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)

A simple method to measure cytosolic calcium binding in intact presynaptic nerve terminals (synaptosomes) from rat brain, which is based on the simultaneous determination of [Ca2+]i and total [45Ca2+] in quin2-loaded synaptosomes undergoing a switch from high- to low-calcium containing medium, is presented. Binding to the cytosolic compartment alone was obtained following depletion of calcium storing organelles in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone/oligomycin plus caffeine. Synaptosomes, as compared to various cells types, have a high calcium binding capacity to the cytosolic compartment; maximum binding, Ca.Bmax, was 4.76 mM and calculated s0.5 was 218 nM. Calcium binding to the cytosolic compartment as a function of aging was also determined; Ca.Bmax was reduced to 1.84 mM and s0.5 increased to 492 nM in 30-month-old rats, indicating that the buffering of high calcium loads is impaired in old animals. The results obtained for binding of calcium to mitochondria and caffeine-sensitive calcium stores are consistent with an age-dependent reduction in calcium bound to mitochondria, whereas caffeine-sensitive calcium stores were unaffected. Finally, we have estimated the net rates of calcium extrusion in intact synaptosomes, and found that calcium efflux through the Na/Ca exchanger and Ca(2+)-ATPase was markedly reduced in old rats.
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PMID:Calcium binding to the cytosol and calcium extrusion mechanisms in intact synaptosomes and their alterations with aging. 153 57

The contribution of sarcoplasmic reticulum (SR) Ca2+ release to evoked tension in rat arterial rings was studied by comparing the effects of ryanodine (an SR Ca2+ channel opener) and thapsigargin and cyclopiazonic acid (CPA) (two Ca(2+)-ATPase inhibitors). Isometric tension was evoked by serotonin (5-HT), 30-50 mM external K+, and 10 mM caffeine in rings of aorta and a small (second-order) branch of the superior mesenteric artery (SMA). Resting tension was unaffected by 10 microM ryanodine or 1-5 microM thapsigargin, but 20 microM CPA raised resting tension in aortic rings and evoked spontaneous contractions in some SMA rings. Ryanodine (10 microM) or 1-5 microM thapsigargin partially depleted the SR Ca2+ stores (indicated by reduced caffeine-evoked contractions) and attenuated 5-HT- and high K(+)-evoked contractions in aortic rings but augmented 5-HT- and high K(+)-evoked contractions in SMA. Caffeine completely emptied the SR Ca2+ stores in the presence of ryanodine but not thapsigargin in both the aorta and SMA; thus, thapsigargin may selectively affect one component of a heterogeneous SR. When the aortic Ca2+ stores were empty (i.e., caffeine contractions were abolished), the 5-HT- and high K(+)-evoked contractions in the aorta were also augmented. CPA rapidly emptied the SR Ca2+ stores in both the aorta and SMA. CPA augmented the 5-HT-evoked contractions in the SMA and in five of nine aortic rings but attenuated evoked contractions in the remaining aortic rings. The attenuation or abolition of the caffeine contractions implies that ryanodine, thapsigargin, and CPA all deplete the SR Ca2+ stores. The attenuated responses to 5-HT and high K+ observed when the aortic SR Ca2+ stores were only partially depleted are consistent with the idea that evoked SR Ca2+ release is a large component of the Ca2+ transient in the aorta. The augmentation of 5-HT- and high K(+P)-evoked responses after partial (SMA) or complete (aorta) depletion of the SR Ca2+ stores suggests that evoked release of SR Ca2+ normally regulates Ca2+ entry by negative feedback and/or that the SR normally buffers the evoked rise in cytosolic Ca2+.
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PMID:Modulation of evoked contractions in rat arteries by ryanodine, thapsigargin, and cyclopiazonic acid. 153 81

