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)

1. Calcium-stimulated ATPase activity was studied in a plasma membrane rich fraction of rat pancreas homogenate. 2. The enzyme is stimulated to the same maximum rate of ATP hydrolysis by either calcium or magnesium, but the apparent requirement for calcium is five-fold lower than for magnesium. 3. Maximum hydrolytic activity of the enzyme is not increased when additional magnesium is added to the optimal amount of calcium. 4. The enzyme does not require Na+ or K+ for its activation by Ca2+ and is not inhibited by ouabain or 2,4-dinitrophenol. 5. Pancreozymin, in concentrations which evoke secretion of zymogen protein, inhibits the calcium-stimulated ATPase. 6. Carbachol and dibutyryl cyclic AMP, in concentrations which increase the release of digestive proteins, do not alter the activity of the calcium-stimulated enzyme. 7. It is suggested that the plasma membrane calcium-activated enzyme, is not involved in the active calcium extrusion, previously reported to occur with use of the various pancreatic secretagogues tested.
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PMID:Calcium-activated ATPase activity in a plasma membrane-rich preparation of rat pancreas. 18 27

In the preceding paper it was shown that an isoform of serum albumin (ASA; active serum albumin) causes a rapid retraction of neurites and increases intracellular content of Ins1,4,5P3 in PC12 cells. Here we examined whether ASA's effects in nerve growth factor-differentiated PC12 cells were mediated through the Ins1,4,5P3/Ca2+ second messenger pathway by monitoring intracellular Ca2+ (Ca2+i) with Fura2. It was found that ASA caused a dose-dependent increase in Ca2+i. In Ca(2+)-free medium, the increase in Ca2+i elicited by ASA was smaller, but the rise in Ins1,4,5P3 content was not appreciably changed. The small Ca2+i increase seen in Ca(2+)-free medium was probably due to the release of Ca2+ from Ins1,4,5P3-sensitive intracellular stores. In Ca(2+)-containing medium the Ca2+ transient induced by ASA was not affected by organic Ca2+ channel blockers, but decreased when Co2+, Mn2+ or Zn2+ were present in the extracellular medium. The effect of other ligands, such as carbachol and bradykinin, whose receptors are coupled to the phosphoinositide system was also investigated. Carbachol at concentrations from 2 to 200 microM, and bradykinin at a concentration of 2 microM did not cause neurite retraction, whereas 200 microM bradykinin caused an approximately 40% decrease in neurite length. Thapsigargin, a Ca(2+)-ATPase inhibitor, caused a sustained elevation of Ca2+i and retraction of neurites, whereas depolarization of the cells by high K+ gave only a transient elevation of Ca2+i, and no neurite retraction. Therefore, a sustained elevation in Ca2+i might be a sufficient trigger to induce neurite retraction in differentiated PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The effect of active serum albumin on PC12 cells: II. Intracellular Ca2+ transients and their role in neurite retraction. 132 93

Calmodulin (CaM) mediates the Ca(2+)-dependent activation of many enzyme systems in accordance with its cellular localization. We have described previously a muscarinic receptor-mediated translocation of CaM from membranes into the cytosol of SK-N-SH human neuroblastoma cells. To explore the potential targets (CaM-binding proteins, CaMBP) for CaM upon translocation, a photoreactive CaM derivative was introduced into living SK-N-SH cells using a scrape-loading technique. Scrape-loading incorporated rhodamine isothiocyanate-labeled CaM with an efficiency of 38%. CaM-diazopyruvamide (CaM-DAP), a Ca(2+)-dependent and CaM-specific probe, was also introduced into the cells. The muscarinic agonist carbachol stimulated a translocation of CaM from membranes into cytosol in CaM-DAP-loaded SK-N-SH cells. Upon photochemical cross-linking, cross-linked adducts of CaM-CaMBP were detected by immunoblotting with anti-CaM antibody. Carbachol stimulated increased photoaffinity labeling of three proteins with relative adduct molecular masses of 70, 120, and 180 kDa. The time course of labeling for the 70- and 120-kDa adducts showed maximal increased by 15-30 min. The 180-kDa adduct displayed a slower time course of maximal labeling, with increases maintained for 2-4 h. Subtracting the molecular mass of CaM, carbachol stimulated binding to CaMBPs of 55, 105, and 163 kDa. Predominant cellular CaMBP were identified using a biotinylated CaM overlay procedure. Western blot analysis indicated the expression of specific CaM-dependent enzymes such as calcineurin, phosphodiesterase, the beta-isoform (rat brain) of CaM kinase II, and Ca(2+)-ATPase. Numerous cytoskeletal CaMBP were expressed such as microtubule-associated protein-2, spectrin, tubulin, caldesmon, adducin, and neuromodulin. Of the CaMBP expressed, phosphodiesterase, calcineurin, caldesmon, and adducin cross-linked with CaM-DAP in the loaded SK-N-SH cells. Carbachol stimulated the time-dependent CaM-DAP labeling of calcineurin and adducin. This study demonstrates the novel incorporation of a photoreactive CaM derivative into living cells, as well as muscarinic receptor-activated CaM-DAP interaction with several cellular CaMBP. We postulate that carbachol-stimulated CaM translocation in SK-N-SH cells may affect the activity of CaM-dependent enzymes and may alter aspects of cytoskeletal function.
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PMID:Carbachol stimulates binding of a photoreactive calmodulin derivative to calmodulin-binding proteins in intact SK-N-SH human neuroblastoma cells. 155 1

