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Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Application of adenosine triphosphate (ATP) induced a depolarizing response in bullfrog sympathetic ganglion cells, which was associated with a decrease in membrane conductance. This depolarizing response was also produced by application of ADP, but not by adenosine, cyclic-AMP or cyclic-GMP. Suramin, an antagonist for the P2-purinoceptor, suppressed the response, while caffeine, an antagonist for the P1-purinoceptor, did not. Application of phospholipase C (PLC) inhibitors such as Li+ and 4-bromophenacyl bromide also suppressed the response. In addition, protein kinase C (PKC) inhibitors such as staurosporine and H-7 had a suppressant effect, while ryanodine, an inhibitor of Ca2 release, did not. These results suggest that ATP and ADP may stimulate P2-purinoceptor coupled with PLC, producing diacylglycerol, which activates PKC, resulting in the closing of K+ channel.
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PMID:Intracellular mechanism of ATP-induced depolarizing response in the bullfrog sympathetic ganglion cells. 890 Feb 16

The effect of angiotensin II (ANG II) on the cytosolic calcium concentration ([Ca2+]i) was studied in freshly (2-8 h) isolated myocytes from the main pulmonary artery of the rat. Myocytes were loaded with the fluorescent indicator indo 1 (1 microM for 30 min) and experiments were performed at room temperature. Short (30 s) applications of ANG II (0.01-10 microM) induced cyclic variations oscillations in [Ca2+]i. The ANG II-induced response was typically composed of three to six oscillations of constant duration (9.8 +/- 0.5 s, n = 40) but of decreasing amplitude. The first oscillation increased [Ca2+]i from 119 +/- 4 to 884 +/- 33 nM (n = 32). ANG II-induced response was concentration dependently inhibited by previous addition to the bathing solution of losartan or SR-47436 (0.01-0.1 microM, each), two specific AT1 receptor-antagonists. In Ca(2+)-free external solutions (containing 0.4-1 mM EGTA), ANG II still produced oscillation in [Ca2+]i. These oscillations disappeared in myocytes pretreated with neomycin (0.1 microM), thapsigargin (1 microM), or phorbol 12,13-dibutyrate (PDBu, 1 microM). In contrast to ANG II, caffeine (o.5-10 mM) induced only one transient rise in [Ca2+]i, which was unaltered by neomycin or PDBu but blocked by thapsigargin. These results show that ANG II produces oscillations in [Ca2+]i in pulmonary arterial myocytes via stimulation of AT1 receptors coupled to phospholipase C activation. ANG II-induced oscillations appear to be related to the cycling of Ca2+ ions from an intracellular store (presumably the sarcoplasmic reticulum) by a primarily inositol trisphosphate-dependent Ca2+ release.
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PMID:Angiotensin II-induced Ca(2+)-oscillations in vascular myocytes from the rat pulmonary artery. 892 24

The functional characteristics of a nonacidic, inositol 1,4,5-trisphosphate- and thapsigargin-insensitive Ca2+ pool have been characterized in mammalian cells derived from the rat pituitary gland (GH3, GC, and GH3B6), the adrenal tissue (PC12), and mast cells (RBL-1). This Ca2+ pool is released into the cytoplasm by the Ca2+ ionophores ionomycin or A23187 after the discharge of the inositol 1,4,5-trisphosphate-sensitive store with an agonist coupled to phospholipase C activation and/or thapsigargin. The amount of Ca2+ trapped within this pool increased significantly after a prolonged elevation of intracellular Ca2+ concentration elicited by activation of Ca2+ influx. This pool was affected neither by caffeine-ryanodine nor by mitochondrial uncouplers. Probing mitochondrial Ca2+ with recombinant aequorin confirmed that this pool did not coincide with mitochondria, whereas its homogeneous distribution across the cytosol, as revealed by confocal microscopy, and its insensitivity to brefeldin A make localization within the Golgi complex unlikely. A proton gradient as the driving mechanism for Ca2+ uptake was excluded since ionomycin is inefficient in releasing Ca2+ from acidic pools and Ca2+ accumulation/release in/from this store was unaffected by monensin or NH4Cl, drugs known to collapse organelle acidic pH gradients. Ca2+ sequestration inside this pool, thus, may occur through a low-affinity, high-capacity Ca2+-ATPase system, which is, however, distinct from classical endosarcoplasmic reticulum Ca2+-ATPases. The cytological nature and functional role of this Ca2+ storage compartment are discussed.
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PMID:Dynamic properties of an inositol 1,4,5-trisphosphate- and thapsigargin-insensitive calcium pool in mammalian cell lines. 901 6

