Inhibitory interaction of the plasma membrane Na+/Ca2+ exchangers with the 14-3-3 proteins.

The three Na+/Ca2+ exchanger isoforms, NCX1, NCX2, and NCX3, contain a large cytoplasmic loop that is responsible for the regulation of activity. We have used 347 residues of the loop of NCX2 as the bait in a yeast two-hybrid approach to identify proteins that could interact with the exchanger and regulate its activity. Screening of a human brain cDNA library identified the epsilon and zeta isoforms of the 14-3-3 protein family as interacting partners of the exchanger. The interaction was confirmed by immunoprecipitation and in vitro binding experiments. The effect of the interaction on the homeostasis of Ca2+ was investigated by co-expressing NCX2 and 14-3-3epsilon in HeLa cells together with the recombinant Ca2+ probe aequorin; the ability of cells expressing both NCX2 and 14-3-3epsilon to dispose of a Ca2+ transient induced by an InsP3-producing agonist was substantially decreased, indicating a reduction of NCX2 activity. The 14-3-3epsilon protein also inhibited the NCX1 and NCX3 isoforms. In vitro binding experiments revealed that all three NCX isoforms interacted with multiple 14-3-3 isoforms. 14-3-3 was bound by both phosphorylated and nonphosphorylated NCX, but the phosphorylated form had much higher binding affinity

Ca2+ handling is altered when arterial myocytes progress from a contractile to a proliferative phenotype in culture

Phenotypic modulation of vascular myocytes is important for vascular development and adaptation. A characteristic feature of this process is alteration in intracellular Ca2+ handling, which is not completely understood. We studied mechanisms involved in functional changes of inositol 1,4,5-trisphosphate (IP3)- and ryanodine (Ry)-sensitive Ca2+ stores, store-operated Ca2+ entry (SOCE), and receptor-operated Ca2+ entry (ROCE) associated with arterial myocyte modulation from a contractile to a proliferative phenotype in culture. Proliferating, cultured myocytes from rat mesenteric artery have elevated resting cytosolic Ca2+ levels and increased IP3-sensitive Ca2+ store content. ATP- and cyclopiazonic acid [CPA; a sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) inhibitor]-induced Ca2+ transients in Ca2+-free medium are significantly larger in proliferating arterial smooth muscle cells (ASMCs) than in freshly dissociated myocytes, whereas caffeine (Caf)-induced Ca2+ release is much smaller. Moreover, the Caf/Ry-sensitive store gradually loses sensitivity to Caf activation during cell culture. These changes can be explained by increased expression of all three IP3 receptors and a switch from Ry receptor type II to type III expression during proliferation. SOCE, activated by depletion of the IP3/CPA-sensitive store, is greatly increased in proliferating ASMCs. Augmented SOCE and ROCE (activated by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol) in proliferating myocytes can be attributed to upregulated expression of, respectively, transient receptor potential proteins TRPC1/4/5 and TRPC3/6. Moreover, stromal interacting molecule 1 (STIM1) and Orai proteins are upregulated in proliferating cells. Increased expression of IP3 receptors, SERCA2b, TRPCs, Orai(s), and STIM1 in proliferating ASMCs suggests that these proteins play a critical role in an altered Ca2+ handling that occurs during vascular growth and remodeling

Activation of c-SRC underlies the differential effects of ouabain and digoxin on Ca2+ 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

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

Ca2+ 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 Ca2+ release-activated Ca2+ 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 Ca2+ 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+/Ca2+ exchanger type 1 (NCX1) and plasma membrane Ca2+ pump isoform 1 (PMCA1). The rate of cytosolic free Ca2+ concentration decay after Ca2+ transients in Ca2+-free medium was also greatly decreased under these conditions. This reduction of Ca2+ 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

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

Prolonged ouabain administration (25 μg·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 Ca2+ signaling in mesenteric artery smooth muscle cells (ASMCs) from normotensive (NT) and OH rats. Resting cytosolic free Ca2+ concentration ([Ca2+]cyt; measured with fura-2) and phenylephrine-induced Ca2+ transients were augmented in freshly dissociated OH ASMCs. Immunoblots revealed that the expression of the ouabain-sensitive α2-subunit of Na+ pumps, but not the predominant, ouabain-resistant α1-subunit, was increased (2.5-fold vs. NT ASMCs) as was Na+/Ca2+ exchanger-1 (NCX1; 6-fold vs. NT) in OH arteries. Ca2+ entry, activated by sarcoplasmic reticulum (SR) Ca2+ store depletion with cyclopiazonic acid (SR Ca2+-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). Ba2+ 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 [Ca2+]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 α2-subunit-NCX1-TRPC6 (ROC) Ca2+ 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

Interaction of extended loop 882-892 of Cp with Mpo.

<p>Cp is shown in blue, heavy chain of Mpo in palegreen, light chain of Mpo in pink. Copper ions are shown as orange spheres. Heme is shown in violet, ferrous is shown in gray sphere. (<b>A</b>) Overall view of Cp monomer contact with Mpo monomer. Selected contact is indicated by dash-line oval (<b>B</b>). Zoom of contact area of Cp with the entrance of heme pocket of Mpo. Cp is shown in cartoon; Mpo is shown in cartoon and semi-transparent surface representation. Ends of loop between domain 5 and 6 are marked. <b>(C)</b> Inhibition of ABTS-peroxidase activity of Mpo by synthetic peptide RPYLKVFNPR corresponding to the 883-892 fragment of Cp sequence.</p