Regulation of cytosolic free Na (Nai) was measured in isolated rabbit gastric glands with the use of a recently developed fluorescent indicator for sodium, SBFI. Intracellular loading of the indicator was achieved by incubation with an acetoxymethyl ester of the dye. Digital imaging of fluorescence was used to monitor Nai in both acid-secreting parietal cells and enzyme-secreting chief cells within intact glands. In situ calibration of Nai with ionophores indicated that SBFI fluorescence (345/385 nm excitation ratio) could resolve 2 mM changes in Nai and was relatively insensitive to changes in K or pH. Measurements on intact glands showed that basal Nai was 8.5 +/- 2.2 mM in parietal cells and 9.2 +/- 3 mM in chief cells. Estimates of Na influx and efflux were made by measuring rates of Nai change after inactivation or reactivation of the Na/K ATPase in a rapid perfusion system. Na/K ATPase inhibition resulting from the removal of extracellular K (Ko) caused Nai to increase at 3.2 +/- 1.5 mM/min and 3.5 +/- 2.7 mM/min in parietal and chief cells, respectively. Na buffering was found to be negligible. Addition of 5 mM Ko and removal of extracellular Na (Nao) caused Nai to decrease rapidly toward 0 mM Na. By subtracting passive Na efflux under these conditions (the rate at which Nai decreased in Na-free solution containing ouabain), an activation curve (dNai/Nai) for the Na/K ATPase was calculated. The pump demonstrated the greatest sensitivity between 5 and 20 mM Nai. At 37 degrees C the pump rate was less than 3 mM/min at 5 mM Nai and 26 mM/min at 25 mM Nai, indicating that the pump has a great ability to respond to changes in Nai in this range. Carbachol, which stimulates secretion from both cell types, was found to stimulate Na influx in both cell types, but did not have detectable effects on Na efflux. dbcAMP+IBMX, potent stimulants of acid secretion, had no effect on Na metabolism.
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PMID:Fluorescence measurements of cytosolic free Na concentration, influx and efflux in gastric cells. 171 35

The tumor promoter thapsigargin releases Ca2+ from intracellular stores by specific inhibition of microsomal Ca-ATPase activity without inositol phosphate formation. Recent studies of the actions of thapsigargin support the concept that the level of Ca2+ within the inositol (1,4,5)-trisphosphate (IP3)-sensitive intracellular pool regulates the Ca2+ permeability of the plasma membrane. We examined the effects of thapsigargin on intracellular Ca2+ concentration ([Ca2+]i) in single rat parotid cells using digital fluorescence microscopy. In the absence of extracellular Ca2+ (Ca2+o), thapsigargin transiently increased [Ca2+]i. Following the thapsigargin-induced [Ca2+]i transient, carbachol in the continued absence of Ca2+o was unable to raise [Ca2+]i, indicating that thapsigargin mobilizes Ca2+ from the IP3-sensitive store. In the converse experiment, carbachol prevented a rise of [Ca2+]i by thapsigargin, suggesting that the IP3- and thapsigargin-sensitive Ca2+ pools are the same. Depletion of Ca2+ from the IP3-sensitive pool by thapsigargin enhanced plasma membrane Ca2+ permeability. Thapsigargin triggered sustained Ca2+ oscillations in Ca2(+)-containing medium which are highly reminiscent of agonist-induced oscillations in these cells. Carbachol addition rapidly raised IP3 levels during oscillations triggered by thapsigargin but did not elevate [Ca2+]i, indicating that the IP3-sensitive pool remains continuously depleted during [Ca2+]i fluctuations. The results from this study rule out the involvement of the IP3-sensitive pool in the mechanisms involved in thapsigargin-induced (and by analogy, agonist-induced) oscillations in parotid cells.
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PMID:Activation of calcium oscillations by thapsigargin in parotid acinar cells. 184 34