Two principal pathways of Ca2+ release from the sarcoplasmic reticulum of excitable and non-excitable cells have been described: one pathway dependent on the second messenger D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), and a second pathway sensitive to Ca2+ and regulated by caffeine and ryanodine. It was found that the Ca(2+)-pump activity of vascular smooth muscle sarcoplasmic reticulum is inhibited by superoxide anion radicals (O2.-); however, the effects of reactive oxygen intermediates on sarcoplasmic reticulum Ca2+ release in vascular muscle cells are not well defined. The purpose of the present study was to evaluate the effects of reactive oxygen intermediates generated from the hypoxanthine/xanthine oxidase reaction system on contractions induced by caffeine, Ins(1,4,5)P3 and norepinephrine in staphylococcal alpha-toxin-permeabilized rabbit mesenteric arteries. This system generates O2.-, H2O2, and hydroxyl radicals. We wished to identify which class of reactive oxygen intermediates is responsible for the associated loss of vascular smooth muscle contractile function. Caffeine and Ins(1,4,5)P3 produced a transient contraction when the sarcoplasmic reticulum of the permeabilized, preparations was preloaded with pCa 7.0 solution for 5 min before washing with 0.5 mM EGTA solution; norepinephrine also produced a transient contraction. Exposure of the preparations to hypoxanthine/xanthine oxidase (for 30 min) attenuated caffeine-induced contraction, but was without effect on Ins(1,4,5)P3-induced contraction. The observed effect of hypoxanthine/xanthine oxidase exposure was superoxide dismutase-inhibitable, suggesting O2.- involvement. Hypoxanthine/xanthine oxidase also inhibited norepinephrine-induced contraction. The effect of hypoxanthine/xanthine oxidase on norepinephrine contraction was protected by catalase, but not by superoxide dismutase and dimethyl sulfoxide; exogenously added H2O2 mimicked the effect of hypoxanthine/xanthine oxidase exposure. H2O2, added exogenously, was without effect on Ins(1,4,5)P3-induced contraction. It is suggested that the pathway of Ca2+ release from the sarcoplasmic reticulum dependent on Ins(1,4,5)P3 is insensitive to O2.-. Instead, caffeine-induced Ca2+ release mechanisms may be susceptible to O2.- and H2O2, rather than O2.- and hydroxyl radicals, may be the active agent in the norepinephrine-induced contraction. Our results are also consistent with the view that the attenuation by H2O2 of the norepinephrine-induced contraction may be linked to the receptor-associated pathway of Ins(1,4,5)P3 formation, but not to degradation processes of Ins(1,4,5)P3.
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PMID:Susceptibility of caffeine- and Ins(1,4,5)P3-induced contractions to oxidants in permeabilized vascular smooth muscle. 904 2

