The role of Na+/Ca2+ exchange in smooth muscle is controversial. Recently we showed that Na+/Ca2+ exchange activity is plentiful, but latent, in smooth muscle cells cultured from rat aorta. The exchanger makes a major contribution to Ca2+ efflux (forward mode) when the cells are stimulated with a hormone that releases stored Ca2+. In the absence of such a stimulus the exchanger makes little or no contribution to the maintenance of basal free Ca2+ in these cells. To produce appreciable Ca2+ influx via the exchanger (reverse mode) in these cells it is necessary to moderately increase cell Na+ as well as substantially decrease the concentration of extracellular Na+. The long-term goal of the proposed research is to elucidate the role of Na+/Ca2+ exchange in vascular smooth muscle from different blood vessels and mammalian species under normal and pathological states. The role of the exchanger in Ca2+ regulation will be defined by coordinated studies of Ca2+ fluxes and cytosolic free Ca2+ in cells with normal or increased Na+ in the absence or presence of a Ca2+ mobilizing hormone. This approach is required because decreasing the concentration of external Na+ triggers the release of stored Ca2+ in some cells and because of the lack of specific inhibitors of Na+/Ca2+ exchange or the plasma membrane Ca2+ ATPase. The mechanism by which angiotensin activates net Ca2+ efflux via Na+/Ca2+ exchange will be investigated in myocytes cultured from rat aorta and in plasma membrane vesicles from these cells. The dependence of forward and reverse mode exchange on intracellular pH, Na+, free Mg2+, and free Ca2+ will be determined. The influence of manipulating the Na+ gradient of cytosolic free Ca2+ and Ca2+ fluxes in stimulated and non-stimulated myocytes from rat and dog brain microvessels and human and dog aorta and coronary arteries will be determined to assess the role of Na+/Ca2+ exchange. The hypothesis that chronic inhibition of forward mode Na+/Ca2+ exchange increases the amount of Ca2+ in the sarcoplasmic reticulum will be tested. Because exchange activity correlates closely with the amount of ATP in intact cells, ATP and a cytosolic fraction will be examined for an influence on exchange activity in membrane vesicles. Plasma membrane vesicles will be used to examine the role of metabolic energy in the regulation of exchange activity. The stoichiometry of the exchanger will be estimated by a thermodynamic method in membrane vesicles. The vesicles will also be used to elucidate the mechanism by which Mg2+ inhibits the exchanger and find out how increasing K+ decreases the potency of Mg2+. Candidate mechanisms of hormonal activation of the exchanger will be tested in vesicle experiments. A close comparison of key properties of exchange activity in membrane vesicles and cells has not previously been done with heart or other cell types.
