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

---

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

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.
...
PMID:Inositol 1,4,5-trisphosphate receptors selectively localized to the acrosomes of mammalian sperm. 764 3

Inside-out vesicles were prepared from the plasma membrane of osteoblasts which had been isolated from the periosteum of 2-3-week-old chicken tibia and cultured for 6-8 days. Calcium uptake was initiated by adding 0.4 mM ATP and detected as a reduction in fluorescence from the reaction medium using the Ca(2+)-specific fluoroprobe, fluo-3. The reaction medium contained ouabain (1 mM) to block Na+, K(+)-ATPase activity and oligomycin (20 micrograms/ml) to block mitochondrial activity. Thapsigargin (5 microM) had little effect, indicating that contributions to Ca2+ uptake by endoplasmic reticulum derived microsomes were minimal. The Ca2+ uptake rate was 9.9 +/- 2.3 nmol/mg protein/min. Trifluoperazine (0.1 mM), which impairs the capacity of calmodulin to activate Ca(2+)-ATPase, substantially inhibited transport, as did quercetin (10 mM) and vanadate (10 microM), inhibitors of Ca(2+)-ATPases. This study has shown the presence of an outwardly directed, calmodulin-sensitive calcium transport system in the primary osteoblast plasma membrane. The pumping rate is substantially less than rates found in the intestine, a tissue which is involved in massive transport of Ca2+, but is similar to rates found in many other tissues. It is concluded that the enzyme does not support calcium translocation to sites of mineralization.
...
PMID:Characterization of calcium efflux by osteoblasts derived from long bone periosteum. 766 10

The synthesis of Calcium Green C18, a lipophilic fluorescent calcium-sensitive dye, and its use as a monitor of Ca2+ efflux from cells is described. This indicator consists of a Calcium Green-1 molecule conjugated to a lipophilic 18-carbon alkyl chain which will intercalate into cell membranes. The Kd of the indicator for Ca2+ in aqueous solution (pH 7.2, 22 degrees C, ionic strength 0.1 M) is 0.23 +/- 0.04 microM and in the presence of liposomes is 0.062 +/- 0.007 microM. Due to its high negativity, the calcium chelating fluorophore faces the cell exterior, when loaded under a defined set of conditions. The dye was found largely on the surface of the cells when loaded at a concentration of 5 microM for 10 min at 37 degrees C. Five minutes after introduction of EGTA, 83-95% fluorescence disappeared, indicating that most of the fluorophore was on the cell surface. Photobleaching was minimal (3-13%). A confocal laser scanning microscope was used to detect and quantify fluorescence. Internalized dye was apparent in cells loaded for longer times (30-60 min) and in membrane-impaired cells, as shown by uptake of propidium iodide. Under defined confocal laser scanning microscope settings, a transient fluorescence at the periphery of approximately 30% of the cells was observed following 10(-8) M parathyroid hormone treatment, indicating the presence of outwardly directed calcium transport across the plasma membrane. Calcium efflux usually lasted 7-10 min, peaking at around 2-3 min. Changes in cell shape were also observed. Calcium efflux was shown to be sensitive to (a) 10 microM quercetin and 10 microM vanadate, partially specific inhibitors of plasma membrane Ca(2+)-ATPase, to (b) 0.1 mM trifluoperazine, an agent which renders calmodulin ineffective, and to (c) 10 mM neomycin sulfate, which blocks release of Ca2+ from intracellular stores. Thapsigargin (5 microM), an inhibitor of Ca(2+)-ATPase of the endoplasmic reticulum, prolonged fluorescence. These observations indicate that cell surface fluorescence was due to the capture of Ca2+ by Calcium Green C18 after Ca2+ had been translocated across osteoblast plasma membranes. Involvement of the plasma membrane Ca(2+)-ATPase, known to be present in osteoblasts in substantial amounts, is implicated.
...
PMID:Characterization of calcium translocation across the plasma membrane of primary osteoblasts using a lipophilic calcium-sensitive fluorescent dye, calcium green C18. 767 32