The effect of thyroid hormone (L-tri-iodothyronine; T3) on the cytosolic free Ca2+ concentration ([Ca2+]i) in L6 myotubes was studied at rest and during activation to explore the possible mediating role of [Ca2+]i in the T3-induced net synthesis of fast-type sarcoplasmic reticulum (SR) Ca(2+)-ATPase. The mean [Ca2+]i at rest was approx. 115 nM in myoblasts, control myotubes and T3-treated myotubes. Therefore it is unlikely that the T3-induced elevation of Ca(2+)-ATPase levels is mediated by [Ca2+]i changes. To investigate the influence of the 4-fold higher Ca(2+)-ATPase levels in T3-treated myotubes (compared with controls) on [Ca2+]i, interventions with caffeine (10 mM) and a high extracellular K+ concentration ([K+]o) (30 mM) were applied which initially mobilize Ca2+ predominantly from the SR. The results showed a lower (caffeine) or not significantly different (high [K+]o) increase in [Ca2+]i in T3-treated myotubes compared with controls. No rise in [Ca2+]i was found in myoblasts with caffeine or high [K+]o. The role of [Ca2+]i in the regulation of Ca(2+)-ATPase levels was investigated by varying [Ca2+]i through exposure of cells to different concentrations of extracellular Ca2+ (0.2-1.8 mM) and ionomycin (0.1-0.25 microM). At subnormal [Ca2+]i (55 nM) the T3-induced net synthesis of Ca(2+)-ATPase was virtually abolished, and at supranormal [Ca2+]i (195 nM) it was greatly depressed. Intermediate stimulation of net Ca(2+)-ATPase synthesis was found at [Ca2+]i of 95 and 165 nM, with an optimum at approx. 125 nM. Similar but less pronounced effects were found for the basal Ca(2+)-ATPase levels. In contracting primary rat myotubes, Ca(2+)-ATPase levels were significantly lower than in tetrodotoxin-arrested myotubes. The same results were obtained in the presence of T3. Since the mean [Ca2+]i in contracting cells is higher than in resting cells, these data agree with those obtained in the L6 cells with ionomycin. A major conclusion of this study is the existence of a [Ca2+]i optimum, near resting levels, for the expression of the fast-type Ca(2+)-ATPase in the L6 muscle cell line.
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PMID:Ca2+ homeostasis and fast-type sarcoplasmic reticulum Ca(2+)-ATPase expression in L6 muscle cells. Role of thyroid hormone. 153 20

Using the rapid filtration technique to investigate Ca2+ movements across the sarcoplasmic reticulum (SR) membrane, we compare the initial phases of Ca2+ release and Ca2+ uptake in malignant hyperthermia susceptible (MHS) and normal (N) pig SR vesicles. Ca2+ release is measured from passively loaded SR vesicles. MHS SR vesicles present a 2-fold increase in the initial rate of calcium release induced by 0.3 microM Ca2+ (20.1 +/- 2.1 vs. 6.3 +/- 2.6 nmol mg-1 s-1). Maximal Ca2+ release is obtained with 3 microM Ca2+. At this optimal concentration, rate of Ca2+ efflux in absence of ATP is 55 and 25 nmol mg-1 s-1 for MHS and N SR, respectively. Ca(2+)-induced Ca2+ release is inhibited by Mg2+ in a dose-dependent manner for both MHS and N pig SR vesicles (K1/2 = 0.2 mM). Caffeine (5 mM) and halothane (0.01% v/v) increase the Ca2+ sensitivity of Ca(2+)-induced Ca2+ release. ATP (5 mM) strongly enhances the rate of Ca2+ efflux (to about 20-40-fold in both MHS and N pig SR vesicles). Furthermore, both types of vesicles do not differ in their high-affinity site for ryanodine (Kd = 12 nM and Bmax = 6 pmol/mg), lipid content, ATPase activity and initial rate of Ca2+ uptake (0.948 +/- 0.034 vs. 0.835 +/- 0.130 mumol mg-1 min-1 for MHS and N SR, respectively). Our results show that MH syndrome is associated to a higher rate of Ca2+ release in the earliest phase of the calcium efflux.
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PMID:Abnormal rapid Ca2+ release from sarcoplasmic reticulum of malignant hyperthermia susceptible pigs. 164 97

Release of Ca2+ from intracellular stores was studied in the parent PC12 cell line and in recently isolated clones sensitive or insensitive to caffeine. In the caffeine-sensitive cells the cytosolic free Ca2+ concentration ([Ca2+]i) responses by the xanthine drug and by stimulants of receptors coupled to inositol 1,4,5-trisphosphate (Ins-P3) generation (bradykinin, ATP) depend on separate pathways because 1) caffeine does not stimulate the hydrolysis of phosphatidylinositol 4,5-bisphosphate and 2) Ca(2+)-induced Ca2+ release, the process activated by caffeine, plays no major role in the Ins-P3-induced Ca2+ mobilization. Although distinct, these two mechanisms converge onto the same Ca2+ store. In fact 1) the [Ca2+]i responses by receptor agonists and caffeine were not additive; 2) either type of agent reduced (up to complete inhibition) the response to a subsequent administration of the same or the other agent; 3) all these responses were prevented by selective Ca2+ ATPase blockers; 4) ryanodine, which affects the intracellular Ca2+ channel sensitive to caffeine, also induced depletion of the receptor-sensitive Ca2+ pool; 5) in the 10 PC12 clones tested, sensitivity to caffeine paralleled ryanodine sensitivity. Therefore, PC12 cells, similar to some smooth muscle fibers but at variance with neurons and other secretory cells, express a single, rapidly exchanging Ca2+ store in which two distinct intracellular Ca2+ channels, i.e. the receptors for caffeine-ryanodine and Ins-P3, appear to be colocalized.
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PMID:Intracellular Ca2+ pools in PC12 cells. A unique, rapidly exchanging pool is sensitive to both inositol 1,4,5-trisphosphate and caffeine-ryanodine. 165 14

