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)

The subcellular distribution of cardiac muscarinic receptors was defined in canine ventricular myocardium, and receptors were solubilized from subcellular fractions enriched in muscarinic receptor content. The subcellular location of muscarinic receptors in cardiac tissue was determined by measurement of the distribution of [3H](+/-)quinuclidinyl benzilate-binding activity in particulate fractions isolated from canine ventricular myocardium. Based upon excellent correlation between [3H](+/-)quinuclidinyl benzilate binding and activity of the sarcolemmal Na+,K+-ATPase throughout the subcellular fractions, muscarinic receptors appeared to be localized to sarcolemma in canine ventricular myocardium. Therefore, membrane fractions enriched in sarcolemma were used as a source of cardiac muscarinic receptors for solubilization. Treatment of membrane vesicle fractions with digitonin (0.6%) resulted in solubilization of [3H](+/-)quinuclidinyl benzilate-binding activity with an extraction yield of 25-35%. Criteria of pharmacological specificity and stereospecificity established the identity of the solubilized binding activity of muscarinic receptors. Solubilization of muscarinic receptors was documented by demonstration of hydrodynamic behavior consistent with molecularly dispersed material. Upon glycerol gradient centrifugation, digitonin-solubilized muscarinic receptors from cardiac tissue sedimented with an apparent sedimentation coefficient of 9S. Pharmacological characterization of the digitonin-solubilized receptors revealed 8- to 39-fold reductions in affinities for muscarinic antagonists compared to the affinities exhibited by receptors in the membrane-bound state. Substantially greater reductions in agonist affinities (reduction of at least 700-fold for all agonists studied) suggested selective loss of ability of the digitonin-solubilized receptors to exhibit high affinity agonist interactions. In contrast to membrane-bound receptors, digitonin-solubilized receptors also demonstrated a loss of guanine nucleotide regulation, as well as steep agonist:radioligand competition curves with slope factors of 1.0, suggesting a homogeneous population of agonist-binding sites. Interpreted within the context of a model of state interconversion for membrane-bound receptors, the results suggested that either muscarinic receptors of a single state were selectively solubilized, or that solubilization induced conversion of all receptors to a single low affinity state, possibly by removal of constituents necessary for assumption of a high affinity agonist conformation.
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PMID:Enrichment, solubilization, and partial characterization of digitonin-solubilized muscarinic receptors derived from canine ventricular myocardium. 630 30

We have previously shown that inositol-1,4,5-trisphosphate (IP3) releases Ca2+ from an intracellular calcium store in permeabilized acinar cells of rat pancreas (H. Streb et al., 1983, Nature (London) 306:67-69). This observation suggests that IP3 might provide the missing link between activation of the muscarinic receptor and Ca2+ release from intracellular stores during stimulation. In order to localize the intracellular IP3-sensitive calcium pool, IP3-induced Ca2+ release was measured in isolated subcellular fractions. A total homogenate was prepared from acinar cells which had been isolated by a collagenase digestion method. Endoplasmic reticulum was separated from mitochondria, zymogen granules and nuclei by differential centrifugation. Plasma membranes and endoplasmic reticulum were separated by centrifugation on a sucrose step gradient or by precipitation with high concentrations of MgCl2. IP3-induced Ca2+ release per mg protein in the total homogenate was the same as in leaky cells and was sufficiently stable to make short separation procedures possible. In fractions obtained by either differential centrifugation at 7000 X g, sucrose-density centrifugation, or MgCl2 precipitation there was a close correlation of Ip3-induced Ca2+ release with the endoplasmic reticulum markers ribonucleic acid (r = 0.96, 1.00, 0.91, respectively) and NADPH cytochrome c reductase (r = 0.63, 0.98, 0.90, respectively). In contrast, there was a clear negative correlation with the mitochondrial markers cytochrome c oxidase (r = -0.64) and glutamate dehydrogenase (r = -0.75) and with the plasma membrane markers (Na+ + K+)-ATPase (r = -0.81) and alkaline phosphatase (r = -0.77) in all fractions analyzed. IP3-induced Ca2+ release was distributed independently of zymogen granule or nuclei content of the fractions as assessed by electron microscopy. The data suggest that inositol-1,4,5-trisphosphate releases Ca2+ from endoplasmic reticulum in pancreatic acinar cells.
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PMID:Effect of inositol-1,4,5-trisphosphate on isolated subcellular fractions of rat pancreas. 633 62

