Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Overexpression of phospholemman (PLM) in normal adult rat cardiac myocytes altered contractile function and cytosolic Ca2+ concentration ([Ca2+]i) homeostasis and inhibited Na+/Ca2+ exchanger (NCX1). In addition, PLM coimmunoprecipitated and colocalized with NCX1 in cardiac myocyte lysates. In this study, we evaluated whether the cytoplasmic domain of PLM is crucial in mediating its effects on contractility, [Ca2+]i transients, and NCX1 activity. Canine PLM or its derived mutants were overexpressed in adult rat myocytes by adenovirus-mediated gene transfer. Confocal immunofluorescence images using canine-specific PLM antibodies demonstrated that the exogenous PLM or its mutants were correctly targeted to sarcolemma, t-tubules, and intercalated discs, with little to none detected in intracellular compartments. Overexpression of canine PLM or its mutants did not affect expression of NCX1, sarco(endo)plasmic reticulum Ca(2+)-ATPase, Na(+)-K(+)-ATPase, and calsequestrin in adult rat myocytes. A COOH-terminal deletion mutant in which all four potential phosphorylation sites (Ser62, Ser63, Ser68, and Thr69) were deleted, a partial COOH-terminal deletion mutant in which Ser68 and Thr69 were deleted, and a mutant in which all four potential phosphorylation sites were changed to alanine all lost wild-type PLM's ability to modulate cardiac myocyte contractility. These observations suggest the importance of Ser68 or Thr69 in mediating PLM's effect on cardiac contractility. Focusing on Ser68, the Ser68 to Glu mutant was fully effective, the Ser63 to Ala (leaving Ser68 intact) mutant was partially effective, and the Ser68 to Ala mutant was completely ineffective in modulating cardiac contractility, [Ca2+]i transients, and NCX1 currents. Both the Ser63 to Ala and Ser68 to Ala mutants, as well as PLM, were able to coimmunoprecipitate NCX1. It is known that Ser68 in PLM is phosphorylated by both protein kinases A and C. We conclude that regulation of cardiac contractility, [Ca2+]i transients, and NCX1 activity by PLM is critically dependent on Ser68. We suggest that PLM phosphorylation at Ser68 may be involved in cAMP- and/or protein kinase C-dependent regulation of cardiac contractility.
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PMID:Serine 68 of phospholemman is critical in modulation of contractility, [Ca2+]i transients, and Na+/Ca2+ exchange in adult rat cardiac myocytes. 1565 56

Rapid and precise control of Na(+)/Ca(2+) exchanger (NCX1) activity is essential in the maintenance of beat-to-beat Ca(2+) homeostasis in cardiac myocytes. Here, we show that phospholemman (PLM), a 15-kDa integral sarcolemmal phosphoprotein, is a novel endogenous protein inhibitor of cardiac NCX1. Using a heterologous expression system that is devoid of both endogenous PLM and NCX1, we first demonstrated by confocal immunofluorescence studies that both exogenous PLM and NCX1 co-localized at the plasma membrane. Reciprocal co-immunoprecipitation studies revealed specific protein-protein interaction between PLM and NCX1. The functional consequences of direct association of PLM with NCX1 was the inhibition of NCX1 activity, as demonstrated by whole-cell patch clamp studies to measure NCX1 current density and radiotracer flux assays to assess Na(+)-dependent (45)Ca(2+) uptake. Inhibition of NCX1 by PLM was specific, because a single mutation of serine 68 to alanine in PLM resulted in a complete loss of inhibition of NCX1 current, although association of the PLM mutant with NCX1 was unaltered. In native adult cardiac myocytes, PLM co-immunoprecipitated with NCX1. We conclude that PLM, a member of the FXYD family of small ion transport regulators known to modulate Na(+)-K(+)-ATPase, also regulates Na(+)/Ca(2+) exchange in the heart.
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PMID:Identification of an endogenous inhibitor of the cardiac Na+/Ca2+ exchanger, phospholemman. 1577 79

