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)

Isolated subplasmalemmal Ca2+ stores ('alveolar sacs') from Paramecium tetraurelia cells sequester 45Ca2+ depending on ATP concentration. 45Ca2+ uptake is sensitive to SERCA-type Ca(2+)-ATPase inhibitors. They cause a slow release of 45Ca2+, as does caffeine. Of some importance are also the negative results we obtained with ryanodine, inositol 1,4,5-trisphosphate (InsP3), cyclic adenosinediphosphoribose (cADPR), 3',5'-cyclic guanosine monophosphate (cGMP, +/- beta-nicotinamide-adenine dinucleotide) or with increased [Ca2+]. These data were corroborated by experiments in vivo, including microinjection studies. Again ryanodine, InsP3, cADPR or cGMP did not trigger exocytosis, the trigger effect of SERCA inhibitors was sluggish, whereas caffeine induced exocytosis in a dose-dependent fashion. We then tested 45Ca2+ release also with isolated cell cortices (cell fragments containing cell membranes with stores and secretory organelles still attached). Under conditions which initiate exocytosis in vitro (depending on [ATP], reduction of [Mg2+] in presence of Ca2+; c.f. Lumpert et al. 1990, Biochem. J. 269, 639) we observed significant 45Ca2+ release with cortices as with isolated alveolar sacs. Our interpretation is as follows. (a) Alveolar sacs have a SERCA-type Ca(2+)-pump. (b) They have some sensitivity to caffeine, but none to ryanodine, InsP3 or cADPR. (c) There might be a direct functional coupling of these subplasmalemmal Ca2+ stores to the plasmalemma to which they are connected via feet-like structures; also like the SR, activation of this store is modulated by Mg2+ and ATP.
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PMID:Subplasmalemmal Ca2+ stores of probable relevance for exocytosis in Paramecium. Alveolar sacs share some but not all characteristics with sarcoplasmic reticulum. 755 86

Intracellular Ca2+ pools contribute to changes in cytosolic [Ca2+] ([Ca2+]i), which play an important role in endothelial cell signaling. Recently, endothelial ryanodine-sensitive Ca2+ stores were shown to regulate agonist-sensitive intracellular Ca2+ pools. Since caffeine binds the ryanodine Ca2+ release channel on the endoplasmic reticulum in a variety of cell types, we examined the effect of caffeine on [Ca2+]i in human aortic endothelial cell monolayers loaded with the fluorescent probe indo 1. Under baseline conditions, 10 mmol/L caffeine induced a small increase in [Ca2+]i from 86 +/- 10 to 115 +/- 17 nmol/L (mean +/- SEM); this effect was similar to that of 5 mumol/L ryanodine and was unaffected by buffer Ca2+ removal. After depletion of an intracellular Ca2+ store by the irreversible endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin (1 mumol/L), ryanodine did not affect [Ca2+]i. In contrast, caffeine induced a large rapid increase in [Ca2+]i (176 +/- 19 to 338 +/- 35 nmol/L, P < .001) after thapsigargin exposure; this effect of caffeine was only observed when extracellular Ca2+ was present. A similar increase in [Ca2+]i was induced by caffeine after depletion of ryanodine- and histamine-sensitive Ca2+ stores or after pretreatment with the endoplasmic reticulum Ca(2+)-ATPase inhibitor cyclopiazonic acid (10 mumol/L). Thus, under baseline conditions the effect of caffeine on [Ca2+]i is similar to that of ryanodine and appears to be due to the release of an intracellular store. However, after depletion of an endoplasmic reticulum Ca2+ store, caffeine, but not ryanodine, stimulates Ca2+ influx, resulting in a large increase in [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Endoplasmic reticulum Ca2+ depletion unmasks a caffeine-induced Ca2+ influx in human aortic endothelial cells. 755 46

