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)

Immune dysfunction has been reported in hypertensive rats, and circulating levels of ouabain are increased in some experimental models of hypertension. Ouabain is an inhibitor of the Na+/K+-ATPase capable of diverse effects on cells of the immune system, but its mode of action on these cells is still unknown. The levels of cytoplasmic calcium ions play an important role in cell signaling, and ouabain may induce an increase in intracellular calcium indirectly through the Na+/Ca2+ exchanger. The current work examined the possibility that this drug could be exerting its effects on thymocytes through calcium mobilization and an increase in the cytosolic calcium concentration. Intracellular calcium was evaluated by using Balb-c mouse thymocytes loaded with FURA-2. Both intracellular and extracellular calcium pools were mobilized by ouabain (3 to 1000 nmol). The influx of extracellular calcium depended on the Na+/Ca2+ exchanger and on voltage-dependent calcium channels, as it was inhibited by amiloride and benzamil, consistent with the inhibition of the Na+/K+ pump. In addition, the increase of calcium from intracellular stores was extremely rapid. Furthermore, an increase in cytosolic calcium levels was obtained with the combination of ouabain and thapsigargin, which was greater than that seen with either drug alone. Our data suggest that low concentrations of ouabain may be acting on thymocytes through a mechanism different from the traditional inhibition of the Na+/K+-ATPase, as the cytosolic calcium rise was partly dependent on the release from intracellular stores.
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PMID:Ca2+ mobilization induced by ouabain in thymocytes involves intracellular and extracellular Ca2+ pools. 1273 88

The aim of this study was to determine whether changes in protein content and/or gene expression of Na+-K+-ATPase subunits underlie its decreased enzyme activity during ischemia and reperfusion. We measured protein and mRNA subunit levels in isolated rat hearts subjected to 30 min of ischemia and 30 min of reperfusion (I/R). The effect of ischemic preconditioning (IP), induced by three cycles of ischemia and reperfusion (10 min each), was also assessed on the molecular changes in Na+-K+-ATPase subunit composition due to I/R. I/R reduced the protein levels of the alpha2-, alpha3-, beta1-, and beta2-isoforms by 71%, 85%, 27%, and 65%, respectively, whereas the alpha1-isoform was decreased by <15%. A similar reduction in mRNA levels also occurred for the isoforms of Na+-K+-ATPase. IP attenuated the reduction in protein levels of Na+-K+-ATPase alpha2-, alpha3-, and beta2-isoforms induced by I/R, without affecting the alpha1- and beta1-isoforms. Furthermore, IP prevented the reduction in mRNA levels of Na+-K+-ATPase alpha2-, alpha3-, and beta1-isoforms following I/R. Similar alterations in protein contents and mRNA levels for the Na+/Ca2+ exchanger were seen due to I/R as well as IP. These findings indicate that remodeling of Na+-K+-ATPase may occur because of I/R injury, and this may partly explain the reduction in enzyme activity in ischemic heart disease. Furthermore, IP may produce beneficial effects by attenuating the remodeling of Na+-K+-ATPase and changes in Na+/Ca2+ exchanger in hearts after I/R.
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PMID:Preconditioning attenuates ischemia-reperfusion-induced remodeling of Na+-K+-ATPase in hearts. 1276 51