The Na(+)/Ca(2+) exchanger (NCX) on the plasma membrane is thought to be the main calcium extrusion system from the cytosol to the extracellular space in many mammalian excitable cells including cardiac myocytes. However, the precise roles of NCX are still unclear because of lack of its specific inhibitors. We generated NCX1-deficient mice by gene targeting to determine the in vivo function of the exchanger. Homozygous mutant died at 9.5 days post coitum. Embryonic hearts did not beat and cardiac myocytes showed apoptosis. These results suggest that NCX1 is required for heart beats and survival of cardiac myocytes in embryos. Heterozygous mutant mice were viable and indistinguishable from wild type mice. mRNA and protein levels in the heart of heterozygous mutant were half as much as wild type mice. In response to pressure overload, mutant mice showed better systolic and diastolic relaxation functions than wild type mice. Intracellular Ca(2+) measurement revealed an increase in calcium content of cytoplasm and sarcoplasmic reticulum (SR) and RNA analysis revealed preserved SR Ca(2+) ATPase expression in the ventricle of mutant mice. These results suggest that NCX plays an important role in cardiac performance in these pathological situations.
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PMID:[Analysis of Na+/Ca2+ exchanger knockout mice]. 1577 78

Previous studies have shown that the newly found endogenous inhibitor (NCX(IF)) of the cardiac Na/Ca exchanger (NCX1) is capable of regulating the muscle strip's contractility and relaxation. Here, the effects of purified NCX(IF) were tested on single cell shortening-lengthening (by using the IR CCD camera coupled with the two-edge video-detector) and [Ca]i-transients (by monitoring the changes in fluo-3 fluorescence). A perfusion of isolated cardiomyocytes (paced at 0.5-1.0 Hz) with NCX(IF) results in 4-6-fold enhancement in the amplitude of cell shortening-lengthening reaching the steady-state levels within 5-8 min (n=20, p<0.009). Simultaneous recordings of cell shortening-lengthening and [Ca]i-transients from the same cell show that the amplitude enhancement is associated with accelerated decay of both signals. Therefore, the NCX(IF)-dependent modulation of the single cell contractility is primarily governed by Ca-related mechanisms. The observed data are consistent with a proposal suggesting that the inhibition of NCX1 by NCX(IF) results in Ca-dependent activation of SERCA (SR Ca ATPase), yielding the accelerated decay of the [Ca]i-transients. The subsequent increase in the SR Ca content may result in enhanced Ca-release reflecting the manifested promotion of [Ca]i-transients. More systematic study is required for confirming this working hypothesis.
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PMID:Purified endogenous inhibitor of the Na/Ca exchanger can enhance the cardiomyocytes contractility and calcium transients. 1678 52

The activity of the cardiac Na(+)-Ca(2+) exchanger (NCX1.1) is allosterically regulated by Ca(2+), which binds to two acidic regions in the cytosolically disposed central hydrophilic domain of the NCX protein. A mutation in one of the regulatory Ca(2+) binding regions (D447V) increases the half-activation constant (K(h)) for allosteric Ca(2+) activation from approximately 0.3 to > 1.8 microm. Chinese hamster ovary cells expressing the D447V exchanger showed little or no activity under physiological ionic conditions unless cytosolic [Ca(2+)] was elevated to > 1 microm. However, when cytosolic [Na(+)] was increased to 20 mm or more (using ouabain-induced inhibition of the Na(+),K(+)-ATPase or the ionophore gramicidin), cells expressing the D447V mutant rapidly accumulated Ca(2+) or Ba(2+) when the reverse (Ca(2+) influx) mode of NCX activity was initiated, although initial cytosolic [Ca(2+)] was < 100 nm. Importantly, the time course of Ca(2+) uptake did not display the lag phase that reflects allosteric Ca(2+) activation of NCX activity in the wild-type NCX1.1; indeed, at elevated [Na(+)], the D447V mutant behaved similarly to the constitutively active deletion mutant Delta(241-680), which lacks the regulatory Ca(2+) binding sites. In cells expressing wild-type NCX1.1, increasing concentrations of cytosolic Na(+) led to a progressive shortening of the lag phase for Ca(2+) uptake. The effects of elevated [Na(+)] developed rapidly and were fully reversible. The activity of the D447V mutant was markedly inhibited when phosphatidylinositol 4,5-bisphosphate (PIP2) levels were reduced. We conclude that when PIP2 levels are high, elevated cytosolic [Na(+)] induces a mode of exchange activity that does not require allosteric Ca(2+) activation.
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PMID:Sodium-calcium exchange does not require allosteric calcium activation at high cytosolic sodium concentrations. 1680 64

