Activation of c-SRC underlies the differential effects of ouabain and digoxin on Ca²⁺ signaling in arterial smooth muscle cells

Abstract

exchanger-1 (NCX1) and TRPC6 gene-encoded receptor-operated channels in mesenteric artery smooth muscle cells (ASMCs) in vivo and in vitro. Here, we test the effects of digoxin on Ca 2ϩ entry and signaling in ASMC. In contrast to ouabain treatment, the in vivo administration of digoxin (30 g·kg Ϫ1 ·day Ϫ1 for 3 wk) did not raise BP and had no effect on resting cytolic free Ca 2ϩ concentration ([Ca 2ϩ ]cyt) or phenylephrine-induced Ca 2ϩ signals in isolated ASMCs. Expression of transporters in the ␣2 Na ϩ pump-NCX1-TRPC6 Ca 2ϩ signaling pathway was not altered in arteries from digoxin-treated rats. Upregulated ␣2 Na ϩ pumps and a phosphorylated form of the c-SRC protein kinase (pY419-Src, ϳ4.5-fold) were observed in ASMCs from rats treated with ouabain but not digoxin. Moreover, in primary cultured ASMCs from normal rats, treatment with digoxin (100 nM, 72 h) did not upregulate NCX1 and TRPC6 but blocked the ouabain-induced upregulation of these transporters. Pretreatment of ASMCs with the c-Src inhibitor PP2 (1 M; 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) but not its inactive analog eliminated the effect of ouabain on NCX1 and TRPC6 expression and ATP-induced Ca 2ϩ entry. Thus, in contrast to ouabain, the interaction of digoxin with ␣2 Na ϩ pumps is unable to activate c-Src phosphorylation and upregulate the downstream NCX1-TRPC6 Ca 2ϩ signaling pathway in ASMCs. The inability of digoxin to upregulate c-Src may underlie its inability to raise long-term BP. cardiotonic steroids; Na ϩ pumps; NCX1; TRPC proteins; receptoroperated Ca 2ϩ entry CARDIOTONIC STEROIDS (CTS) have a long history in the treatment of heart failure and certain cardiac arrhythmias (3). Besides their role in regulation of cardiac function, endogenous CTS that are structurally related to the strophanthus family and that include ouabain are also implicated in control of salt metabolism and blood pressure (BP) (11,49). Plasma levels of endogenous ouabain are significantly elevated in ϳ45% of patients with essential hypertension (48, 55) and in animals with several forms of salt-dependent hypertension (16,21,26). Moreover, in rodents, the prolonged administration of low doses of ouabain induces ouabain-induced hypertension (OH) (29,30,39,40,46,61).Endogenous ouabain influences arterial tone and peripheral vascular resistance (10,12,20) by activating Ca 2ϩ signaling pathways in arterial smooth muscle cells (ASMCs) (46). These "signature" pathways involve inhibition of high-affinity ␣2 Na ϩ pumps and increased Ca 2ϩ entry via the Na ϩ /Ca 2ϩ exchanger-1 (NCX1), and TRPC gene-encoded receptor-and store-operated channels (ROCs and SOCs, respectively) (46). Rodent ASMCs express ␣2 Na ϩ pumps that mostly modulate Ca 2ϩ homeostasis and Ca 2ϩ signaling (18). Prior work has shown that the expression of ␣2 Na ϩ pumps, NCX1 and TRPC1 and TRPC6 is upregulated in ASMCs from OH rats (46). These transporters are involved in control of agonistmediated vasoconstriction (51, 63), myogenic tone, and BP (25,57,62,63). The arterial expression o...

Expression of rat Na-K-ATPase α2 enables ion pumping but not ouabain-induced signaling in α1-deficient porcine renal epithelial cells

“…Furthermore, it is important to note the well-established role of ␣2 in the control of smooth muscle contraction and the development of hypertension via its interaction with the Na ϩ /Ca 2ϩ exchanger (17,53). Moreover, recent studies from the University of Maryland highlighted the potential cross-talk between ␣1-and ␣2-induced signal transduction (44,55). In the present study, we mutated the rat ␣2 into a ouabain-resistant form to use ouabain selection for the generation of the LX-␣2-4 cell line.…”

Rostafuroxin Protects from Podocyte Injury and Proteinuria Induced by Adducin Genetic Variants and Ouabain

“…This hypothesis is further reinforced by the evidence that, in mesenteric arteries of OHR rats, ouabain increases the activity of a2 Na,K-ATPase, Na-CA exchanger 1, and TRPC6 channels in a Src-dependent manner, being responsible for the increased intracellular Ca 21 (Zulian et al, 2013). Since rostafuroxin blocks adducin-and ouabain-induced effects, we can envisage that the compound may also affect Ca 21 signaling in glomerular podocytes, thus preventing mutant b-adducin-and ouabain-mediated nephrin cleavage induced by calpain or, possibly, the TRPC6-PLC-g1-mediated pathway for nephrin signal transduction regulation.…”

Arterial α2-Na+pump expression influences blood pressure: lessons from novel, genetically engineered smooth muscle-specific α2mice

“…In the aforementioned hypertension models, however, the Ca 2ϩ transporter protein reprogramming can be expected to enhance Ca 2ϩ gain and signaling by myocytes (29,45,59) and thereby promote the rise in BP. The implication is that this protein reprogramming, which we attribute to a centrally regulated, sustained increase in circulating EO (7,15) that acts directly on myocytes (28,45,60), contributes significantly to the elevated BP in all those hypertension models.…”

“…In numerous rodent hypertension models, including the Dahl, spontaneously hypertensive rat, ANG II, DOCA-salt, Milan, and ouabain-induced models, these same Ca 2ϩ transporters (e.g., NCX1, SERCA2, and/or TRPC6) are markedly upregulated (2,15,44,45,52,59,60). In both the ␣ 2 SM-Tg and ␣ 2 SM-DN models, the Ca 2ϩ transporter reprogramming appears to be "protective": it either minimizes Ca 2ϩ depletion and attenuates BP decline (␣ 2 SM-Tg mice) or minimizes Ca 2ϩ overload and attenuates BP elevation (␣ 2 SM-DN mice).…”