Abstract 525: Nanomolar Ouabain Increases Na/Ca Exchanger-1 Expression and Enhances Ca2+ Signaling in Human Arterial Myocytes: A Mechanism That Links Salt to Increased Vascular Resistance?
The mechanisms by which excess dietary NaCl raises blood pressure (BP) in hypertension are unresolved. Much evidence indicates that endogenous ouabain (EO, an adrenocortical hormone and Na+ pump blocker) plays a role. 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 ≈ 20 nM). Therefore, we studied the expression, distribution and function of Na+ pump α subunit isoforms in ASMCs from surgically-recovered human artery segments. Immunoblots and immunocytochemistry reveal that human ASMCs (hASMCs) express α1 and α2 Na+ pumps. As in rodents, α2, but not α1, pumps are confined to plasma membrane (PM) microdomains adjacent to sarcoplasmic reticulum, SR (identified with ER Tracker, an SR stain, and anti-SR Ca2+ pump-2, SERCA2, antibodies). Na/Ca exchanger-1 (NCX1) and TRPC6 (component of receptor-operated channels, ROCs) co-localize with α2 in the PM microdomains. Incubation (72 hr) with 100 nM ouabain (blocks nearly all α1 and α2 pumps) was toxic to most cultured hASMCs, but 10 nM ouabain (blocks 90-95% of α2 and <<50% of α1 pumps) increased NCX1 and SERCA2 expression by 45±10% and 31±6%, respectively (P<0.05; n=5 patients). Ca2+ transients were measured with fura 2: The 72 hr, 10 nM ouabain pre-treatment increased 10 μM ATP-induced SR Ca2+ release in 0Ca2+ media by 27±8% (P<0.05; n=3), and increased Ca2+ influx when external Ca2+ was restored (ATP still present) by 96±15% (P<0.01; n=3 patients). This Ca2+ influx was likely mediated primarily by ROCs and store-operated Ca2+ channels. Thus, α2 Na+ pumps, NCX1, ROCs, and the SR are structurally and functionally linked. Freshly-isolated myocytes from the arteries of several rat hypertension models display comparable protein expression and Ca2+ signaling changes. We conclude that the same molecular mechanisms regulate long-term Ca2+ homeostasis and signaling in human and rodent ASMCs. These ouabain/EO-modulated mechanisms underlie the ‘whole body autoregulation’ associated with increased vascular resistance and elevation of BP in human hypertension.
- © 2012 by American Heart Association, Inc.