Using fluorescent Ca2+ indicator fura-2 and whole-cell patch-clamp techniques, we examined the effect of 2-nicotinamidoethyl nitrate (nicorandil) on the intracellular free Ca2+ concentration ([Ca2+]i) and electrical properties in single guinea pig ventricular myocytes. Nicorandil (10 nM approximately 1 mM) reduced the resting level [Ca2+]i monitored as fura-2 fluorescence ratio in a concentration-dependent manner. Dibutyryl guanosine 3':5'-cyclic monophosphate (cyclic GMP), a membrane permeable cyclic GMP analogue, mimicked the nicorandil action. Neither application of caffeine (10 mM) nor deprivation of extracellular Na+ ions could prevent the nicorandil action on [Ca2+]i. In contrast, the nicorandil effect was virtually blocked by sodium orthovanadate (40 microM), a Ca2+ pumping ATPase inhibitor. During electrophysiological experiments, nicorandil shortened action potential durations (205 +/- 80 ms to 153 +/- 76 ms) by increasing a glibenclamide-sensitive outward K+ conductance. However, the drug produced little hyperpolarization (approximately 2 mV) because the resting potential of ventricular myocytes was close to the K+ equilibrium potential. The involvement of voltage-dependent Ca-channel current and Na-Ca exchanger was considered to be minimal under physiological conditions. It is thus concluded that nicorandil decreases basal [Ca2+]i via cyclic GMP-mediated activation of the plasma membrane Ca2+ pump in guinea pig ventricular myocytes.
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PMID:Nicorandil reduces the basal level of cytosolic free calcium in single guinea pig ventricular myocytes. 166 5

Addition of dantrolene 8.5 x 10(-5) M caused a mono-exponential decay of the depolarization contractures caused by inhibition of the sarcolemmal Na,K-ATPase with propranolol 1 mM or by depolarization of the sarcolemma and T tubular membranes with KCl 100 mM. The half-times of the inhibitory effects were 6 s for the propranolol contracture and 11 s for the KCl contracture. The inhibition of both contractures was complete. Inhibition of the caffeine (10 mM) contracture was bi-exponential with half-times of 45 s and 9.5 min. Inhibition was incomplete; 29.6 +/- 5.0% of the contracture tension could not be inhibited. The inhibition of twitch contractions was similar to that of the caffeine contracture, with half-times of 48 s and 9.1 min, and 20.6 +/- 1.2% of the initial twitch tension could not be inhibited. The contracture tensions induced by release of Ca from the mitochondria with dicumarol, and by actin-myosin binding with the sulfhydryl inhibitor, N-ethyl-maleimide, could not be inhibited by dantrolene. The present results indicate that dantrolene inhibits depolarization signals from the sarcolemma and the T tubular membranes, in addition to inhibition of the coupling between the T tubules and the sarcoplasmic reticulum, and of the release of Ca from the sarcoplasmic reticulum. All these effects of dantrolene may contribute to its therapeutic effect in malignant hyperthermia.
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PMID:Separate sites for the dantrolene-induced inhibition of contracture of the rat diaphragm preparation due to depolarization or to caffeine. 172 87

