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)

The effect of cAMP on active Ca2+ extrusion across the plasma membrane of intact human platelets was studied using quin2, a fluorimetric indicator of free Ca2+ in the cytoplasmic compartment ([Ca2+]cyt). Elevations of cAMP were achieved by incubation with dibutyryl-cAMP or by forskolin, which was found to selectively elevate cAMP without affecting cGMP levels. Progress curves of Ca2+ extrusion from quin2-overloaded platelets were measured. The rate vs. [Ca2+]cyt characteristic was calculated as previously described (Johansson, J.S. and Haynes, D.H. (1988) J. Membr. Biol. 104, 147-163). Forskolin, at a maximally effective concentration of 10 microM, was shown to stimulate Ca2+ extrusion by increasing by a factor of 1.6 +/- 0.5 the Vm of a saturable component, previously identified with a Ca(2+)-Mg(2+)-ATPase located in the plasma membrane. Neither the Km (80 nM) or Hill coefficient (1.7 +/- 0.3) of the Ca(2+)-ATPase was affected. Forskolin had no effect on the linear, non-saturable component of extrusion (previously identified with a Na+/Ca2+ exchanger) over the [Ca2+]cyt range examined (50-1500 nM). Dibutyryl-cAMP (Bt2-cAMP, 1 mM) stimulated the Ca(2+)-Mg(2+)-ATPase component of Ca2+ extrusion by a factor of 2.0 +/- 0.6. Separate experiments showed that 10 microM forskolin reduces the resting [Ca2+]cyt from 112 nM to 96 nM. Mathematical analysis showed that this can be accounted for by the above-mentioned increase in Vm of the pump, countered by a 37-74% increase in the rate constant for passive Ca2+ leakage across the plasma membrane. The results suggest two mechanisms by which prostacyclin-induced elevation of cAMP inhibits platelet aggregation: (a) lowering of resting [Ca2+]cyt and (b) increasing the rate of Ca2+ extrusion after the initial influx or triggered release event.
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PMID:Cyclic AMP stimulates Ca(2+)-ATPase-mediated Ca2+ extrusion from human platelets. 131 70

Sodium nitroprusside (SNP) and other agents that elevate cGMP levels are known to inhibit the aggregation of human platelets. Published data suggest that cGMP attenuation of agonist-induced Ca2+ transients is involved in this effect. The present study shows that elevation of cGMP increases the rate of the Ca2+ extrusion pump located in the plasma membrane (PM) but does not have a direct effect on the Ca2+ accumulating pump of the dense tubules (DT). The study verifies that SNP can specifically elevate the cGMP level in the platelet. The kinetics of the Ca2+ extrusion system were studied in situ in platelets overloaded with the cytoplasmic Ca2+ indicator quin2 according to a published protocol developed in this laboratory. Elevation of cGMP by means of (10 microM) SNP increased the Vm of the Ca(2+)-ATPase pump by 63%, without affecting its Km (66-80 nM) or Hill coefficient (1.6-1.8). Dibutyryl-cGMP (Bt2-cGMP), preincubated for 45 min at 1 mM, increased the Vm by a factor of 2.2 +/- 0.4. The experiments did not give any indication that SNP or Bt2-cGMP change the rate of the Na+/Ca2+ exchanger which makes a minor contribution to Ca2+ extrusion in the studied [Ca2+]cyt range. The rate constant for passive leakage of Ca2+ across the PM was increased by 32 +/- 4% by SNP and 90 +/- 34% by Bt2-cGMP. The net result is that the free Ca2+ in the cytoplasm ([Ca2+]cyt) at 'rest' is lowered from control values of 112 nM to 89 nM or 80 nM, respectively. The kinetics of Ca2+ uptake by the dense tubules were determined in situ using the fluorescence of chlorotetracycline (CTC) according to protocols developed in this laboratory. Analysis showed that SNP and Bt2-cGMP had no effect on the Vm or Km of the dense tubular pump, and did not affect the rate constant for passive leakage. The agents did decrease resting [Ca2+]dt by 25% or 30%, respectively, but this result can be explained purely in terms of the reduced [Ca2+]cyt. The effects of cGMP (vs. cAMP) on the PM and DT pumps are closely correlated with reported effects of cGMP/cAMP induced phosphorylation of a protein of the molecular weight of the PM pump and a 22 kDa activator of the DT pump. Cyclic AMP increases the rate of both the PM and the DT pumps, whereas cGMP increases the rate of the PM pump only.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Cyclic GMP increases the rate of the calcium extrusion pump in intact human platelets but has no direct effect on the dense tubular calcium accumulation system. 131 72