1. The aim of the present study was to identify the sources of Ca2+ contributing to acetylcholine (ACh)-induced release of endothelium-derived hyperpolarizing factor (EDHF) from endothelial cells of rat mesenteric artery and to assess the pathway involved. The changes in membrane potentials of smooth muscles by ACh measured with the microelectrode technique were evaluated as a marker for EDHF release. 2. ACh elicited membrane hyperpolarization of smooth muscle cells in an endothelium-dependent manner. The hyperpolarizing response was not affected by treatment with 10 microM indomethacin, 300 microM NG-nitro-L-arginine or 10 microM oxyhaemoglobin, thereby indicating that the hyperpolarization is not mediated by prostanoids or nitric oxide but is presumably by EDHF. 3. In the presence of extracellular Ca2+, 1 microM ACh generated a hyperpolarization composed of the transient and sustained components. By contrast, in Ca(2+)-free medium, ACh produced only transient hyperpolarization. 4. Pretreatment with 100 nM thapsigargin and 3 microM cyclopiazonic acid, endoplasmic reticulum Ca(2+)-ATPase inhibitors, completely abolished ACh-induced hyperpolarization. Pretreatment with 20 mM caffeine also markedly attenuated ACh-induced hyperpolarization. However, the overall pattern and peak amplitude of hyperpolarization were unaffected by pretreatment with 1 microM ryanodine. 5. In the presence of 5 mM Ni2+ or 3 mM Mn2+, the hyperpolarizing response to ACh was transient, and the sustained component of hyperpolarization was not observed. On the other hand, 1 microM nifedipine had no effect on ACh-induced hyperpolarization. 6. ACh-induced hyperpolarization was nearly completely eliminated by 500 nM U-73122 or 200 microM 2-nitro-4-carboxyphenyl-N, N-diphenylcarbamate, inhibitors of phospholipase C, but was unchanged by 500 nM U-73343, an inactive form of U-73122. Pretreatment with 20 nM staurosporine, an inhibitor of protein kinase C, did not modify ACh-induced hyperpolarization. 7. These results indicate that the ACh-induced release of EDHF from endothelial cells of rat mesenteric artery is possibly initiated by Ca2+ release from inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pool as a consequence of stimulation of phospholipid hydrolysis due to phospholipase C activation, and maintained by Ca2+ influx via a Ni(2+)- and Mn(2+)-sensitive pathway distinct from L-type Ca2+ channels. The Ca(2+)-influx mechanism seems to be activated following IP3-induced depletion of the pool.
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PMID:Sources of Ca2+ in relation to generation of acetylcholine-induced endothelium-dependent hyperpolarization in rat mesenteric artery. 910 9

1. Cytosolic Ca2+ concentration ([Ca2+]i) during exposure to acetylcholine or caffeine was measured in mouse duodenal myocytes loaded with fura-2. Acetylcholine evoked a transient increase in [Ca2+]i followed by a sustained rise which was rapidly terminated after drug removal. Although L-type Ca2+ currents participated in the global Ca2+ response induced by acetylcholine, the initial peak in [Ca2+]i was mainly due to release of Ca2+ from intracellular stores. 2. Atropine, 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, a muscarinic M3 antagonist), pirenzepine (a muscarinic M1 antagonist), methoctramine and gallamine (muscarinic M2 antagonists) inhibited the acetylcholine-induced Ca2+ release, with a high affinity for 4-DAMP and atropine and a low affinity for the other antagonists. Selective protection of muscarinic M2 receptors with methoctramine during 4-DAMP mustard alkylation of muscarinic M3 receptors provided no evidence for muscarinic M2 receptor-activated [Ca2+]i increase. 3. Acetylcholine-induced Ca2+ release was blocked by intracellular dialysis with a patch pipette containing either heparin or an anti-phosphatidylinositol antibody and by external application of U73122 (a phospholipase C inhibitor). 4. Acetylcholine-induced Ca2+ release was insensitive to external pretreatment with pertussis toxin, but concentration-dependently inhibited by intracellular dialysis with a patch pipette solution containing an anti-alpha q/alpha 11 antibody. An antisense oligonucleotide approach revealed that only the Gq protein was involved in acetylcholine-induced Ca2+ release. 5. Intracellular applications of either an anti-beta com antibody or a peptide corresponding to the G beta gamma binding domain of the beta-adrenoceptor kinase 1 had no effect on acetylcholine-induced Ca2+ release. 6. Our results show that, in mouse duodenal myocytes, acetylcholine-induced release of Ca2+ from intracellular stores is mediated through activation of muscarinic M3 receptors which couple with a Gq protein to activate a phosphatidylinositol-specific phospholipase C.
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PMID:Specific Gq protein involvement in muscarinic M3 receptor-induced phosphatidylinositol hydrolysis and Ca2+ release in mouse duodenal myocytes. 917 86