The mechanism of the potentiating effect of vasoactive intestinal peptide (VIP) on cholecystokinin (CCK-8)-induced amylase release was studied in isolated and perifused pancreatic acini of the guinea pig. VIP (30 pM-10 nM) potentiated CCK-8 (100 pM)-induced amylase release. Unexpectedly, VIP inhibited CCK-8-induced intracellular Ca2+ oscillations. Forskolin (10 microM), an activator of adenylate cyclase, potentiated CCK-8 (100 pM)-induced amylase release with a time course similar to that observed with VIP. Caffeine (20 mM) inhibited both amylase release and Ca2+ oscillations in response to CCK-8, suggesting that inhibition of Ca2+ oscillations does not necessarily lead to a potentiation of amylase release. When intracellular Ca2+ concentration ([Ca2+]c) was raised by thapsigargin (10 microM), a selective inhibitor of Ca(2+)-ATPase in the endoplasmic reticulum (ER), VIP (10 nM) induced significantly greater amylase release than that induced by VIP alone. When [Ca2+]c was lowered by preincubation with BAPTA-AM (25 microM), a cell-permeant Ca2+ chelator, VIP-induced amylase release was completely abolished. These results suggest that VIP, in spite of its inhibitory action on Ca2+ oscillations, facilitates a Ca(2+)-dependent process distal to the increase in [Ca2+]c to potentiate CCK-8-induced amylase release.
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PMID:Potentiation of cholecystokinin-induced amylase release by peptide VIP in guinea pig pancreatic acini. 756 61

We investigated the effect of selective blocker of Ca(2+)-ATPase of sarcoplasmic reticulum (SR), thapsigargin (TG), on the responses of single myocytes of guinea-pig heart to high [Ca2+]o and noradrenaline (NA). The cells were loaded with acetomethylester of Indo 1 and free [Ca2+]i monitored as the ratio of fluorescence at 405 and 495 nm. The changes in cell length were monitored by a TV displacement system. In the normal cells increase in [Ca2+]o from 2.0 to 5.0 mM increased amplitude of contraction by 154 +/- 8%, decreased the time to peak contraction from 343 +/- 25 ms to 328 +/- 20 ms and decreased the total duration of contraction from 813 +/- 81 ms to 800 +/- 47 ms (not significant). The rate of rise of rapid phase of Ca2+ transients and their amplitude was increased and decay accelerated. The 10(-9) mM NA increased the amplitude of contraction by 154 +/- 9%, decreased time to peak contraction from 343 +/- 25 ms to 273 +/- 17 ms and the total duration of contraction from 813 +/- 81 ms to 603 +/- 28 ms. The rate of rise of the rapid phase of Ca2+ transients was greatly accelerated and their amplitude was increased. The 10(-7) M(TG) blocked completely the rapid phase of Ca2+ transients, increased time to peak [Ca2+]i and delayed decay. Total amplitude of the transients was hardly affected. Accordingly, time to peak contraction was prolonged to 583 +/- 50 ms but the amplitude of contraction was only slightly decreased. Calcium stores in the SR were completely depleted as assessed by caffeine Ca2+ transients and contractures. In the cells pretreated with TG 10(-9) M NA but not 5.0 mM Ca2+ increased amplitude of the transients. Amplitude of contractions was increased by both agents more than in the control cells. The 5.0 mM Ca2+ slightly decreased time to peak contraction and NA tended to increase it. Both inotropic agents accelerated relaxation. It is concluded that amplitude of contractions may be increased by stimulation of Ca2+ influx, however, the control of kinetics of contraction results from altered handling of the influx by the functioning SR.
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PMID:Thapsigargin inhibits the effects of noradrenaline and high [Ca2+]o on kinetics but not on amplitude of contraction in the single myocytes of guinea-pig heart. 759 37

We investigated the mechanism by which caffeine influences myofilament responsiveness to Ca2+ by measuring isometric force, Ca2+ binding, and ATPase activity of dog cardiac myofilament proteins. Caffeine (20 mM) increased submaximal and depressed maximal force in skinned fiber bundles. Although the Ca2+ sensitivity of myofilament activity was increased by caffeine, there was no effect on Ca2+ binding to troponin C (TnC) in skinned fiber bundles. To determine if caffeine altered actin-myosin interaction or affected myosin directly, myofibrillar, actomyosin, and myosin ATPase activities were measured. Maximal Ca(2+)-activated myofibrillar Mg(2+)-ATPase activity was depressed by 20 mM caffeine, whereas submaximal Mg(2+)-ATPase activities were not changed. Actomyosin Mg(2+)-ATPase activity was significantly depressed by caffeine concentrations > or = 15 mM. Myosin Ca(2+)-ATPase activity was depressed by caffeine, whereas Mg(2+)-ATPase and K(EDTA)-ATPase activities were not affected. These data suggest that caffeine affects myofilament function via a mechanism that is independent of TnC-Ca2+ binding but that may involve direct effects on actin-cross-bridge interaction.
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PMID:Caffeine alters cardiac myofilament activity and regulation independently of Ca2+ binding to troponin C. 761 52