1. A method has been developed for calculating the flux of Ca2+ across the sarcoplasmic reticulum (SR) during excitation-contraction coupling in mammalian heart cells. FSR will symbolize the net rate of movement of Ca2+, per litre of accessible cytoplasm, into or out of the sarcoplasmic reticulum. FSR has the units MS-1. 2. A theory of the cytoplasmic [Ca2+]i transient in mammalian heart cells is presented in which the [Ca2+]i transient results from the various cellular processes that tend to increase or decrease cytoplasmic [Ca2+]i. According to the theory, FSR can be calculated if all cellular processes that contribute to the [Ca2+]i transient (other than Ca2+ fluxes across the SR) are either eliminated or are known quantitatively. 3. To obtain the measurements required to apply this theory, [Ca2+]i transients and membrane currents were recorded in guinea-pig single ventricular myocytes subjected to whole-cell voltage clamp and internal perfusion. [Ca2+]i transients were recorded through the use of the Ca2+ indicator, Fura-2 (pentapotassium salt). 4. Ca2+ fluxes through the sodium-calcium exchanger were eliminated in all experiments, by perfusing the cells, internally and externally with Na(+)-free solutions. Ca2+ flux through the sarcolemmal L-type Ca2+ channel was measured as the verapamil-sensitive current. Influx of Ca2+ through all other voltage-dependent pathways was found to be negligible for the calculation of FSR over the time course of a single [Ca2+]i transient. 5. In the combined absence of Ca2+ current, Na(+)-Ca2+ exchange and fluxes across the SR (10 mM-caffeine), the net rate of removal of Ca2+ from the cytoplasm, which includes presumed contributions from sarcolemmal Ca(2+)-ATPase and mitochondrial Ca2+ transport, was found to be a negligible quantity in the calculation of FSR, over the time course of a single [Ca2+]i transient. 6. Calculation of FSR requires that the Ca(2+)-binding capacity of cytoplasm be known. [Ca2+]i transients recorded during measurable total Ca2+ influx into the cytoplasm (verapamil-sensitive current in the absence of fluxes across the SR) were compared with theoretical Ca2+ transients computed on the assumption that the entering Ca2+ could bind only to intracellular ligands (values for ligands taken from literature) and to Fura-2 (30 microM). The slope of the regression line relating calculated total change in [Ca2+]i to the measured total Ca2+ influx was 0.99, not different from the perfect theoretical slope of 1.0 (correlation coefficient, 0.81; standard deviation of slope, 0.14; n = 7).4+ the SR and FSR had a similar time course to that on depolarization. 10. The unidirectional efflux of Ca2+ from the SR, symbolized FSR, rel was calculated utilizing assumed characteristics of the Ca2+ pump of the SR. The value of FSR, rel was not affected by repolarization from voltage-clamp pulses greater than 150 ms in duration.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Flux of Ca2+ across the sarcoplasmic reticulum of guinea-pig cardiac cells during excitation-contraction coupling. 177 Apr 53

We investigated the effect of 2-methyl-1,4-naphtoquinone (Menadione) on sarcoplasmic reticulum (SR) Ca2+ content and electrically stimulated contractions (ESCs) of single isolated myocytes of guinea-pig ventricular myocardium. The contractures initiated by means of microinjections of caffeine into the close vicinity of the cell were used as an indirect index of the SR Ca2+ content. Superfusion of the cells for 45 min with Menadione resulted in gradual disappearance of contractile responses to caffeine, prolongation of time to peak amplitude of ESCs by 48 +/- 15% and complete inhibition of postrest and postextrasystolic potentiation. These results are consistent with those of Floreani and Carpenedo (7) who found that Menadione strongly inhibits the SR Ca2+ ATPase. Despite depletion of the SR Ca2+ the amplitude of ESCs did not change which suggests that contractions were initiated in the cells treated with Menadione by Ca2+ derived from the sources other than the SR.
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PMID:The effect of menadione on sarcoplasmic reticulum Ca2+ and contractions of single guinea-pig cardiomyocytes. 178 17

The microsomal Ca-ATPase inhibitor thapsigargin induces in rat salivary acinar cells [Ca2+]i oscillations which, though similar to those activated by agonists, are independent of inositol phosphates or inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular Ca2+ stores (Foskett, J. K., Roifman, C., and Wong, D. (1991) J. Biol. Chem. 266, 2778-2782). To examine whether the oscillation mechanism resides in another, thapsigargin- and IP3-insensitive intracellular store, we examined the effects of caffeine and ryanodine, known modulators of Ca2+ release from sarcoplasmic reticulum in excitable cells. Oscillations were induced by caffeine (1-20 mM) in nonoscillating thapsigargin-treated acinar cells, which required the continued presence of caffeine, whereas caffeine was without effect or reduced oscillation amplitude in oscillating cells. Ryanodine (10-50 microM) inhibited oscillations in most of the cells. These results suggest that Ca2+ oscillations in parotid acinar cells are driven by periodic Ca2+ release from an IP3-insensitive Ca2+ store with properties similar to sarcoplasmic reticulum of excitable cells.
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PMID:Free cytoplasmic Ca2+ concentration oscillations in thapsigargin-treated parotid acinar cells are caffeine- and ryanodine-sensitive. 183 May 87


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