We investigated the muscarinic activation of Ca(2+)-activated Cl- currents [ICl(Ca)] in voltage-clamped equine tracheal myocytes. The threshold of cytosolic free Ca2+ concentration ([Ca2+]i) required for activation of ICl(Ca) was 202 +/- 22 nM, and full activation of the current occurred at 771 +/- 31 nM. Hexahydro-sila-difenidol (M3 antagonist) inhibited the methacholine-induced phasic [Ca2+]i increase and ICl(Ca) in a concentration-dependent manner, whereas methoctramine (M2 antagonist) only slightly attenuated the [Ca2+]i increase and ICl(Ca) (14.8 and 21.4%, respectively), consistent with incomplete selectivity. Dialysis of heparin (10 mg/ml) blocked methacholine-induced [Ca2+]i and ICl(Ca) but had no effect on the caffeine-induced Ca2+ release or ICl(Ca); inositol 1,4,5-trisphosphate (100 microM) induced ICl(Ca) and blocked the methacholine current. Conversely, ruthenium red (50 microM) prevented the caffeine-induced [Ca2+]i release and ICl(Ca) but had no effect on methacholine-induced [Ca2+]i or current. Intracellular dialysis of the calmodulin antagonist N-(6-aminohexyl)-1-naphthalenesulfonamide (W-7, 500 microM) or the Ca2+/calmodulin-dependent protein kinase inhibitor KN93 (5 microM) had no effect on the [Ca2+]i increase or ICl(Ca). Pertussis toxin (0.5 mg/ml) did not affect the increase in [Ca2+]i or ICl(Ca). Dialysis with antibodies directed against the alpha-subunit of Gq/G11 (Gq alpha/ G alpha 11) blocked the methacholine-induced ICl(Ca) in a concentration-dependent manner, whereas anti-G alpha i-1/G alpha 1-2 antibodies (1:35) and anti-G alpha i-3/G(o) alpha antibodies (1:35) were without effect. The results indicate that stimulation of phospholipase C via M3/Gq proteins is the predominant signaling pathway for the activation of ICl(Ca); at high agonist concentrations, Ca(2+)-induced Ca2+ release does not appear to play a prominent role in muscarinic signaling.
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PMID:Muscarinic signaling pathway for calcium release and calcium-activated chloride current in smooth muscle. 927 48

1. This study was aimed at characterizing ATP-induced rises in cytosolic free calcium ion, [Ca2+]i, in a population of rat striatal astrocytes loaded with the fluorescent Ca2+ probe Fura2, by means of fluorescence spectrometry. 2. ATP triggered a fast and transient elevation of [Ca2+]i in a concentration-dependent manner. The responses of the purine analogues 2-methylthio-ATP (2-meSATP), adenosine-5'-O-(2-thiodiphosphate) (ADP beta S), as well as uridine-5'-triphosphate (UTP) resembled that of ATP, while alpha, beta-methylene-ATP (alpha, beta-meATP) and beta, gamma-methylene-ATP (beta, gamma-meATP) were totally ineffective. 3. Suramin (50 microM) had only a minor effect on the ATP response, whereas pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (5 microM) significantly depressed the maximum response. 4. Extracellular Ca2+ did not contribute to the observed [Ca2+]i rise: removing calcium from the extracellular medium (with 1 mM EGTA) or blocking its influx by means of either Ni2+ (1 mM) or Mn2+ (1 mM) did not modify the nucleotide responses. 5. Furthermore, after preincubation with 10 microM thapsigargin, the nucleotide-evoked [Ca2+]i increments were completely abolished. In contrast, 10 mM caffeine did not affect the responses, suggesting that thapsigargin-, but not caffeine/ryanodine-sensitive stores are involved. 6. Both application of the G-protein blocker guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) (1 mM) and preincubation with pertussis toxin (PTx) (350 ng ml-1) partially inhibited the nucleotide-mediated responses. Moreover, the phospholipase C (PLC) inhibitor U-73122, but not its inactive stereoisomer U-73343 (5 microM), significantly reduced the ATP-evoked [Ca2+]i rise. 7. In conclusion, our results suggest that, in rat striatal astrocytes, ATP-elicited elevation of [Ca2+]i is due solely to release from intracellular stores and is mediated by a G-protein-linked P2Y receptor, partially sensitive to PTx and coupled to PLC.
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PMID:Characterization of the Ca2+ responses evoked by ATP and other nucleotides in mammalian brain astrocytes. 928 6