The effect of staurosporine on the Ca2+ signalling induced by the muscarinic receptor agonist carbachol (CCh) was studied in Fura-2-loaded rat parotid acinar cells. At concentrations > 1 nM, staurosporine dose-dependently enhanced the sustained increase in cytosolic free Ca2+ concentration ([Ca2+]i), but did not affect the peak [Ca2+]i seen just after stimulation. The enhancement of the sustained increase in [Ca2+]i was not attenuated by the protein kinase C activator, 4 beta-phorbol 12-myristate 13-acetate, and not mimicked by another inhibitor of protein kinase C, K-252a, suggesting that the effect of staurosporine on the CCh-induced Ca2+ signalling may be due to a mechanism independent of the inhibitory action on protein kinase C. Staurosporine also enhanced the increases in [Ca2+]i induced by the microsomal Ca(2+)-ATPase inhibitor thapsigargin (TG) and the Ca2+ ionophore ionomycin (Iono). When the cells were stimulated by CCh, TG, or Iono in the absence of extracellular Ca2+, a transient increase in [Ca2+]i due to Ca2+ release from intracellular stores was observed. This increase in [Ca2+]i was unaffected by preincubation with staurosporine. However, when Ca2+ was added to the extracellular medium after [Ca2+]i had returned to the resting level, the increase in [Ca2+]i was significantly enhanced by staurosporine. In addition, staurosporine accelerated the Mn2+ influx following the addition of CCh, TG, or Iono. These results suggest that staurosporine modulates the Ca2+ entry system activated by depletion of intracellular Ca2+ stores in rat parotid acinar cells.
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PMID:Staurosporine enhances Ca2+ entry induced by depletion of intracellular Ca2+ stores in rat parotid acinar cells. 755 79

The Drosophila proteins, Trp and Trpl, are suggested to be cation channels responsible for depolarization of the receptor potential associated with stimulation of insect photoreceptor cells by light. Consistent with this hypothesis, we recently showed that recombinant Trpl forms Ca(2+)- and Ba(2+)-permeable non-selective cation channels when expressed in Sf9 cells using the baculovirus expression vector. As Trpl may be activated in the photoreceptor cell after stimulation of phospholipase C, we hypothesized that a similar regulation of recombinant Trpl may be observed in the Sf9 cell after activation of heterologous membrane receptors linked to Ca(2+)-signal-transduction pathways. To test this hypothesis, Ca2+ signalling was examined in Fura-2-loaded Sf9 cells infected with baculovirus containing cDNA for the M5 muscarinic receptor alone (M5 cells) or in cells co-infected with both M5 and Trpl-containing baculoviruses (M5-Trpl cells). Addition of carbachol (100 microM) to M5 cells produced an increase in cytosolic free Ca2+ concentration ([Ca2+]i) (mean +/- S.D.; n = 17) from 101 +/- 20 to 762 +/- 178 nM which declined to a sustained elevated level of 384 +/- 102 nM after 3 min. The sustained component was eliminated by removal of extracellular Ca2+ or by addition of La3+ or Gd3+ (10 microM). In M5-Trpl cells, basal [Ca2+]i increased as a function of time after infection. To evaluate the contribution of Ca2+ influx to the overall profile observed, Ba2+, a Ca2+ surrogate that is not a substrate for the Ca2+ pump, was used. The increase in basal [Ca2+]i seen in M5-Trpl cells was associated with an increase in basal Ba2+ influx. Addition of carbachol to M5-Trpl cells at 30-36 h after infection produced a large increase in [Ca2+]i to a sustained value of 677 +/- 143 nM. This change in [Ca2+]i was (1) blocked by atropine, (2) attenuated in the absence of extracellular Ca2+, and (3) relatively insensitive to La3+, but blocked by Gd3+ in the 0.1-1 mM range. In the presence of 10 microM Gd3+ to block the endogenous-receptor-mediated Ca(2+)-influx in M5-Trpl cells. In sharp contrast increase in Ba2+ influx in M5-Trpl cells. In sharp contrast, neither Ca2+ nor Ba2+ influx through Trpl was affected by thapsigargin, a selective inhibitor of the endoplasmic reticulum Ca(2+)-ATPase pump.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Receptor-mediated activation of recombinant Trpl expressed in Sf9 insect cells. 783 80

