Ouabain augments Ca2+ transients in arterial smooth muscle without raising cytosolic Na+

Abstract

Ouabain and other cardiotonic steroids (CTS) inhibit Na+ pumps and are widely believed to exert their cardiovascular effects by raising the cytosolic Na+ concentration ([Na+]cyt) and Ca2+. This view has not been rigorously reexamined despite evidence that low-dose CTS may act without elevating [Na+]cyt; also, it does not explain the presence of multiple, functionally distinct isoforms of the Na+ pump in many cells. We investigated the effects of Na+ pump inhibition on [Na+]cyt(with Na+ binding benzofuran isophthalate) and Ca2+ transients (with fura 2) in primary cultured arterial myocytes. Low concentrations of ouabain (3–100 nM) or human ouabain-like compound or reduced extracellular K+ augmented hormone-evoked mobilization of stored Ca2+ but did not increase bulk [Na+]cyt. Augmentation depended directly on external Na+, but not external Ca2+, and was inhibited by 10 mM Mg2+ or 10 μM La3+. Evoked Ca2+ transients in pressurized small resistance arteries were also augmented by nanomolar ouabain and inhibited by Mg2+. These results suggest that Na+ enters a tiny cytosolic space between the plasmalemma (PL) and the adjacent sarcoplasmic reticulum (SR) via an Mg2+- and La3+-blockable mechanism that is activated by SR store depletion. The Na+ and Ca2+ concentrations within this space may be controlled by clusters of high ouabain affinity (α3) Na+ pumps and Na/Ca exchangers located in PL microdomains overlying the SR. Inhibition of the α3 pumps by low-dose ouabain should raise the local concentrations of Na+ and Ca2+ and augment hormone-evoked release of Ca2+ from SR stores. Thus the clustering of small numbers of specific PL ion transporters adjacent to the SR can regulate global Ca2+ signaling. This mechanism may affect vascular tone and blood flow and may also influence Ca2+ signaling in many other types of cells.