Excess salt intake is a major risk factor for hypertension. However, the molecular mechanisms underlying salt-dependent hypertension remain obscure. Our recent studies using selective Na(+)/Ca(2+) exchange inhibitors and genetically engineered mice provide compelling evidence that salt-dependent hypertension is triggered by Ca(2+) entry through Na(+)/Ca(2+) exchanger type 1 (NCX1) in arterial smooth muscle. Endogenous cardiac glycosides, which may contribute to salt-dependent hypertension, seem to be necessary for NCX1-mediated hypertension. Intriguingly, recent studies by Dostanic-Larson et al. using knock-in mice with modified cardiac glycoside binding affinity of Na(+),K(+)-ATPases demonstrate that this binding site plays an important physiological role in blood pressure control. Thus, when cardiac glycosides inhibit Na(+),K(+)-ATPase in arterial smooth muscle cells, the elevation of local Na(+) on the submembrane area is believed to facilitate Ca(2+) entry through NCX1, resulting in vasoconstriction. This proposed pathway may have enabled us to explain how to link dietary salt to hypertension.
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PMID:Topics on the Na+/Ca2+ exchanger: role of vascular NCX1 in salt-dependent hypertension. 1696 Apr 23

Both beta-adrenergic blockade and bradycardia may contribute to the therapeutic effect of beta-blockers in chronic heart failure (CHF). This study tested the relative importance of bradycardia by comparing cilobradine (Cilo), a sinus node inhibitor, with a beta-blocker, metoprolol (Meto), in an established canine model of CHF. Dogs were chronically instrumented for hemodynamic and left ventricular (LV) volume measurements. CHF was created by daily coronary embolization via a chronically implanted coronary (left anterior descending coronary artery) catheter. After establishment of CHF, control (n=6), Meto (30 mg/day, n=5), Cilo (low) (1 mg/kg/day, n=5), or Cilo (high) (3 mg/kg/day, n=5) was given orally for 12 weeks. Systemic hemodynamics, echocardiography, and pressure volume analysis were measured at baseline, at CHF, and 3 months after treatment in an awake state. Protein levels of cardiac sarcoplasmic reticulum calcium-ATPase (SERCA2a), ryanodine receptor (RyR2), and Na+-Ca2+ exchanger (NCX1) were measured by Western blot. RyR2 protein kinase A (PKA) phosphorylation was determined by back-phosphorylation. After 12 weeks, Meto and Cilo (high and low) produced similar bradycardic effects, accompanied by a significantly improved LV dP/dt versus control [Meto, 2602+/-70; Cilo (low), 2517+/-45; Cilo (high), 2579+/-78; control, 1922+/-115 mm Hg/s; p<0.05]. Both Meto and Cilo (high) normalized protein levels of SERCA2a and NCX1 and reversed PKA hyperphosphorylation of RyR2, in contrast to controls. High-dose cilobradine effectively produced bradycardia and improved cardiac function after CHF, comparable with metoprolol. Restored protein levels of SERCA2a and improved function of RyR2 may be important mechanisms associated with cilobradine therapy.
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PMID:Bradycardic therapy improves left ventricular function and remodeling in dogs with coronary embolization-induced chronic heart failure. 1727 96

The various cardiac regions have specific action potential properties appropriate to their electrical specialization, resulting from a specific pattern of ion-channel functional expression. The present study addressed regionally defined differential ion-channel expression in the non-diseased human heart with a genomic approach. High-throughput real-time RT-PCR was used to quantify the expression patterns of 79 ion-channel subunit transcripts and related genes in atria, ventricular epicardium and endocardium, and Purkinje fibres isolated from 15 non-diseased human donor hearts. Two-way non-directed hierarchical clustering separated atria, Purkinje fibre and ventricular compartments, but did not show specific patterns for epicardium versus endocardium, nor left- versus right-sided chambers. Genes that characterized the atria (versus ventricles) included Cx40, Kv1.5 and Kir3.1 as expected, but also Cav1.3, Cav3.1, Cav alpha2 delta2, Nav beta1, TWIK1, TASK1 and HCN4. Only Kir2.1, RyR2, phospholamban and Kv1.4 showed higher expression in the ventricles. The Purkinje fibre expression-portrait (versus ventricle) included stronger expression of Cx40, Kv4.3, Kir3.1, TWIK1, HCN4, ClC6 and CALM1, along with weaker expression of mRNA encoding Cx43, Kir2.1, KChIP2, the pumps/exchangers Na(+),K(+)-ATPase, NCX1, SERCA2, and the Ca(2+)-handling proteins RYR2 and CASQ2. Transcripts that were more strongly expressed in epicardium (versus endocardium) included Cav1.2, KChIP2, SERCA2, CALM3 and calcineurin-alpha. Nav1.5 and Nav beta1 were more strongly expressed in the endocardium. For selected genes, RT-PCR data were confirmed at the protein level. This is the first report of the global portrait of regional ion-channel subunit-gene expression in the non-diseased human heart. Our data point to significant regionally determined ion-channel expression differences, with potentially important implications for understanding regional electrophysiology, arrhythmia mechanisms, and responses to ion-channel blocking drugs. Concordance with previous functional studies suggests that regional regulation of cardiac ion-current expression may be primarily transcriptional.
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PMID:Regional and tissue specific transcript signatures of ion channel genes in the non-diseased human heart. 1754 Jun 96

