Upregulation of Na⁺and Ca²⁺transporters in arterial smooth muscle from ouabain-induced hypertensive rats

Abstract

Prolonged ouabain administration (25 microg kg(-1) day(-1) for 5 wk) induces "ouabain hypertension" (OH) in rats, but the molecular mechanisms by which ouabain elevates blood pressure are unknown. Here, we compared Ca(2+) signaling in mesenteric artery smooth muscle cells (ASMCs) from normotensive (NT) and OH rats. Resting cytosolic free Ca(2+) concentration ([Ca(2+)](cyt); measured with fura-2) and phenylephrine-induced Ca(2+) transients were augmented in freshly dissociated OH ASMCs. Immunoblots revealed that the expression of the ouabain-sensitive alpha(2)-subunit of Na(+) pumps, but not the predominant, ouabain-resistant alpha(1)-subunit, was increased (2.5-fold vs. NT ASMCs) as was Na(+)/Ca(2+) exchanger-1 (NCX1; 6-fold vs. NT) in OH arteries. Ca(2+) entry, activated by sarcoplasmic reticulum (SR) Ca(2+) store depletion with cyclopiazonic acid (SR Ca(2+)-ATPase inhibitor) or caffeine, was augmented in OH ASMCs. This reflected an augmented expression of 2.5-fold in OH ASMCs of C-type transient receptor potential TRPC1, an essential component of store-operated channels (SOCs); two other components of some SOCs were not expressed (TRPC4) or were not upregulated (TRPC5). Ba(2+) entry activated by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol [a measure of receptor-operated channel (ROC) activity] was much greater in OH than NT ASMCs. This correlated with a sixfold upregulation of TRPC6 protein, a ROC family member. Importantly, in primary cultured mesenteric ASMCs from normal rats, 72-h treatment with 100 nM ouabain significantly augmented NCX1 and TRPC6 protein expression and increased resting [Ca(2+)](cyt) and ROC activity. SOC activity was also increased. Silencer RNA knockdown of NCX1 markedly downregulated TRPC6 and eliminated the ouabain-induced augmentation; silencer RNA knockdown of TRPC6 did not affect NCX1 expression but greatly attenuated its upregulation by ouabain. Clearly, NCX1 and TRPC6 expression are interrelated. Thus, prolonged ouabain treatment upregulates the Na(+) pump alpha(2)-subunit-NCX1-TRPC6 (ROC) Ca(2+) signaling pathway in arterial myocytes in vitro as well as in vivo. This may explain the augmented myogenic responses and enhanced phenylephrine-induced vasoconstriction in OH arteries (83) as well as the high blood pressure in OH rats.

A role of the sodium pump in spreading ischemia in rats

“…87 Another limitation is that the concept of intracellular Ca 2þ regulation by high affinity NaKAs has been partially based on other vascular beds than the cerebral circulation. 25,56 More research is thus needed to investigate these mechanisms in the brain.…”

Knockout of Na+/Ca2+ exchanger in smooth muscle attenuates vasoconstriction and L-type Ca2+ channel current and lowers blood pressure

“…A likely possibility is that receptoroperated channels (ROCs), which are an important source of Ca 2ϩ during receptor activation (44), especially at high catecholamine concentrations (30), may be downregulated in NCX1 SMϪ/Ϫ arteries. In primary cultured arterial myocytes, a knockout of NCX1 markedly decreases the expression of transient receptor potential canonical 6 channel protein (a component of ROCs) and reduces ROC activity (33).…”

“…Moreover, the SM-specific overexpression of NCX elevates BP and induces salt-dependent hypertension, and SEA0400 lowers BP in several salt-dependent animal models (21). Indeed, NCX type-1 (NCX1) is upregulated in mesenteric arteries from rats with ouabain-induced hypertension (33) and pulmonary arteries from humans with primary pulmonary hypertension (49). Whether NCX is involved in maintaining normal BP, however, has not been resolved.…”

Attenuated renal vascular responses to acute angiotensin II infusion in smooth muscle-specific Na+/Ca2+exchanger knockout mice

“…These findings indicate that salt-sensitive hypertension is linked to Ca 2ϩ entry through NCX1 in arterial smooth muscle (14). Indeed, NCX1 protein is upregulated in arteries in several types of hypertension (23,31,36,39).…”

“…Renovascular function plays a pivotal role in salt-dependent hypertension (9,34), and it has been reported that Na ϩ /Ca 2ϩ exchanger mechanisms are disrupted in saltsensitive hypertension (2,21,32). In contrast, other groups observed enhanced expression of NCX1 in arterial smooth muscle in several forms of hypertension (10,23,39). This might imply that NCX-mediated Ca 2ϩ transport is enhanced under these circumstances.…”