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.