Modulation of [Ca2+]i in response to receptor activation is a critical determinant of vascular smooth muscle tone. In this study, we examined the effect of continuous stimulation of alpha 1-adrenoceptors with phenylephrine (PE) on [Ca2+]i in single pulmonary artery smooth muscle cells (PASMCs) cultured from explants of canine intrapulmonary artery. Fura 2-loaded PASMCs pretreated with propranolol (5 mumol/L) were continuously superfused with PE at 37 degrees C on the stage of an inverted fluorescence microscope, and [Ca2+]i was measured using a dual-wavelength spectrofluorometer. Resting values of [Ca2+]i were 96 +/- 4 nmol/L. PE (10 mumol/L) stimulated oscillations in [Ca2+]i at a frequency of 1.35 +/- 0.07/min, which reached a peak [Ca2+]i of 650 +/- 26 nmol/L (n = 69 cells). The oscillations lasted for > 30 minutes and were constant in amplitude and frequency. Both the amplitude and frequency of PE-induced [Ca2+]i oscillations increased in a dose-dependent (3 x 10(-8) to 10(-4) mol/L) manner. Pretreatment with the alpha 1-adrenoceptor antagonist prazosin (50 nmol/L) or removal of extracellular Ca2+ abolished the repetitive [Ca2+]i oscillations induced by PE. The voltage-operated Ca2+ channel blockers nifedipine (1 mumol/L) and verapamil (1 mumol/L) had no effect on the [Ca2+]i oscillations. In contrast, inhibition of phospholipase C with U73122 (10(-7) to 10(-5) mol/L) attenuated the oscillations in a dose-dependent fashion. The nonselective protein kinase inhibitor staurosporine (10(-9) to 10(-7) mol/L) had a minimal inhibitory effect on the oscillations. Caffeine (30 mmol/L) and thapsigargin (1 mumol/L) abolished the oscillations, whereas pretreatment with ryanodine (1 to 100 mumol/L) had no effect. In freshly dispersed PASMCs, PE (10 mumol/L) induced oscillations in [Ca2+]i similar to those observed in cultured cells, and patch-clamp experiments revealed oscillations in membrane potential. These results indicate that PE induces [Ca2+]i oscillations in PASMCs via stimulation of alpha 1-adrenoceptors coupled to phospholipase C activation. Voltage-operated Ca2+ channels and protein kinases are not required for the oscillations. The requirement for extracellular Ca2+ and intracellular Ca2+ stores indicates that both Ca2+ influx and intracellular Ca2+ release play a role in the maintenance of the oscillations.
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PMID:Phenylephrine-induced Ca2+ oscillations in canine pulmonary artery smooth muscle cells. 935 55

In vitro differentiation of mouse embryonic stem cells within three-dimensional cell aggregates called embryoid bodies parallels the development of postimplantation embryos at the egg cylinder stage, where visceral and parietal endoderm diverge from the primitive endoderm. We have investigated spontaneous [Ca2+]i oscillations by means of confocal laser-scanning microscopy in primitive endodermal cell layers of embryoid bodies during their differentiation to parietal and visceral endoderm. The frequency of [Ca2+]i oscillations increased from day 4 to day 19 of development, whereas their duration decreased from day 3 to days 16-17. Oscillations depended on both extracellular Ca2+ and Ca2+ release from intracellular stores as they were abolished in Ca(2+)-free solution and in the prescence of Ni2+ and thapsigargin. Signal transduction operated via the phospholipase C (PLC)-mediated inositol 1,4,5-triphosphate (InsP3) pathway with a negative feedback loop via protein kinase C (PKC) as U73,122, a blocker of PLC; bisindolylmaleimide 1, staurosporine, and H-7, blockers of PKC; and 10 mM caffeine totally inhibited [Ca2+]i spiking. Thimerosal, which hypersensitizes the InsP3 receptor, as well as vasopressin and bradykinin, which act via the InsP3 pathway, increased the frequency of [Ca2+]i spikes. In the prescence of brefeldin A (50 microM) or monensin (20 microM), which both inhibit endo/exocytotic vesicle pathways, an immediate transient increase in spiking activity was followed by a decline within 1 to 2 h. In the presence of brefeldin A or thapsigargin or in the absence of extracellular Ca2+, endocytotic vesicles were absent, suggesting that oscillating [Ca2+]i transients are involved in the exo/endocytotic vesicle shuttle.
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PMID:Spontaneous calcium oscillations in embryonic stem cell-derived primitive endodermal cells. 945 52


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