The human neuroblastoma cell line SH-SY5Y, maintained at confluence for 14 days, released [3H]-noradrenaline ([3H]NA) when stimulated with either the muscarinic receptor agonist methacholine or bradykinin. The major fraction of release was rapid, occurring in < 10 s, whereas nicotine-evoked release was slower. When the extracellular [Ca2+]e) was buffered to approximately 50-100 nM, release evoked by nicotine was abolished, whereas that in response to methacholine or bradykinin was reduced by approximately 50% with EC50 values of -5.46 +/- 0.05 M and -7.46 +/- 0.06 M (log 10), respectively. Methacholine and bradykinin also produced rapid elevations of both inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and intracellular free [Ca2+] ([Ca2+]i). These elevations were reduced at low [Ca2+]e and under these conditions the EC50 values for peak elevation of [Ca2+]i were -6.00 +/- 0.14 M for methacholine and -7.95 +/- 0.34 M for bradykinin (n = 3 for all EC50 determinations). At low [Ca2+]e, depletion of nonmitochondrial intracellular Ca2+ stores with the Ca(2+)-ATPase inhibitor thapsigargin produced a transient small elevation of [Ca2+]i and a minor release of [3H]NA. At low [Ca2+]e, thapsigargin abolished elevation of [Ca2+]i in response to methacholine and bradykinin and completely inhibited their stimulation of [3H]NA release. It is proposed, therefore, that Ca2+ release from Ins (1,4,5)P3-sensitive stores is a major trigger of methacholine- and bradykinin-evoked [3H]NA release in SH-SY5Y cells.
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PMID:Mobilization of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores supports bradykinin- and muscarinic-evoked release of [3H] noradrenaline from SH-SY5Y cells. 786 Nov 49

Initial findings from our laboratory have indicated that muscarinic enhancement of K(+)-evoked release of dopamine from perifused striatal slices is reduced after exposure to 56Fe-particle irradiation. This finding suggested that there is a radiation-induced deficit in muscarinic receptor sensitivity. Subsequent findings have indicated that at least part of the loss in sensitivity may occur as a result of alterations in the initial steps of the signal transduction process and involve muscarinic receptor-G protein coupling/uncoupling. The present study was carried out to localize this deficit further by determining carbachol-stimulated low-Km guanosine triphosphatase (GTPase) activity in striatal and hippocampal tissue obtained from rats exposed to 0, 0.1 or 1.0 Gy of 56Fe-particle irradiation. In addition, to examine the specificity of the effect of 56Fe-particle irradiation, alpha 1-adrenergic-stimulated low-Km GTPase activity was also examined in these tissues. The results showed that there was a high degree of specificity in the effects of 56Fe particles. Decrements were observed in muscarinic-stimulated low-Km GTPase in striatum but not in hippocampus, and 56Fe-particle irradiation did not affect alpha 1-adrenergic low-Km GTPase activity in either brain tissue.
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PMID:Iron-56 irradiation diminishes muscarinic but not alpha 1-adrenergic-stimulated low-Km GTPase in rat brain. 797 91

The purpose of the present study was to examine Ca2+ signaling mechanisms in Sf9 cells and to demonstrate expression and functional linkage of a mammalian receptor to changes in cytosolic free Ca2+ concentration ([Ca2+]i). Addition of p-octopamine (50 microM to fura 2-loaded Sf9 cells produced a small transient increase in [Ca2+]i from a basal level of 58 +/- 10 to 194 +/- 7.6 (SD) nM. The response to octopamine was inhibited by both cyproheptadine and chlorpromazine and was mimicked by clonidine. In contrast, [Ca2+]i did not change in response to dopamine (50 microM), substance P (50 nM), histamine (50 microM), ATP (50 microM), acetylcholine (10 or 100 microM), carbachol (10 or 100 microM), serotonin (50 microM), epinephrine (10 microM), or bradykinin (50 nM). The Ca(2+)-adenosinetriphosphatase inhibitors thapsigargin (200 nM) and 2,5-di-tert-butylhydroquinone (BHQ; 10 microM) increased [Ca2+]i to 307 +/- 13 and 137 +/- 20 nM, respectively. In contrast to BHQ, the response to thapsigargin was attenuated by La3+ or removal of extracellular Ca2+ and increased by elevation of extracellular Ca2+. These results suggest that thapsigargin but not BHQ stimulates Ca2+ influx. The rat brain muscarinic receptor (subtype M5) was incorporated into the baculovirus by homologous recombination. Addition of carbachol (100 microM) increased [Ca2+]i from 92.7 +/- 6.4 to 480 +/- 26 nM in Sf9 cells infected with recombinant virus containing the M5 receptor cDNA. The effect of carbachol on [Ca2+]i was concentration dependent with a 50% effective concentration of approximately 30 microM and was blocked by atropine (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Ca2+ signaling in Sf9 insect cells and the functional expression of a rat brain M5 muscarinic receptor. 802 3

