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Abstract
Na$\sp+$-Ca$\sp{2+}$ exchange is a reversible transport mechanism in the plasma membrane of certain eukaryotic cells. The magnitude and direction of Ca$\sp{2+}$ transport via the exchanger are determined by the magnitude and orientation of electrical and chemical ion gradients (Na$\sp+$ and Ca$\sp{2+}$) as well as the degree of saturation of kinetic and allosteric sites. Using $\sp{45}$Ca$\sp{2+}$ or $\sp{22}$Na$\sp+$ flux experiments, or fluorescent dyes to measure cytoplasmic free Ca$\sp{2+}$ and Na$\sp+,$ it was observed that the exchanger is not active in unstimulated renal epithelial cells (LLC-MK$\sb2$) and aortic myocytes. However, activation of Ca$\sp{2+}$ efflux mode of Na$\sp+$-Ca$\sp{2+}$ exchange was observed after raising cytoplasmic free Ca$\sp{2+}$ by treating the cells with angiotensin or ionomycin. The Ca$\sp{2+}$ influx mode of Na$\sp+$-Ca$\sp{2+}$ exchange occurred in both renal epithelial cells and immortalized aortic myocytes only when intracellular Na$\sp+$ was increased and extracellular Na$\sp+$ was partially or completely replaced by another monovalent cation.
The kinetic properties of the exchanger in renal epithelial cells and in aortic myocytes were similar. The dependence of exchange activity on internal Na$\sp+$ was sigmoidal. Additionally, external Mg$\sp{2+}$, like external Na$\sp+$, competitively inhibits Ca$\sp{2+}$ influx via the exchanger. The presence of external K$\sp+$ increased the selectivity of Ca$\sp{2+}$ influx mode of Na$\sp+$-Ca$\sp{2+}$ exchanger for Ca$\sp{2+}$. Hence, K$\sp+$ decreased the potency of Mg$\sp{2+}$ without affecting the K$\sb{\rm m}$ for Ca$\sp{2+}$. ATP depletion in these cells decrease the exchange activity.
In Na$\sp+$-loaded immortalized aortic myocytes, K$\sp+$ depolarized the plasma membrane and produced a large free Ca$\sp{2+}$ transient that was followed by a small sustained rise in free Ca$\sp{2+}$. It appears that a moderate increase in intracellular Ca$\sp{2+}$ inhibits the Ca$\sp{2+}$ influx mode of Na$\sp+$-Ca$\sp{2+}$ exchange by a kinetic rather than a thermodynamic mechanism because a 3-fold increase in cytosolic Ca$\sp{2+}$ decreases the calculated driving force for Ca$\sp{2+}$ influx via the exchanger by only $\sim$10%. Studies of exchange activity in membrane vesicles from aortic myocytes suggest that the exchanger may have an autoinhibitory domain. Endogenous proteolysis partially restored activity lost during vesicle preparation. In conclusion, the results of the above studies suggest that Na$\sp+$-Ca$\sp{2+}$ exchange makes a prominent contribution to Ca$\sp{2+}$ efflux in aortic myocytes and renal epithelial cells.