The sarcoplasmic reticulum (SR) of cardiac myocytes loses Ca during rest. In the present study, we estimated the rest-dependent unidirectional Ca efflux from the SR in intact rabbit and rat ventricular myocytes. We determined the time course of depletion of the SR Ca content (assessed as the amount of Ca released by caffeine) after inhibition of the SR Ca-ATPase by thapsigargin. Before rest intervals in Na-containing, Ca-free solution, a 3-min preperfusion with 0Na,0Ca solution was performed to deplete Nai but keep the SR Ca content constant. The decrease in Nai should stimulate Ca efflux via Na/Ca exchange when Nao is reintroduced. Thapsigargin treatment was limited to the last 2 min of preperfusion with 0Na,0Ca solution to minimize SR Ca loss before addition of Na, while attaining complete block of the SR Ca pump. Total SR Ca content was estimated from the [Ca]i transient evoked by caffeine, taking into account passive cellular Ca buffering. The time constants for SR Ca loss after thapsigargin were 385 and 355 s, whereas the pre-rest SR Ca content was estimated to be 106 and 114 microM (mumol/l nonmitochondrial cell volume) in rabbit and rat myocytes, respectively. The unidirectional Ca efflux from the SR was similar in the two cell types (rabbit: 0.27 microM s-1; rat: 0.32 microM s-1). These values are also comparable with that estimated from elementary Ca release events ("Ca sparks," 0.2-0.8 microM s-1). Thus, resting leak of Ca from SR may be primarily via occasional openings of SR Ca release channels. Finally, this flux is very slow compared with other Ca transporters in ventricular myocytes.
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PMID:Rate of diastolic Ca release from the sarcoplasmic reticulum of intact rabbit and rat ventricular myocytes. 761 43

A new 31.5-kD ouabain receptor protein (NORP), which is independent of Na(+)-K+ ATPase, was recently isolated selectively from transverse tubule membrane-junctional sarcoplasmic reticulum (TTM-JSR) complexes of cat cardiac muscle. We investigated the role of this NORP in cardiac function with special reference to the positive inotropic effect (PIE) of ouabain, preparing and using a monoclonal antibody (MoAB, immunoglobulin) raised against the receptor protein. Electrically stimulated papillary muscles were immersed in a Tyrode solution containing the anti-NORP MoAB (40 microM), of which the binding potency was high enough for immunological use, for 60 min and then washed out. Thirty minutes after removal of the MoAB, both twitch and K-contracture were still inhibited, but both resting and action potentials and caffeine-induced contracture were unchanged, indicating that NORP plays a key role in excitation (E)-contraction (C) coupling. The intracellular localization of the protein was investigated by immunohistochemical electron microscopy, and the protein was shown to be located on the TTM, the location being probably its external surface and opposite to feet which occupy the TTM-JSR gap. These results indicate that E-C coupling of cardiac muscle cells is mediated through NORP and that ouabain-PIE occurs through the influence of ouabain on NORP in the E-C coupling process.
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PMID:An immunohistochemical study of the significance of a new 31.5-kD ouabain receptor protein isolated from cat cardiac muscle. 761 87

1. The effects of depletion of intracellular Ca2+ stores on muscle tension and the intracellular Ca2+ concentration ([Ca2+])i were studied in fura-2 loaded longitudinal smooth muscle cells of the rat ileum. 2. After exposure to a Ca(2+)-free solution, application of Ca2+ caused a small contraction and a rise in [Ca2+]i, both of which were potentiated when the muscle was challenged with carbachol or caffeine before the addition of Ca2+. 3. Cyclopiazonic acid (CPA), a specific inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase, dose-dependently decreased tension development and the rises in [Ca2+]i induced by carbachol and caffeine in the Ca(2+)-free solution, but conversely increased the Ca(2+)-induced responses even in the presence of the voltage-dependent Ca2+ channel blockers, methoxyverapamil and nifedipine. 4. The contraction and rise in [Ca2+]i evoked by Ca2+ gradually declined with time after removal of CPA, while the reverse was the case for the responses to carbachol and caffeine. 5. The Ca(2+)-induced contraction and rise in [Ca2+]i in the presence of CPA were inhibited by the replacement of Na+ with K+ or Cs+, and by the addition of Cd2+, Ba2+, Ni2+ or La3+. 6. The influx of Mn2+ was much greater in extent in the presence of CPA than in its absence. 7. These results suggest that the emptying of intracellular Ca2+ stores may activate Ca2+ influx not associated with voltage-dependent Ca2+ channels in the rat ileal smooth muscle.
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PMID:Ca2+ entry activated by emptying of intracellular Ca2+ stores in ileal smooth muscle of the rat. 762 Jul 6