The effect of Thapsigargin (TG) shown to block selectively the Ca(2+)-ATPase of sarcoplasmic reticulum (SR) on the SR Ca2+ content and contractions of single rat cadiomyocytes was investigated. In 80% of cells 10(-6) M TG blocked Ca2+ uptake by SR at rest, depleted the SR Ca2+, inhibited postrest potentiation and changed the pattern of response to premature contractions. Amplitude of steady state contractions was reduced to 11 +/- 4% of pre-TG control. In 20% of cells TG had similar effect on the SR function, however, amplitude of contractions was reduced to only 50 +/- 7% of control. It is concluded that the results obtained in 80% of the cells are compatible with the wide-spread notion that contractions of rat cardiac myocytes are activated predominantly by Ca2+ released from SR. However, population of myocytes of ventricular myocardium of this species is not homogeneous in this respect. In 20% of them contractions are activated by both sarcolemmal and SR Ca2+ contribution being approximately equal. These cells occupy intermediate position between guinea-pig cells and the bulk of rat cells.
...
PMID:The effect of thapsigargin on sarcoplasmic reticulum Ca2+ content and contractions of single myocytes of rat ventricular myocardium. 769 89

1. The mechanism of hydralazine-induced vasorelaxation was investigated in rabbit isolated aorta, by determining its ability to interfere with force development under a variety of conditions. 2. Hydralazine relaxed phenylephrine-contracted aorta with half maximal relaxation at 17 microM and maximal relaxation above 100 microM. At 200 microM, hydralazine had little effect on contractions induced by 25 mM or 50 mM K+. 3. Hydralazine was equally effective at inhibiting contractile responses to phenylephrine in the absence or presence of extracellular Ca2+. Responses to phenylephrine in Ca(2+)-free solution were blocked to the same degree whether hydralazine was applied during filling of the sarcoplasmic reticulum (SR) Ca2+ stores or after filling was complete. Caffeine-induced contractions were less sensitive to block by hydralazine. 4. Thapsigargin, cyclopiazonic acid, ryanodine, nifedipine and diltiazem all failed to block the inhibitory effect of hydralazine on tonic contractions to phenylephrine in the presence of extracellular Ca2+. However, when cyclopiazonic acid was applied either with diltiazem or ryanodine, substantial inhibition of the hydralazine response was observed. 5. We propose that tonic contractions to phenylephrine are largely maintained by Ca2+ cycling through the SR, with Ca2+ entering the smooth muscle cell being sequestered by the SR eventually to leak out through IP3-activated channels close to the contractile proteins. Sequestration of Ca2+ would employ two pathways, one sensitive to inhibitors of the SR Ca(2+)-ATPase and the other to Ca antagonists. We further suggest that, in the presence of extracellular Ca2+ and phenylephrine, the leakage of Ca2+through IP3-activated channels is significantly reduced only if both routes for SR Ca2+ accumulation are blocked or the Ca2+-ATPase is blocked while the SR is made leaky with ryanodine.6. We conclude that the main action of hydralazine is to block the IP3-dependent release of Ca2+ from the sarcoplasmic reticulum. Thus conditions that diminish the contribution of IP3-induced Ca2+ release to tension can inhibit the hydralazine-induced vasorelaxation.
...
PMID:Inhibition of calcium release from the sarcoplasmic reticulum of rabbit aorta by hydralazine. 771 24