The inhibition of Ca(2+)-ATPase, (Na+ + K+)-ATPase and Na+/Ca2+ exchange by Cd2+ was studied in fish intestinal basolateral plasma membrane preparations. ATP driven 45Ca2+ uptake into inside-out membrane vesicles displayed a Km for Ca2+ of 88 +/- 17 nM, and was extremely sensitive to Cd2+ with an IC50 of 8.2 +/- 3.0 pM Cd2+, indicating an inhibition via the Ca2+ site. (Na+ + K+)-ATPase activity was half-maximally inhibited by micromolar amounts of Cd2+, displaying an IC50 of 2.6 +/- 0.6 microM Cd2+. Cd2+ ions apparently compete for the Mg2+ site of the (Na+ + K+)-ATPase. The Na+/Ca2+ exchanger was inhibited by Cd2+ with an IC50 of 73 +/- 11 nM. Cd2+ is a competitive inhibitor of the exchanger via an interaction with the Ca2+ site (Ki = 11 nM). Bepridil, a Na+ site specific inhibitor of Na+/Ca2+ exchange, induced an additional inhibition, but did not change the Ki of Cd2+. Also, Cd2+ is exchanged against Ca2+, albeit to a lesser extent than Ca2+. The exchanger is only partly blocked by the binding of Cd2+. In vivo cadmium that has entered the enterocyte may be shuttled across the basolateral plasma membrane by the Na+/Ca2+ exchanger. We conclude that intracellular Cd2+ ions will inhibit plasma membrane proteins predominantly via a specific interaction with divalent metal ion sites.
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PMID:Actions of cadmium on basolateral plasma membrane proteins involved in calcium uptake by fish intestine. 132 93

To study the diastolic properties of the heart includes examining active relaxation, passive ventricular stiffness and atrial contraction. (i) The main determinant of active relaxation is the adenosine triphosphate (ATP) concentration. Relaxation needs to occur so that the ATP content of the cell can be decreased by activation of the myosin ATPase, which in turn depends upon an intracellular messenger, elevation of the calcium transient. In a model of cardiac hypertrophy active relaxation is always slower. This slowing accompanies a slowing of the calcium transient, a diminution in the activity of the Na+/Ca2+ exchanger, a change in the properties of Na+, K+ ATPase and a decreased concentration of Ca2+ ATPase in the sarcoplasmic reticulum. (ii) Chamber stiffness is likely to be increased only in relation to the degree of ventricular hypertrophy. The main, if not unique, determinant of ventricular diastolic tissue stiffness is the structure and concentration of the collagen. Consequently tissue stiffness is augmented in cardiac hypertrophy in which the ventricular collagen concentration is elevated. It is important that both clinically and experimentally cases of cardiac hypertrophy, even those resulting from pressure overload in which myocardial stiffness and cardiac collagen concentration remain unchanged, have been documented. A good example of this is the DOCA-salt model of arterial hypertension. (iii) Atrial contraction is normally more rapid than ventricular contraction, the biological basis for which is the difference in isomyosin content.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Biological basis of diastolic dysfunction of the hypertensive heart. 139 55

This communication reports the kinetics of the Na+/Ca2+ exchanger and of the plasma membrane (PM) Ca2+ pump of the intact human platelet. The kinetic properties of these two systems were deduced by studying the rate of Ca2+ extrusion and its Na+ dependence for concentrations of cytoplasmic free Ca2+ ([Ca2+]cyt) in the 1-10-microM range. The PM Ca(2+)-ATPase was previously characterized (Johansson, J.S. Haynes, D.H. 1988. J. Membrane Biol. 104:147-163) for [Ca2+]cyt < or = 1.5 microM with the fluorescent Ca2+ indicator quin2 (Kd = 115 nM). That study determined that the PM Ca2+ pump in the basal state has a Vmax = 0.098 mM/min, a Km = 80 nM and a Hill coefficient = 1.7. The present study extends the measurable range of [Ca2+]cyt with the intracellular Ca2+ probe, rhod2 (Kd = 500 nM), which has almost a fivefold lower affinity for Ca2+. An Appendix also describes the Mg2+ and pH dependence of the Kd and fluorescence characteristics of the commercially available dye, which is a mixture of two molecules. Rates of active Ca2+ extrusion were determined by two independent methods which gave good agreement: (i) by measuring Ca2+ extrusion into a Ca(2+)-free medium (above citation) or (ii) by the newly developed "ionomycin short-circuit" method, which determines the ionomycin concentration necessary to short circuit the PM Ca2+ extrusion systems. Absolute rates of extrusion were determined by knowledge of how many Ca2+ ions are moved by ionomycin per minute. The major findings are as follows: (i) The exchanger is saturable with respect to Ca2+ with a Km = 0.97 +/- 0.31 microM and Vmax = 1.0 +/- 0.6 mM/min. (ii) At high [Ca2+]cyt, the exchanger works at a rate 10 times as large as the basal Vmax of the PM Ca2+ extrusion pump. (iii) The exchanger can work in reverse after Na+ loading of the cytoplasm by monensin. (iv) The PM Ca2+ extrusion pump is activated by exposure to [Ca2+]cyt > or = 1.5 microM for 20-50 sec. Activation raises the pump Vmax to 1.6 +/- 0.6 mM/min and the Km to 0.55 +/- 0.24 microM. (v) The Ca2+ buffering capacity of the cytoplasm is 3.6 mM in the 0.1 to 3 microM range of [Ca2+]cyt. In summary, the results show that the human platelet can extrude Ca2+ very rapidly at high [Ca2+]cyt. Both the Na+/Ca2+ exchanger and Ca2+ pump activation may prevent inappropriate platelet activation by marginal stimuli.
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PMID:Rapid Ca2+ extrusion via the Na+/Ca2+ exchanger of the human platelet. 146 6