1. We have used both Ca(2+)-sensitive microelectrodes and fura-2 to measure the intracellular free calcium ion concentration ([Ca2+]i or its negative log, pCai) of snail neurones voltage clamped to -50 or -60 mV. Using Ca(2+)-sensitive microelectrodes, [Ca2+]i was found to be approximately 174 nM and pCai, 6.76 +/- 0.09 (mean +/- S.E.M.; n = 11); using fura-2, [Ca2+]i was approximately 40 nM and pCai, 7.44 +/- 0.06 (mean +/- S.E.M., n = 10). 2. Depolarizations (1-20 s) caused an increase in [Ca2+]i which was abolished by removal of extracellular Ca2+, indicating that the rise in [Ca2+]i was due to Ca2+ influx through voltage-activated Ca2+ channels. 3. Caffeine (10-20 mM) caused an increase in [Ca2+]i in the presence or absence of extracellular Ca2+. The effects of caffeine on [Ca2+]i could be prevented by ryanodine. 4. Thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a small increase in resting [Ca2+]i and slowed the rate of recovery from Ca2+ loads following 20 s depolarizations. 5. Neither replacement of extracellular sodium with N-methyl-D-glucamine (NMDG), nor loading the cells with intracellular sodium, had any effect on resting [Ca2+]i or the rate of recovery of [Ca2+]i following depolarizations. 6. The mitochondrial uncoupling agent carbonyl cyanide m-chlorophenylhydrazone (CCmP) caused a small gradual rise in resting [Ca2+]i. Removal of extracellular sodium during exposure to CCmP had no further effect on [Ca2+]i. 7. Intracellular orthovanadate caused an increase in resting [Ca2+]i and prevented the full recovery of [Ca2+]i following small Ca2+ loads, but removal of extracellular sodium did not cause a rise in [Ca2+]i. We conclude that there is no Na(+)-Ca2+ exchanger present in the cell body of these neurones and that [Ca2+]i is maintained by an ATP-dependent Ca2+ pump.
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PMID:Intracellular calcium and its sodium-independent regulation in voltage-clamped snail neurones. 762 74

Calcium flux is required for the mammalian sperm acrosome reaction, an exocytotic event triggered by egg binding, which results in a dramatic rise in sperm intracellular calcium. Calcium-dependent membrane fusion results in the release of enzymes that facilitate sperm penetration through the zona pellucida during fertilization. We have characterized inositol 1,4,5-trisphosphate (IP3)-gated calcium channels and upstream components of the phosphoinositide signaling system in mammalian sperm. Peptide antibodies colocalized G alpha q/11 and the beta 1 isoform of phospholipase C (PLC beta 1) to the anterior acrosomal region of mouse sperm. Western blotting using a polyclonal antibody directed against purified brain IP3 receptor (IP3R) identified a specific 260 kD band in 1% Triton X-100 extracts of rat, hamster, mouse and dog sperm. In each species, IP3R immunostaining localized to the acrosome cap. Scatchard analysis of [3H]IP3 binding to rat sperm sonicates revealed a curvilinear plot with high affinity (Kd = 26 nM, Bmax = 30 pmol/mg) and low affinity (Kd = 1.6 microM, Bmax = 550 pmol/mg) binding sites, reflecting among the highest receptor densities in mammalian tissue. Immunoelectron microscopy confirmed the acrosomal localization in rat sperm. The IP3R fractionated with acrosomes by discontinuous sucrose gradient centrifugation and was enriched in the medium of acrosome-reacted sperm. ATP-dependent 45Ca2+ loading of digitonin permeabilized rat sperm was decreased by 45% in the presence of 10 microM IP3. The IP3-mediated release of calcium was blocked by heparin. Thapsigargin, a sequiterpene lactone inhibitor of the microsomal Ca(2+)-ATPase, stimulated the acrosome reaction of mouse sperm to the same extent as the Ca2+ ionophore, A23187. The failure of caffeine and ryanodine to affect calcium accumulation suggested that thapsigargin acted through an IP3-sensitive store. The presence of G alpha q/11, PLC beta 1 and a functional IP3R in the anterior acrosomal region of mammalian sperm, as well as thapsigargin's induction of the acrosome reaction, implicate IP3-gated calcium release in the mammalian acrosome reaction.
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PMID:Inositol 1,4,5-trisphosphate receptors selectively localized to the acrosomes of mammalian sperm. 764 3


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