Na, K-ATPase and Mg-ATPase activities were measured in the synaptosomes of the temporal auditory projection area and the frontal association area. Moreover, the effects of carbacholine and serotonin on those activities were investigated. Na, K-ATPase activity in the synaptosomes of the association area was shown to be reliably higher that in the synaptosomes of the projection area (11.02 +/- 0.45 vs 8.40 +/- 0.55 microM Pi/mg of protein hr; P less than 0.05). Mg-ATPase activity was higher in the second case as compared to the first one (11.40 +/- 0.38 vs 9.04 +/- 0.35; p less than 0.05). Carbacholine and serotonin (10(-8)-10(-3) M) were found to induce equal inhibition of Na, K-ATPase activity in the synaptosomes of both cortices (1 max = 25-30%, 1C50 = 0.2-0.3 microM) which is blocked respectively with atropine (10(-6) M) and methysergide (10(-6) M) and enhanced in presence of GTP (5.10(-5) M). The enzyme activity is also inhibited by the non-hydrolysable guanine nucleotide, GTP gamma S (10(-8)-10(-4) M), in the absence of the antagonists (1 max = 35-40%, 1 C50 = 0.02 microM). In the methysergide-containing medium serotonin exerts a dose-dependent stimulatory effect on Na, K-ATPase which is more pronounced in the synaptosomes of the association area (A max = 25%, A C50 = 0.05 microM). Mg-ATPase activity of membrane preparations is liable to be stimulated by both serotonin and carbacholine, stimulation being more pronounced in the synaptosomes of the association cortex as well (A max = 35%, A C50 = 0.2-0.3 microM). This effect is insensitive either to the antagonists of the corresponding receptors or to GTP. GTP gamma S does not cause alterations in the enzymatic activity. Na, K-ATPase is suggested to be coupled to muscarine and serotonin receptors in the synaptic membranes of both projection and association cortical areas via a GTP-binding protein. At the same time, the agonists of receptors mentioned above are presumably also capable to effect Mg-ATPase activity by the receptor-independent way.
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PMID:[Effect of carbacholine and serotonin on the ATPase activity in synaptosomes of the projection and association areas of the feline cerebral cortex]. 185 44

The rabbit gastric gland model was used to study the nature of the muscarinic cholinergic and gastrin responses of parietal cells. Carbachol (100 microM) stimulation of acid secretion, as measured by the accumulation of aminopyrine, was inhibited by the M1 antagonist pirenzepine with an IC50 of 13 microM; by the M2 antagonist 11,2-(diethylamino)methyl-1-piperidinyl acetyl-5,11-dihydro-6H-pyrido 2,3-b 1,4-benzodiazepin-6-one (AF-DX 116) with an IC50 of 110 microM; and by the M3 antagonist diphenylacetoxy-4-methylpiperidinemethiodide (4-DAMP) with an IC50 of 35nM. The three antagonists displayed similar IC50 values for the inhibition of carbachol-stimulated production of 14CO2 from radiolabeled glucose, which is a measure of the turnover of the H(+)-H(+)-ATPase. Intracellular calcium levels wer measured in gastric glands loaded with FURA2. Carbachol was shown both to release calcium from an intracellular pool and to promote calcium entry across the plasma membrane. The calcium entry was inhibitable by 20 microM La3+. The relative potency of the three muscarinic antagonists for inhibition of calcium entry was essentially the same as for inhibition of acid secretion or metabolism. However, the rise in cell calcium due to release of calcium from intracellular stores was inhibited by 4-DAMP with an IC50 of 1.7 nM. Image analysis confirmed that the effect of carbachol and of the antagonists on intracellular calcium was occurring in the partial cell. In particular, the high-affinity inhibition of calcium release by 4-DAMP occurs in the parietal cell. Accordingly, it appears that the secretory receptor of the parietal cell is of the M3 type, and acid secretion depends on the entry of calcium rather than on calcium release from intracellular stores. In parallel experiments gastrin (G-17-sulfated) produced a dose-dependent increase in intracellular calcium (EC50, 0.14 +/- 0.013 microM). No stimulation of acid secretion was observed, but pepsinogen secretion was stimulated dose-dependently (EC50 = 1.17 +/- 0.21 microM).
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PMID:Second messengers in the gastric gland: a focus on calcium. 204 37