The effect of thapsigargin, an inhibitor of the sarco-endoplasmic reticulum Ca(2+)-ATPase, on voltage-dependent Ca2+ channels has been investigated in the A7r5 cell line and in membrane preparations from rat aorta, heart and brain. Patch-clamp technique showed that, at micromolar concentrations, thapsigargin inhibited the L-type Ca2+ channel current in A7r5 cells. It depressed the current at all voltages without change in the steady state inactivation curve. The rates of inactivation of the Ca2+ current were highly variable among the cells suggesting that more than one component of L-type Ca2+ current coexist in A7r5 cells, differing in the kinetics of inactivation. Thapsigargin appeared to be more potent on the slower-inactivating Ca2+ current than on the faster-inactivating one. In the same range of concentrations, thapsigargin inhibited the specific binding of 3H(+)-isradipine in intact cells while 45Ca2+ uptake in intracellular stores of skinned cells was inhibited at nanomolar concentrations. The equilibrium dissociation constant of 3H(+)-isradipine was increased in the presence of thapsigargin as a result of an increase of the dissociation rate constant indicating that the inhibitory effect of the antagonist cannot be attributed to a simple competitive interaction with the dihydropyridine binding site. Maximum binding capacity was unaffected. A similar pattern of inhibition of 3H(+)-isradipine binding was observed in membrane preparations from rat aorta, heart and brain. Those results indicate that, at micromolar concentrations, thapsigargin inhibits the voltage-dependent Ca2+ current by a direct interaction with the L-type Ca2+ channels.
...
PMID:Evidence for a direct interaction of thapsigargin with voltage-dependent Ca2+ channel. 771 40

To clarify the agonist-induced Ca2+ entry mechanism, effects of thapsigargin and cyclopiazonic acid, selective inhibitors of endoplasmic reticulum Ca(2+)-ATPase, on intracellular free Ca2+ concentration ([Ca2+]i) were studied in cultured human aortic endothelial cells loaded with the fluorescent Ca2+ indicator fura-2. Thapsigargin (1-1000 nM) and cyclopiazonic acid (0.1-100 microM) produced a biphasic change in [Ca2+]i, which consisted of a transient peak elevation followed by a long-lasting decline of [Ca2+]i in a concentration-dependent manner. In the presence of thapsigargin or cyclopiazonic acid, the rapid transient elevation of [Ca2+]i elicited by histamine was attenuated in a time-dependent manner. The slow declining phase of the response to thapsigargin and cyclopiazonic acid was completely eliminated by removal of extracellular Ca2+, and it was also prevented by reduction of the extracellular Cl- concentration to 40 mM or by the Cl- channel blocker N-phenylanthranilic acid. These findings suggest that the initial transient rising phase and the slow declining phase of [Ca2+]i in response to thapsigargin and cyclopiazonic acid reflect a blockade of Ca2+ uptake into the endoplasmic reticulum and the Cl(-)-sensitive Ca2+ entry activated by the depletion of agonist-sensitive intracellular Ca2+ stores, respectively, in human aortic endothelial cells.
...
PMID:Modulation of cytosolic Ca2+ concentration by thapsigargin and cyclopiazonic acid in human aortic endothelial cells. 772 Jul 74

Intracellular free calcium ([Ca2+]i) has multiple functional roles in renal epithelia, including mediating ligand- and volume-activated K+ and Cl- channels, modulating the permeability of apical membrane to Na+, and regulating tubuloglomerular feedback. We investigated glucose effects on intracellular pH (pHi) and [Ca2+]i in Madin-Darby canine kidney (MDCK) cells using fluorescent probes, SNARF-1 and fura 2, respectively. The addition of glucose decreased both pHi and [Ca2+]i in a dose-dependent fashion. Thapsigargin (TG) and cyclopiazonic acid (CPA), well-known endoplasmic reticulum (ER) Ca(2+)-adenosinetriphosphatase (Ca(2+)-ATPase) inhibitors, abolished the glucose-induced [Ca2+]i decrease. Without glucose, 1 microM TG induced a sustained elevation in [Ca2+]i, which increased further with glucose addition, whereas 15 microM CPA induced a transient increase in [Ca2+]i that was not affected by further addition of glucose. The sustained elevation in [Ca2+]i induced by TG was dependent on extracellular Ca2+. TG-induced [Ca2+]i increase was modulated by glucose, i.e., at higher glucose concentrations, TG induced a larger and more rapid rise in [Ca2+]i. We conclude that glucose has dual effects on [Ca2+]i regulation. Glucose alone reduces [Ca2+]i by activating ER-type Ca(2+)-ATPase, since this phenomenon is TG and CPA sensitive. In the presence of TG, glucose increases [Ca2+]i probably by increasing Ca2+ entry. Our data suggest a model in which TG activates capacitative Ca2+ entry by depletion of the ER Ca2+ pool. Glucose increases TG-induced [Ca2+]i elevation by further enhancing capacitative Ca2+ entry.
...
PMID:Modulation of intracellular Ca2+ by glucose in MDCK cells: role of endoplasmic reticulum Ca(2+)-ATPase. 773 24