The properties of muscarinic acetylcholine receptors in the cell line MCM1, derived from an SV40 T-antigen-induced atrial tumor in a transgenic mouse, were determined. Binding studies using the nonselective muscarinic antagonist [3H]quinuclidinyl benzilate, the M1-selective antagonist pirenzepine, and the M2-selective antagonist AFDX-116 indicate that the receptors have the pharmacological properties of the cardiac (M2) receptor subtype. The receptors could be immunoprecipitated with a monoclonal antibody specific for the cardiac receptor, thus confirming the identity of the receptors expressed in these cells. The types of G proteins expressed in the cells were determined by Northern blot analyses: mRNA encoding the alpha subunits of Gs, G(o), and Gi-2, but not Gi-1 or Gi-3, were detected, consistent with previous observations of neonatal mammalian atria. The muscarinic receptors were functionally active, as demonstrated by the ability of the agonist to stimulate phosphoinositide turnover and to inhibit adenylyl cyclase activity. The availability of a mammalian atrial cell line that continues to express the appropriate functionally coupled subtype of muscarinic receptor may provide a useful system for the investigation of the regulation of expression and function of cardiac muscarinic receptors.
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PMID:Characterization of muscarinic acetylcholine receptors expressed by an atrial cell line derived from a transgenic mouse tumor. 813 11

The mechanism of TCR-stimulated Ca2+ influx was studied in the Jurkat human T cell line using Ca2+ indicator dyes and whole-cell patch clamp. Ca2+ influx induced by inositol 1,4,5-triphosphate (IP3)-coupled surface receptors (either the TCR or a heterologous muscarinic receptor) was compared with Ca2+ influx induced by inhibitors of the microsomal Ca(2+)-ATPase (thapsigargin, cyclopiazonic acid, di-tert-butylhydroquinone), which release stored Ca2+ without production of IP3. The same Ca2+ influx pathway could be activated by IP3-dependent or IP3-independent means, and therefore appeared to be regulated by the fullness of the microsomal Ca2+ stores rather than by the direct action of IP3. Depletion of stored Ca2+ by either receptor stimulation or microsomal Ca(2+)-ATPase inhibition activated a low conductance, Ca(2+)-selective, non-voltage-activated membrane current. Ca2+ currents induced by receptor stimulation and Ca(2+)-ATPase inhibition were not additive. Several properties of the depletion-activated Ca2+ current suggest that it is carried by a novel type of Ca2+ channel rather than an electrogenic carrier or pump. The conductance saturated when external Ca2+ was raised (Kd approximately 2 mM) and became highly permeable to monovalent cations when external Ca2+ was lowered to below 100 nM, much as has been observed for some voltage-gated Ca2+ channels. The Ca2+ current was reversibly blocked by > 90% with 0.3 mM Cd2+, whereas the same concentration of Ni2+ or Co2+ blocked only 50 to 60% of the current. However, the absence of voltage-dependent activation, relative conductance sequence for divalent cations (Ca2+ > Ba2+ approximately Sr2+ >> Mn2+), and lack of inhibition by nifedipine, D600, diltiazem, delta-conotoxin, or aga-IVa were unlike that of voltage-gated Ca2+ channels.
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PMID:Activation of Ca2+ current in Jurkat T cells following the depletion of Ca2+ stores by microsomal Ca(2+)-ATPase inhibitors. 818 45

Recently, it was reported that muscarinic-type cholinergic receptors coupled to the phosphoinositide messenger system are present in the rabbit inner medullary collecting duct and Madin-Darby canine kidney (MDCK) cells. The receptor density in MDCK cells is 50 times more than that in inner medullary collecting duct cells. To examine if muscarinic receptor activation influences Na-K-ATPase, the effects of a cholinergic agonist, carbachol, on Na-K-ATPase activity in MDCK cells were measured. Carbachol inhibited Na-K-ATPase activity in a time- and concentration-dependent manner. A maximum of approximately 80% of the enzyme activity was inhibited in 160 min with an EC50 of 5 microM carbachol. The inhibition of Na-K-ATPase activity was reversible; up to 80% of the enzyme activity was recovered within 4 h after carbachol was removed. The inhibitory effect of carbachol was blocked by a muscarinic antagonist atropine and by inhibitors of protein kinase C (PKC), 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine HCl, and N-(2-(methylamino)ethyl)-5-isoquinoline sulfonamide HCl. Direct activators of PKC, phorbol 12-myristate 13-acetate, N(n-heptyl)-5-chloro-1-naphthalene sulfonamide, and phosphatidyl serine, also inhibited Na-K-ATPase activity in MDCK cells, and their effect was also blocked by PKC inhibitors. These results indicate that cholinergic agonists inhibit Na-K-ATPase activity in MDCK cells by the activation of PKC. It is concluded that the inhibition of Na-K-ATPase by PKC may, in part, be responsible for the natriuretic action of cholinergic agonists, which have been shown to stimulate phosphoinositide hydrolysis in renal collecting duct cells.
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PMID:Cholinergic inhibition of Na-K-ATPase via activation of protein kinase C in Madin-Darby canine kidney cells. 840 83


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