The effects of the cholinergic agonist carbachol on ouabain-sensitive K(+)-activated 4-nitrophenylphosphatase (K(+)-O2NPhPase) activity of rabbit and pig ventricular sarcolemma were examined. Carbachol (0.01-1000 microM) alone had no effect on K(+)-O2NPase. However, in the presence of GTP (100 microM) or its analog guanosine 5'-[gamma-thio]triphosphate (GTP[S], 1 microM) the agonist reduced this enzymatic activity (IC50 = 0.3 microM) by about 45% in a concentration-dependent manner. The GTP[S]-dependent effect of carbachol was blocked by 10 microM atropine, an antagonist of muscarinic acetylcholine receptor (mAcChoR). In the presence of micromolar concentrations of ATP or the GDP analog guanosine 5'-[beta-thio]diphosphate, carbachol did not change sarcolemmal K(+)-O2NPhPase activity. GTP[S] alone reduced this activity (IC50 = 2 microM) by about 40% in a concentration-dependent manner with a lag period of about 3 min. This lag disappeared in the presence of carbachol. Treatment of sarcolemmal membranes with 20 micrograms/ml pertussis toxin, which catalyzed ADP-ribosylation of the 40-41-kDa alpha-subunits of inhibitory GTP-binding protein (Gi), abolished the GTP[S]-promoted inhibitory effect of carbachol. Immunochemically, these alpha-subunits were identified as alpha 12- and alpha i3-subunits. It is suggested that the carbachol-induced inhibition of ouabain-sensitive K(+)-O2NPhPase activity of mammalian myocardial sarcolemma is a result of a negative coupling between mAcChoR and Na+/K(+)-ATPase via Gi protein.
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PMID:Involvement of pertussis-toxin-sensitive G protein in muscarinic-receptor-mediated inhibition of K(+)-activated 4-nitrophenylphosphatase activity of cardiac sarcolemma. 217 73

The effect of vasoactive intestinal peptide (VIP) and forskolin on carbachol-induced K+ release from superfused rat submandibular and parotid gland fragments was examined using a K(+)-sensitive electrode. Carbachol (0.1, 1, and 10 microM) superfused over the glandular fragments for 15 min caused a concentration-dependent, transient elevation of K+ efflux, with a peak value after approximately 5 min. The carbachol-induced release of K+ could be divided into two distinct components, one transient peak lasting 5-8 min independent of extracellular Ca2+ and a second component of K+ release dependent on Ca2+ in the perfusion medium. VIP (1 microM) lacked effect on K+ efflux on its own but increased the carbachol (1 microM)-evoked K+ release. The VIP effects on K+ efflux were mimicked by forskolin (10 microM). Omission of Ca2+ from the medium totally abolished the augmenting effect of VIP and forskolin on carbachol-evoked K+ efflux. The Ca2+ ionophore A23187 (1 or 10 microM) induced a prolonged low-rate efflux of K+, which was dependent on Ca2+ in the medium. This effect of A23187 on K+ secretion was potentiated by forskolin (10 microM). The Na(+)-K(+)-ATPase blocker ouabain did not affect K+ release on its own, a lack of effect which remained following pretreatment with forskolin. It is concluded that VIP, by increasing the intracellular levels of cAMP in the glandular cell, potentiates carbachol-evoked Ca2(+)-dependent K+ efflux. These results may help to explain the synergistic effects of the coexisting transmitters VIP and acetylcholine.
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PMID:VIP and forskolin enhance carbachol-induced K+ efflux from rat salivary gland fragments by a Ca2(+)-sensitive mechanism. 226 Jun 41

The involvement of Ca2+ and cyclic nucleotides in neurohormonal regulation of Na+-K+-ATPase (Na+-K+ pump) activity in guinea pig pancreatic acinar cells was investigated. Changes in Na+-K+ pump activity elicited by secretagogues were assessed by [3H]ouabain binding and by ouabain-sensitive 86Rb+ uptake. Carbachol (CCh) and cholecystokinin octapeptide (CCK-8) each stimulated both ouabain-sensitive 86Rb+ uptake and equilibrium binding of [3H]ouabain by approximately 60%. Secretin increased both indicators of Na+-K+ pump activity by approximately 40% as did forskolin, 8-bromo- and dibutyryl cAMP, theophylline, and isobutylmethylxanthine. Incubation of acinar cells in Ca2+-free HEPES-buffered Ringer (HR) with 0.5 mM EGTA reduced the stimulatory effects of CCh and CCK-8 by up to 90% but caused only a small reduction in the effects of secretin, forskolin, and cAMP analogues. In addition, CCh, CCK-8, secretin, and forskolin each stimulated ouabain-insensitive 86Rb+ uptake by acinar cells. The increase elicited by CCh and CCK-8 was greatly reduced in the absence of extracellular Ca2+, while that caused by the latter two agents was not substantially altered. The effects of secretagogues on free Ca2+ levels in pancreatic acinar cells also were investigated with quin-2, a fluorescent Ca2+ chelator. Basal intracellular Ca2+ concentration ([Ca2+]i) was 161 nM in resting cells and increased to 713 and 803 nM within 15 s after addition of 100 microM CCh or 10 nM CCK-8, respectively. Forskolin, secretin, and cAMP analogues had no effect on [Ca2+]i, nor did they either reduce or potentiate the rise in [Ca2+]i evoked by CCh.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Intracellular mediators of Na+-K+ pump activity in guinea pig pancreatic acinar cells. 241 68


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