This study was aimed to examine whether the changes of protein expression of sodium transporters in the ischemic penumbra are associated with the pathogenesis of ischemia-induced brain edema and/or brain cell injury. An experimental model of cerebral ischemia was made by permanent middle cerebral artery occlusion (pMCAO) in rats and the changes of protein expression of sodium transporters in the ischemic penumbra were examined by immunoblotting. Extensive infarction was observed in the frontal and parietal cortical and subcortical areas at 3 and 6h after pMCAO. Immunoblotting analyses revealed significantly increased expressions of electrogenic NBC (241 +/- 11% at 3 h and 154 +/- 9% at 6 h, P < 0.05) and NHE1 (144 +/- 3% at 3 h and 170 +/- 9% at 6 h, P < 0.05), compared with sham-operated controls. In contrast, Na-K-ATPase expression (78 +/- 6% at 3 h and 85 +/- 3% at 6 h, P < 0.05) was significantly decreased. The expression of NCX1 was unchanged at 3 h, but was significantly increased at 6 h (141 +/- 3%, P < 0.05). In addition, the expressions of neuronal (NeuN) and astroglial cell (GFAP) proteins were decreased, whereas the expression of oligodendrocyte protein (CNPase) was unchanged. Taken together, the selectively increased expressions of NHE1, electrogenic NBC, and NCX1 and decreased expression of Na-K-ATPase in the ischemic penumbra are likely to contribute to the secondary brain cell damages presumably through intracellular Na(+) accumulation, cell swelling, and intracellular Ca(2+) overload.
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PMID:Altered expression of sodium transporters in ischemic penumbra after focal cerebral ischemia in rats. 1766 98

Phospholemman (PLM) regulates cardiac Na(+)/Ca(2+) exchanger (NCX1) and Na(+)-K(+)-ATPase in cardiac myocytes. PLM, when phosphorylated at Ser(68), disinhibits Na(+)-K(+)-ATPase but inhibits NCX1. PLM regulates cardiac contractility by modulating Na(+)-K(+)-ATPase and/or NCX1. In this study, we first demonstrated that adult mouse cardiac myocytes cultured for 48 h had normal surface membrane areas, t-tubules, and NCX1 and sarco(endo)plasmic reticulum Ca(2+)-ATPase levels, and retained near normal contractility, but alpha(1)-subunit of Na(+)-K(+)-ATPase was slightly decreased. Differences in contractility between myocytes isolated from wild-type (WT) and PLM knockout (KO) hearts were preserved after 48 h of culture. Infection with adenovirus expressing green fluorescent protein (GFP) did not affect contractility at 48 h. When WT PLM was overexpressed in PLM KO myocytes, contractility and cytosolic Ca(2+) concentration ([Ca(2+)](i)) transients reverted back to those observed in cultured WT myocytes. Both Na(+)-K(+)-ATPase current (I(pump)) and Na(+)/Ca(2+) exchange current (I(NaCa)) in PLM KO myocytes rescued with WT PLM were depressed compared with PLM KO myocytes. Overexpressing the PLMS68E mutant (phosphomimetic) in PLM KO myocytes resulted in the suppression of I(NaCa) but had no effect on I(pump). Contractility, [Ca(2+)](i) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the PLMS68E mutant were depressed compared with PLM KO myocytes overexpressing GFP. Overexpressing the PLMS68A mutant (mimicking unphosphorylated PLM) in PLM KO myocytes had no effect on I(NaCa) but decreased I(pump). Contractility, [Ca(2+)](i) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the S68A mutant were similar to PLM KO myocytes overexpressing GFP. We conclude that at the single-myocyte level, PLM affects cardiac contractility and [Ca(2+)](i) homeostasis primarily by its direct inhibitory effects on Na(+)/Ca(2+) exchange.
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PMID:Regulation of cardiac myocyte contractility by phospholemman: Na+/Ca2+ exchange versus Na+ -K+ -ATPase. 1870 46


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