To investigate Ca2+/cation entry pathway in vascular endothelial cells, we examined the effects of thapsigargin on [Ca2+]i and Mn2+ entry in cultured porcine aortic endothelial cells. Thapsigargin inhibits the activity of endoplasmic reticulum (ER, intracellular Ca2+ pool) Ca(2+)-ATPase, and stimulates Ca2+ entry from extracellular space by depleting intracellular Ca2+ pool. Cultured endothelial cells were loaded with fura-2/AM, and [Ca2+]i was measured by the ratios of fluorescence at 340/380 nm excitation, and Mn2+ entry was observed by the quenching of fluorescence at 360 nm excitation. Thapsigargin elevated [Ca2+]i in a time- and dose-dependent manner. The increase in [Ca2+]i caused by thapsigargin was lowered in Ca(2+)-free solution containing 3 mM EGTA. Verapamil (10(-5) M) and equimolar replacement of extracellular NaCl by LiCl had no effects on the maximum elevation of [Ca2+]i by thapsigargin. The increase in [Ca2+]i by thapsigargin was significantly inhibited by either NiCl2 (10(-3) M) or membrane depolarization using 50 mM KCl. Thapsigargin stimulated Mn2+ entry concomitantly with the increase in [Ca2+]i. Mn2+ entry was augmented in Ca(2+)-free solution. These results suggested that (1) the increase in [Ca2+]i by thapsigargin consisted of both Ca2+ release from ER and Ca2+ entry from extracellular space, and (2) thapsigargin also stimulated Mn2+ entry, which was interfered with in the presence of extracellular Ca2+.
...
PMID:A study on thapsigargin-induced calcium ion and cation influx pathways in vascular endothelial cells. 774 81

Thapsigargin is a high affinity inhibitor of sarco- and endoplasmic reticulum (SERCA) type ATPases. We have used kinetics to determine the dissociation constant of thapsigargin-sarcoplasmic reticulum Ca(2+)-ATPase interaction in the absence and presence of non-ionic detergent. The observed "off" rate constant was measured as 0.0052 s-1 at 26 degrees C by the kinetics of inhibition of ATPase activity following transfer from an inactivated thapsigargin-ATPase complex to native ATPase. Inactive ATPase was produced by cross-linking the active site with glutaraldehyde. The observed dissociation rate constant was increased 7-fold by 0.1% Triton X-100, indicating that perturbation of the transmembrane and stalk region by detergent altered the binding parameters of the inhibitor. In addition, thapsigargin stabilized the ATPase against inactivation caused by detergent in the absence of Ca2+. The observed "on" rate constant of thapsigargin was measured at 26 degrees C as 25 s-1 irrespective of thapsigargin concentration, by the kinetics of thapsigargin- induced change in intrinsic fluorescence. An Arrhenius plot showed a temperature dependence of this rate constant, indicative of a conformational change in the protein with an activation energy of 9.5 kcal/mol for thapsigargin binding. The affinity of the Ca(2+)-ATPase for thapsigargin was calculated to be greater than 2 pM at pH 7.0 and 26 degrees C.
...
PMID:Kinetics of thapsigargin-Ca(2+)-ATPase (sarcoplasmic reticulum) interaction reveals a two-step binding mechanism and picomolar inhibition. 774 17


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---