In this study we investigated the release of Ca2+ in brain microsomes after Ca2+ loading by the Ca(2+)-ATPase or by the Na+/Ca2+ exchanger. The results show that in microsomes loaded with Ca2+ by the Ca(2+)-ATPase, Ins(1,4,5)P3 (5 microM) released 21 +/- 2% of the total Ca2+ accumulated, and that in the microsomes loaded with Ca2+ by the Na+/Ca2+ exchanger, Ins(1,4,5)P3 released 28 +/- 3% of the total Ca2+ accumulated. These results suggest that receptors of Ins(1,4,5)P3 may be co-localized with the Na+/Ca2+ exchanger in the endoplasmic reticulum membrane or that there are Ins(1,4,5)P3 receptors in the plasma membrane where the Na+/Ca2+ exchanger is normally present, or both. We also found that Ins(1,4,5)P3 inhibited the Ca(2+)-ATPase by 33.7%, but that it had no significant effect on the Na+/Ca2+ exchanger.
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PMID:Ins(1,4,5)P3 induces Ca2+ release from brain microsomes loaded either by the Ca2+ ATPase or by the Na+/Ca2+ exchanger. 148 60

We examined the effect of cGMP on Na+/Ca2+ exchange in rat aortic smooth muscle cells (VSMCs) in primary culture. The intracellular Ca2+ concentration [( Ca2+]i) was raised by adding ionomycin to VSMCs incubated at high extracellular pH (pH0) (pH0 = 8.8) and high extracellular Mg2+ (Mg2+0) (Mg2+0 = 20 mM), conditions that inhibit activity of the sarcolemmal Ca2+ pump. 45Ca2+ efflux observed under these conditions was mostly extracellular Na+ (Na+0)-dependent and thus presumably catalyzed by the Na+/Ca2+ exchanger. Brief treatment of VSMCs with 8-bromo-cGMP or atrial natriuretic peptide increased this Na+0-dependent 45Ca2+ efflux by about 50%. The 8-bromo-cGMP treatment did not significantly influence total cell Na+, membrane potential, and cell pH. Conversely, when VSMCs were loaded with Na+ and then exposed to a Na+0-free medium, the rate of 45Ca2+ uptake into VSMCs increased as cell Na+ increased. Prior treatment of VSMCs with 8-bromo-cGMP accelerated 45Ca2+ uptake by up to 60% without influencing Na+ loading itself. Treatment of VSMCs with 25 microM 2,5-di-(tert-butyl)-1,4-benzohydroquinone, an inhibitor of the sarcoplasmic reticulum Ca(2+)-ATPase, induced a transient elevation of [Ca2+]i. 8-Bromo-cGMP stimulated the rate of recovery phase of this Ca2+ transient measured in the high pHo/high Mg2+o medium. All these results indicate that cGMP stimulates Na+/Ca2+ exchange in VSMCs.
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PMID:Cyclic GMP stimulates Na+/Ca2+ exchange in vascular smooth muscle cells in primary culture. 164 93