To clarify the correlation between intracellular Ca2+ dynamics and level of Ca2+-regulatory proteins, changes in Ca2+ handling and these proteins were investigated in a whole-heart experimental model of pressure-overload hypertrophy. We used 17-18-week-old male Dahl salt-sensitive rats (DS) and Dahl salt-resistant rats (DR) fed a high-salt diet. We monitored the fura-2 fluorescence ratio, an index of cytoplasmic Ca2+ concentration ([Ca2+]i), using a Ca2+ analyzer in a retrograde perfused heart. Left ventricular pressure (LVP) and an electrocardiogram were simultaneously recorded. Ca2+ handling was assessed by exposing the hearts to 2 min of low-Na+ (70 mmol/l) perfusion to produce an increase in [Ca2+]i (n = 6), which was sensitive to Ni2+, a blocker of the Na+/Ca2+ exchanger (NCX). In another series, the hearts were stimulated at 2.5 to 5 Hz to determine the Ca2+-force-frequency relationship (n = 6). DS rats showed marked cardiac hypertrophy without any signs of failure. The time-to-peak Ca2+ transient was prolonged in DS compared with that in DR during normal beating. During low-Na+ exposure, the time-to-peak diastolic [Ca2+]i (TTP) and the decay-time from peak [Ca2+]i (DT) were prolonged in DS compared with DR (TTP, 43.3 +/- 4.0 vs. 32.5 +/- 2.5 s, p < 0.05; DT, 70.0 +/- 8.8 vs. 29.2 +/- 2.7 s, p < 0.005). Following pretreatment with 10 mmol/l caffeine to inhibit sarcoplasmic reticulum (SR) function, TTP and DT were still prolonged in DS compared with DR (TTP, 64.2 +/- 9.7 vs. 37.0 +/- 5.8 s, p < 0.05; DT, 55.8 +/- 12.6 vs. 26.0 +/- 5.7 s, p < 0.05). The force (LVP)-frequency relationship was initially positive in DR but was negative at all times in DS (%LVP/2.5 Hz: DS, 90.3 +/- 2.0%; DR, 112.2 +/- 4.5%; p < 0.05). Elevation of diastolic [Ca2+]i (percent increase of baseline) was greater in DS than in DR with increased stimulation (5 Hz: DS, 80.7 +/- 6.7%; DR, 52.1 +/- 5.9%; p < 0.05). In Western blot analysis, the protein level of NCX was equivalent, whereas that of SR Ca2+ ATPase (SERCA2) was significantly decreased in DS compared with DR. These results suggest that slowing of cellular Ca2+ mobilization and removal is related to impaired Ca2+ handling in late-phase cardiac hypertrophy. Both the activity of the NCX and that of the SR may be affected. The SR dysfunction may be associated with change in protein level of SERCA2.
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PMID:Impaired Ca2+ handling in perfused hypertrophic hearts from Dahl salt-sensitive rats. 1456 4

Smooth muscle cell calcium dynamics and diameter were measured in intact pressurized rat mesenteric artery segments during vasoconstriction and vasomotion. Arteries showed a certain norepinephrine (NE) threshold (0.3-0.4 microM) for the onset of vasomotion, during a cumulative NE concentration-response curve. This was due to a necessary [Ca2+]i threshold (increase over basal level of 22.2 +/- 2.6%) to elicit oscillations. The calcium oscillations obtained were synchronous over the entire vessel length and phase-shifted (in advance by 1.7 +/- 0.3 seconds) with respect to the diameter oscillations. A similar result was obtained using a KCl depolarization to contract the arteries, even though the [Ca2+]i threshold was much smaller in this case (increase over basal level of 9.9 +/- 4.3%), as compared with the NE-elicited vasomotion. Blockade of the Na+/K+-ATPase with 1 microM ouabain, or of the Na+/Ca2+ exchanger (NCX) with 1 microM KB-R 7943, did not abolish the calcium oscillations, thus showing that these two pumps are only modulatory elements, while on the other hand, voltage-gated calcium channels have been found to be important in the vasomotion mechanism.
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PMID:Calcium dynamics and vasomotion in rat mesenteric arteries. 1508 65

