Activation of c-SRC underlies the differential effects of ouabain and digoxin on Ca(2+) signaling in arterial smooth muscle cells.

Cardiotonic steroids (CTS) of the strophanthus and digitalis families have opposing effects on long-term blood pressure (BP). This implies hitherto unrecognized divergent signaling pathways for these CTS. Prolonged ouabain treatment upregulates Ca(2+) entry via Na(+)/Ca(2+) 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(2+) entry and signaling in ASMC. In contrast to ouabain treatment, the in vivo administration of digoxin (30 µg·kg(-1)·day(-1) for 3 wk) did not raise BP and had no effect on resting cytolic free Ca(2+) concentration ([Ca(2+)](cyt)) or phenylephrine-induced Ca(2+) signals in isolated ASMCs. Expression of transporters in the α2 Na(+) pump-NCX1-TRPC6 Ca(2+) signaling pathway was not altered in arteries from digoxin-treated rats. Upregulated α2 Na(+) pumps and a phosphorylated form of the c-SRC protein kinase (pY419-Src, ~4.5-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 µM; 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(2+) entry. Thus, in contrast to ouabain, the interaction of digoxin with α2 Na(+) pumps is unable to activate c-Src phosphorylation and upregulate the downstream NCX1-TRPC6 Ca(2+) signaling pathway in ASMCs. The inability of digoxin to upregulate c-Src may underlie its inability to raise long-term BP.

Cross talk between plasma membrane Na(+)/Ca (2+) exchanger-1 and TRPC/Orai-containing channels: key players in arterial hypertension.

Arterial smooth muscle (ASM) Na(+)/Ca(2+) exchanger type 1 (NCX1) and TRPC/Orai-containing receptor/store-operated cation channels (ROC/SOC) are clustered with α2 Na(+) pumps in plasma membrane microdomains adjacent to the underlying junctional sarcoplasmic reticulum. This arrangement enables these transport proteins to function as integrated units to help regulate local Na(+) metabolism, Ca(2+) signaling, and arterial tone. They thus influence vascular resistance and blood pressure (BP). For instance, upregulation of NCX1 and TRPC6 has been implicated in the pathogenesis of high BP in several models of essential hypertension. The models include ouabain-induced hypertensive rats, Milan hypertensive rats, and Dahl salt-sensitive hypertensive rats, all of which exhibit elevated plasma ouabain levels. We suggest that these molecular mechanisms are key contributors to the increased vascular resistance ("whole body autoregulation") that elevates BP in essential hypertension. Enhanced expression and function of ASM NCX1 and TRPC/Orai1-containing channels in hypertension implies that these proteins are potential targets for pharmacological intervention.

Dysregulation of Ca2+ signaling in astrocytes from mice lacking amyloid precursor protein.

The relationship between altered metabolism of the amyloid-ß precursor protein (APP) and Alzheimer's disease is well established but the physiological roles of APP still remain unclear. Here, we studied Ca(2+) signaling in primary cultured and freshly dissociated cortical astrocytes from APP knockout (KO) mice and from Tg5469 mice overproducing by five- to sixfold wild-type APP. Resting cytosolic Ca(2+) (measured with fura-2) was not altered in cultured astrocytes from APP KO mice. The stored Ca(2+) evaluated by measuring peak amplitude of cyclopiazonic acid [CPA, endoplasmic reticulum (ER) Ca(2+) ATPase inhibitor]-induced Ca(2+) transients in Ca(2+)-free medium was significantly smaller in APP KO astrocytes than in wild-type cells. Store-operated Ca(2+) entry (SOCE) activated by ER Ca(2+) store depletion with CPA was also greatly reduced in APP KO astrocytes. This reflected a downregulated expression in APP KO astrocytes of TRPC1 (C-type transient receptor potential) and Orai1 proteins, essential components of store-operated channels (SOCs). Indeed, silencer RNA (siRNA) knockdown of Orai1 protein expression in wild-type astrocytes significantly attenuated SOCE. SOCE was also essentially reduced in freshly dissociated APP KO astrocytes. Importantly, knockdown of APP with siRNA in cultured wild-type astrocytes markedly attenuated ATP- and CPA-induced ER Ca(2+) release and extracellular Ca(2+) influx. The latter correlated with downregulation of TRPC1. Overproduction of APP in Tg5469 mice did not alter, however, the stored Ca(2+) level, SOCE, and expression of TRPC1/4/5 in cultured astrocytes from these mice. The data demonstrate that the functional role of APP in astrocytes involves the regulation of TRPC1/Orai1-encoded SOCs critical for Ca(2+) signaling.

Upregulation of Na+/Ca2+ exchanger and TRPC6 contributes to abnormal Ca2+ homeostasis in arterial smooth muscle cells from Milan hypertensive rats.