The acute administration of thiazides results in a decrease in the urinary Ca2+/Na+ ratio, whereas chronic administration of these diuretics decreases calciuria. In both situations, Ca2+ transport is enhanced in the early part of the distal tubule. The purpose of our study was to determine whether the hypocalciuric action of thiazides was due to a change in the active transport of Ca2+ through the basolateral membrane of the nephron or to an effect (direct or indirect) on the permeability of the distal tubule luminal membrane to calcium. In order to detect intrinsic differences between membranes of the proximal and distal tubules, the effect of the diuretic was examined in proximal and distal tubule preparations, and in basolateral and luminal membranes from the two segments separately. Preincubation of microdissected distal tubules in hypotonic solution containing 500 microM hydrochlorothiazide (HCTZ) did not influence the Ca2(+)-dependent ATP hydrolysis (Ca2+ = 1 microM) nor the Mg2(+)-dependent ATP hydrolysis (Mg2+ = 100 microM). Similarly 100 microM HCTZ did not change the Ca2+ ATPase activity in intact proximal and distal tubule suspensions, at Ca2+ concentrations ranging from 0.05 microM to 1 microM. ATP-dependent Ca2+ transport was present in basolateral membrane vesicles from proximal and distal tubule suspensions. Preincubation of the membranes with 100 microM HCTZ did not influence this transport. A Na+/Ca2+ exchanger, present in the basolateral membranes from the distal tubule, was also insensitive to HCTZ. In contrast, preincubation of luminal membranes from the distal tubules (but not proximal tubules) with 500 microM HCTZ significantly increased the Ca2+ uptake by these membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The hypocalciuric effect of thiazides: subcellular localization of the action. 201 69

Measurements of unidirectional calcium fluxes in stripped intestinal epithelium of the tilapia, Oreochromis mossambicus, in the presence of ouabain or in the absence of sodium indicated that calcium absorption via the fish intestine is sodium dependent. Active Ca2+ transport mechanisms in the enterocyte plasma membrane were analyzed. The maximum capacity of the ATP-dependent Ca2+ pump (Vm: 0.63 nmol.min-1.mg-1, Km:27 nM Ca2+) is calculated to be 2.17 nmol.min-1.mg-1, correcting for 29% inside-out oriented vesicles in the membrane preparation. The maximum capacity of the Na+/Ca2+ exchanger with high affinity for Ca2+ (Vm:7.2 nmol.min-1.mg-1, Km:181 nM Ca2+) is calculated to be 13.6 nmol.min-1.mg-1, correcting for 53% resealed vesicles and assuming symmetrical behavior of the Na+/Ca2+ exchanger. The high affinity for Ca2+ and the sixfold higher capacity of the exchanger compared to the ATPase suggest strongly that the Na+/Ca2+ exchanger will contribute substantially to Ca2+ extrusion in the fish enterocyte. Further evidence for an important contribution of Na+/Ca2+ exchange to Ca2+ extrusion was obtained from studies in which the simultaneous operation of ATP- and Na(+)-gradient-driven Ca2+ pumps in inside-out vesicles was evaluated. The fish enterocyte appears to present a model for a Ca2+ transporting cell, in which Na+/Ca2+ exchange activity with high affinity for Ca2+ extrudes Ca2+ from the cell.
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PMID:Calcium absorption by fish intestine: the involvement of ATP- and sodium-dependent calcium extrusion mechanisms. 213 39

Stimulation of macrophages with platelet-activating factor (PAF) elicits an increase of intracellular calcium concentration, Ca2+i, which was monitored here at the single cell level with the calcium-sensitive dye Fura-2. The sustained component of this Ca2+i increase reflects the dynamic balance achieved between enhanced Ca2+ influx and efflux. In macrophages where a steady increase of Ca2+i has been evoked by 50 nM thapsigargin (a molecule known to empty Ca2+ stores and elevate Ca2+i in various cell types), PAF activates Ca2+ efflux, without causing a preceding increase in Ca2+i. This result shows that in this case, Ca2+ extrusion is not merely a consequence of a Ca2+i increase. PAF-evoked Ca2+ extrusion does not result from the activation of the Na+/Ca2+ exchanger. Exogenous arachidonic acid (10-100 microM) elicits Ca2+ efflux in macrophages where Ca2+i has been previously elevated by either PAF or thapsigargin. PAF-induced Ca2+ extrusion is blocked by 4-bromophenacylbromide, an inhibitor of arachidonic acid production by phospholipase A2. Together, these results suggest that arachidonic acid, which is produced in PAF-stimulated macrophages, contributes to the regulation of a Ca2+ extrusion system, which is presumably a Ca2(+)-ATPase.
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PMID:Arachidonic acid activates Ca2+ extrusion in macrophages. 214 78


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