. During the forthcoming project period, characterization of the Na/Ca exchange system in barnacle muscle should be completed, including direct determination of the exchange stoichiometry, and the comparative properties of the forward (Na entry/Ca exit) and reverse (Ca entry/Na exit) modes of operation of the exchanger as well as the """"""""futile modes"""""""" (Ca/Ca and Na/Na) exchange. Of particular interest is the role of internal Ca and Na in the regulation of exchanger turnover. The information obtained from this model system will be applied to the study of Ca transport and metabolism in arterial smooth muscle (ASM) cells. Preliminary studies on rings of bovine tail artery (Progress Report) indicate that Na/Ca exchange plays a prominent role in the regulation of (Ca2+)i, the intracellular free Ca concentration, in this tissue. In the proposed experiments, a Ca-sensitive fluorescent dye (fura-2) and digital imaging methods will be used to measure (Ca2+)i in single, dissociated bovine tail ASM cells. (Ca2+)i will be measured in resting and activated cells, and following reduction of Na electrochemical gradient. The latter should provide direct information about the Na/Ca exchange system in these cells. Patch clamp methods, in conjunction with the imaging methods, will be used to measure the ionic currents associated with Na/Ca exchange, and to determine the factors that regulate Na/Ca exchange in ASM. The results of these experiments should provide new information about the relative importance of Na/Ca exchange and other (parallel) Ca transport systems in the regulation of (Ca2+)i in these cells. The results may be applicable to pathophysiological processes such as those associated with hypertension.