Mouse embryonic stem (mES) cells have the potential to differentiate into all types of cells, but the physiological properties of undifferentiated mES cells, including Ca2+ signaling systems, are not fully understood. In this study, we investigated Ca2+ signaling pathways in mES cells by using confocal Ca2+ imaging systems, patch clamp techniques and RT-PCR. The stimulations with ATP and histamine (His) induced a transient increase of intracellular Ca2+ concentration ([Ca2+]i), which were prevented by the pretreatment of 2-amino-ethoxydiphenyl borate (2-APB), a blocker for inositol-1,4,5-triphosphate receptors (InsP3Rs). The application of caffeine (Caff) or ryanodine (Ry) did not change [Ca2+]i. When stores were depleted with Ca2+ -ATPase blocker, thapsigargin (TG), or histamine, the capacitative Ca2+ entry (CCE) was observed. In whole cell patch clamp mode, store-operated Ca2+ currents could be recorded in cells treated with histamine and thapsigargin. On the other hand, voltage-operated Ca2+ channels (VOCCs) could not be elicited. The application of blockers for plasma membrane Ca2+ pump (PMCAs) (carboxeosin or caloxin2A1) induced a large increase of [Ca2+]i. When the Na+/Ca2+ exchangers (NCXs) were blocked by Na+ free solution or KBR7943, [Ca2+]i was also elevated. Using RT-PCR, mRNAs for InsP3Rs type-1, -2, and -3, PMCA-1 and -4, NCX-1, -2, and -3 could be detected. From these results, we conclude that Ca2+ release from ER is mediated by InsP3Rs in mES cells before differentiation and Ca2+ entry through plasma membrane is mainly mediated by the store-operated Ca2+ channels (SOCs). For the Ca2+ extrusion systems, both NCXs and PMCAs play important roles for maintaining the low level of [Ca2+]i.
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PMID:Functional expression of Ca2+ signaling pathways in mouse embryonic stem cells. 1519 61

This study uses genetically altered mice to examine the contribution of the Na(+)-K(+)-ATPase alpha2 catalytic subunit to resting potential, excitability, and contractility of the perinatal diaphragm. The alpha2 protein is reduced by 38% in alpha2-heterozygous and absent in alpha2-knockout mice, and alpha1-isoform is upregulated 1.9-fold in alpha2-knockout. Resting potentials are depolarized by 0.8-4.0 mV in heterozygous and knockout mice. Action potential threshold, overshoot, and duration are normal. Spontaneous firing, a developmental function, is impaired in knockout diaphragm, but this does not compromise its ability to fire evoked action potential trains, the dominant mode of activation near birth. Maximum tetanic force, rate of activation, force-frequency and force-voltage relationships, and onset and magnitude of fatigue are not changed. The major phenotypic consequence of reduced alpha2 content is that relaxation from contraction is 1.7-fold faster. This finding reveals a distinct cellular role of the alpha2-isoform at a step after membrane excitation, which cannot be restored simply by increasing alpha1 content. Na+/Ca2+ exchanger expression decreases in parallel with alpha2-isoform, suggesting that Ca2+ extrusion is affected by the altered alpha2 genotype. There are no major compensatory changes in expression of sarcoplasmic reticulum Ca(2+)-ATPase, phospholamban, or plasma membrane Ca(2+)-ATPase. These results demonstrate that the Na(+)-K(+)-ATPase alpha1-isoform alone is able to maintain equilibrium K+ and Na+ gradients and to substitute for alpha2-isoform in most cellular functions related to excitability and force. They further indicate that the alpha2-isoform contributes significantly less at rest than expected from its proportional content but can modulate contractility during muscle contraction.
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PMID:The Na(+)-K(+)-ATPase alpha2-subunit isoform modulates contractility in the perinatal mouse diaphragm. 1525 93

Epidemiological data document that regular exercise protects against the morbidity and mortality associated with ischemic heart disease. Therefore, we tested the hypothesis that daily exercise (DE) increases the ventricular arrhythmia threshold (VAT) induced by coronary artery occlusion and alters the expression of calcium regulatory proteins. The VAT was defined as the time from coronary occlusion to sustained ventricular tachycardia resulting in a reduction in arterial pressure. To test this hypothesis, we recorded the VAT in conscious sedentary normotensive, sedentary hypertensive, and DE hypertensive rats, and we associated these thresholds with the protein expression of the L-type calcium channel, Na+/Ca2+ exchanger, phospholamban, and sarco(endo)plasmic reticulum Ca(2+)-ATPase. Results document a significantly reduced time to ventricular arrhythmias (sedentary hypertensive, 3.7 +/- 0.3 min vs. sedentary normotensive, 4.8 +/- 0.3 min), an increased Na+/Ca2+ exchanger protein expression (47%), and a decreased phospholamban protein expression (-34%) in conscious hypertensive rats. DE increased the VAT (5.9 +/- 0.2 min), decreased the protein expression of the Na+/Ca2+ exchanger, and normalized the protein expression of phospholamban in the hypertensive rats. Thus DE may be a primary prevention approach for reducing the incidence of arrhythmias by altering calcium regulatory proteins in hypertensive rats.
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PMID:Daily exercise-induced cardioprotection is associated with changes in calcium regulatory proteins in hypertensive rats. 1547 72

