Nanomolar ouabain increases NCX1 expression and enhances Ca2+signaling in human arterial myocytes: a mechanism that links salt to increased vascular resistance?

Abstract

The mechanisms by which NaCl raises blood pressure (BP) in hypertension are unresolved, but much evidence indicates that endogenous ouabain is involved. In rodents, arterial smooth muscle cell (ASMC) Na+pumps with an α2-catalytic subunit (ouabain EC50≤1.0 nM) are crucial for some hypertension models, even though ≈80% of ASMC Na+pumps have an α1-subunit (ouabain EC50≈ 5 μM). Human α1-Na+pumps, however, have high ouabain affinity (EC50≈ 10–20 nM). We used immunoblotting, immunocytochemistry, and Ca2+imaging (fura-2) to examine the expression, distribution, and function of Na+pump α-subunit isoforms in human arteries and primary cultured human ASMCs (hASMCs). hASMCs express α1- and α2-Na+pumps. Further, α2-, but not α1-, pumps are confined to plasma membrane microdomains adjacent to sarcoplasmic reticulum (SR), where they colocalize with Na/Ca exchanger-1 (NCX1) and C-type transient receptor potential-6 (receptor-operated channels, ROCs). Prolonged inhibition (72 h) with 100 nM ouabain (blocks nearly all α1- and α2-pumps) was toxic to most cultured hASMCs. Treatment with 10 nM ouabain (72 h), however, increased NCX1 and sarco(endo)plasmic reticulum Ca2+-ATPase expression and augmented ATP (10 μM)-induced SR Ca2+release in 0 Ca2+, ouabain-free media, and Ca2+influx after external Ca2+restoration. The latter was likely mediated primarily by ROCs and store-operated Ca2+channels. These hASMC protein expression and Ca2+signaling changes are comparable with previous observations on myocytes isolated from arteries of many rat hypertension models. We conclude that the same structurally and functionally coupled mechanisms (α2-Na+pumps, NCX1, ROCs, and the SR) regulate Ca2+homeostasis and signaling in hASMCs and rodent ASMCs. These ouabain/endogenous ouabain-modulated mechanisms underlie the whole body autoregulation associated with increased vascular resistance and elevation of BP in human, salt-sensitive hypertension.