The Milan hypertensive strain (MHS) of rats is a model for hypertension in humans. Inherited defects in renal function have been well studied in MHS rats, but the mechanisms that underlie the elevated vascular resistance are unclear. Altered Ca(2+) signaling plays a key role in the vascular dysfunction associated with arterial hypertension. Here we compared Ca(2+) signaling in mesenteric artery smooth muscle cells from MHS rats and its normotensive counterpart (MNS). Systolic blood pressure was higher in MHS than in MNS rats (144 +/- 2 vs. 113 +/- 1 mmHg, P < 0.05). Resting cytosolic free Ca(2+) concentration (measured with fura-2) and ATP-induced Ca(2+) transients were augmented in freshly dissociated arterial myocytes from MHS rats. Ba(2+) entry activated by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (a measure of receptor-operated channel activity) was much greater in MHS than MNS arterial myocytes. This correlated with a threefold upregulation of transient receptor potential canonical 6 (TRPC6) protein. TRPC3, the other component of receptor-operated channels, was marginally, but not significantly, upregulated. The expression of TRPC1/5, components of store-operated channels, was not altered in MHS mesenteric artery smooth muscle. Immunoblots also revealed that the Na(+)/Ca(2+) exchanger-1 (NCX1) was greatly upregulated in MHS mesenteric artery (by approximately 13-fold), whereas the expression of plasma membrane Ca(2+)-ATPase was not altered. Ca(2+) entry via the reverse mode of NCX1 evoked by the removal of extracellular Na(+) induced a rapid increase in cytosolic free Ca(2+) concentration that was significantly larger in MHS arterial myocytes. The expression of alpha(1)/alpha(2) Na(+) pumps in MHS mesenteric arteries was not changed. Immunocytochemical observations showed that NCX1 and TRPC6 are clustered in plasma membrane microdomains adjacent to the underlying sarcoplasmic reticulum. In summary, MHS arteries exhibit upregulated TRPC6 and NCX1 and augmented Ca(2+) signaling. We suggest that the increased Ca(2+) signaling contributes to the enhanced vasoconstriction and elevated blood pressure in MHS rats.

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

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.

Orai1, a critical component of store-operated Ca2+ entry, is functionally associated with Na+/Ca2+ exchanger and plasma membrane Ca2+ pump in proliferating human arterial myocytes.

Ca(2+) entry through store-operated channels (SOCs) in the plasma membrane plays an important role in regulation of vascular smooth muscle contraction, tone, and cell proliferation. The C-type transient receptor potential (TRPC) channels have been proposed as major candidates for SOCs in vascular smooth muscle. Recently, two families of transmembrane proteins, Orai [also known as Ca(2+) release-activated Ca(2+) channel modulator (CRACM)] and stromal interacting molecule 1 (STIM1), were shown to be essential for the activation of SOCs mainly in nonexcitable cells. Here, using small interfering RNA, we show that Orai1 plays an essential role in activating store-operated Ca(2+) entry (SOCE) in primary cultured proliferating human aortic smooth muscle cells (hASMCs), whereas Orai2 and Orai3 do not contribute to SOCE. Knockdown of Orai1 protein expression significantly attenuated SOCE. Moreover, inhibition of Orai1 downregulated expression of Na(+)/Ca(2+) exchanger type 1 (NCX1) and plasma membrane Ca(2+) pump isoform 1 (PMCA1). The rate of cytosolic free Ca(2+) concentration decay after Ca(2+) transients in Ca(2+)-free medium was also greatly decreased under these conditions. This reduction of Ca(2+) extrusion, presumably via NCX1 and PMCA1, may be a compensation for the reduced SOCE. Immunocytochemical observations indicate that Orai1 and NCX1 are clustered in plasma membrane microdomains. Cell proliferation was attenuated in hASMCs with disrupted Orai1 expression and reduced SOCE. Thus Orai1 appears to be a critical component of SOCE in proliferating vascular smooth muscle cells, and may therefore be a key player during vascular growth and remodeling.

Calcium signaling mediated by P2Y receptors in mouse taste cells.

Evidence implicates a number of neuroactive substances and their receptors in mediating complex cell-to-cell communications in the taste bud. Recently, we found that ATP, a ubiquitous neurotransmitter/neuromodulator, mobilizes intracellular Ca2+ in taste cells by activating P2Y receptors. Here, P2Y receptor-cellular response coupling was characterized in detail using single cell ratio photometry and the inhibitory analysis. The sequence of underlying events was shown to include ATP-dependent activation of PLC, IP3 production, and IP3 receptor-mediated Ca2+ release followed by Ca2+ influx. Data obtained favor SOC channels rather than receptor-operated channels as a pathway for Ca2+ influx that accompanies Ca2+ release. Intracellular Ca2+ mobilized by ATP is apparently extruded by the plasma membrane Ca2+-ATPase, while a contribution of the Na+/Ca2+ exchange and other mechanisms of Ca2+ clearance is negligible. Cyclic AMP-dependent phosphorylation is likely to control a gain of the phosphoinositide cascade involved in ATP transduction. ATP-responsive taste cells are abundant in circumvallate, foliate, and fungiform papillae. Taken together, our observations point to a putative role for ATP as a neurotransmitter operative in the taste bud.