Transient elevations of cytosolic Ca2+ are a common mechanism of cellular signaling. In striated muscle, the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) plays an important role in terminating Ca2+ transients by returning cytosolic Ca2+ to intracellular stores. Stored Ca2+ can then be released again for subsequent signaling. We down-regulated SERCA2 gene expression in cultured cardiac myocytes by means of endogenous transcription of small interfering RNA encoded by an exogenous cDNA template. The cDNA template was delivered by adenovirus vector. Reduction of SERCA expression in all myocytes in culture was documented by immunochemistry, real-time RT-PCR, and determination of ATP-dependent Ca2+ transport. The reduction of SERCA2 expression was associated with the up-regulation of transient receptor potential (TRP) channel proteins (TRPC4 and TRPC5) and Na+/Ca2+ exchanger, indicating that intracellular store deficiency was compensated for by Ca2+ fluxes through the plasma membrane. In fact, SERCA silencing was followed by increased transcription of Na+/Ca2+ exchanger, TRPC4, TRPC5, and related transcriptional factors, such as stimulating protein 1, myocyte enhancer factor 2, and nuclear factor of activated cells 4, through activation of calcineurin. This finding demonstrates that the observed compensation occurs through transcriptional crosstalk and the remodeling of Ca2+ signaling pathways. The wide significance of this regulatory mechanism is related to its general involvement in Ca2+ signaling dynamics and in cardiac development and hypertrophy.
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PMID:Sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) gene silencing and remodeling of the Ca2+ signaling mechanism in cardiac myocytes. 1554 97

Cardiomyocytes derived from mouse embryonic stem (mES) cells have been demonstrated to exhibit a time-dependent expression of ion channels and signal transduction pathways in electrophysiological studies. However, ion transporters, such as Na+/K+ ATPase (Na+ pump) or Na+/Ca2+ exchanger, which play crucial roles for cardiac function, have not been well studied in this system. In this study, we investigated the functional expression of Na+/K+ ATPase and Na+/Ca2+ exchanger in mES cells during in vitro differentiation into cardiomyocytes, as well as the functional coupling between the two transporters. By measuring [Na+]i and Na+ pump current (Ip), it was shown that an ouabain-high sensitive Na+/K+ ATPase was expressed functionally in undifferentiated mES cells and these activities increased during a time course of differentiation. Using RT-PCR, the expression of mRNA for alpha1-subunit and alpha3-subunit of the Na+/K+ ATPase could be detected in both undifferentiated mES cells and derived cardiomyocytes. In contrast alpha2-subunit mRNA could be detected only in derived cardiomyocytes but not in undifferentiated mES cells. mRNA for the Na+/Ca2+ exchanger 1 isoform (NCX1) could be detected in undifferentiated mES cells and its expression levels seemed to gradually increase throughout the differentiation accompanied by increasing its Ca2+ extrusion function. At the middle stages of differentiation (after 10-day induction), more than 75% derived cardiomyocytes exhibited [Ca2+]i oscillations by blocking of Na+/K+ ATPase, suggesting the functional coupling with Na+/Ca2+ exchanger. From these results and RT-PCR analysis, we conclude that alpha2-subunit Na+/K+ ATPase mainly contributes to establish the functional coupling with NCX1 at the middle stages of differentiation of cardiomyocytes.
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PMID:Na+/K+ ATPase and its functional coupling with Na+/Ca2+ exchanger in mouse embryonic stem cells during differentiation into cardiomyocytes. 1558 94

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


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