Gene/Protein Disease Symptom Drug Enzyme Compound
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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)

We have microinjected a mAb specifically directed to phosphatidylinositol 4,5-bisphosphate (PIP2) into one blastomere of two-cell stage Xenopus laevis embryos. This antibody binds to endogenous PIP2 and reduces its rate of hydrolysis by phospholipase C. Antibody-injected blastomeres undergo partial or complete arrest of the cell cycle whereas the uninjected sister blastomeres divided normally. Since PIP2 hydrolysis normally produces diacylglycerol (DG) and inositol 1,4,5-triphosphate (Ins[1,4,5]P3), we attempted to measure changes in the levels of DG following stimulation of PIP2 hydrolysis in antibody-injected oocytes. The total amount of DG in antibody-injected oocytes was significantly reduced compared to that of water-injected ones following stimulation by either acetylcholine or progesterone indicating that the antibody does indeed suppress PIP2 hydrolysis. We also found that the PIP2 antibodies greatly reduced the amount of intracellular Ca2+ released in the egg cortex during egg activation. As an indirect test for Ins(1,4,5)P3 involvement in the cell cycle we injected heparin which competes with Ins(1,4,5)P3 for binding to its receptor, and thus inhibits Ins(1,4,5)P3-induced Ca2+ release. Microinjection of heparin into one blastomere of the two-cell stage embryo caused partial or complete arrest of the cell cycle depending upon the concentration of heparin injected. We further investigated the effect of reducing any [Ca2+]i gradients by microinjecting dibromo-BAPTA into the blastomere. Dibromo-BAPTA injection completely blocked mitotic cell division when a final concentration of 1.5 mM was used. These results suggest that PIP2 turnover as well as second messenger activity influence cell cycle duration during embryonic cell division in frogs.
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PMID:Reducing inositol lipid hydrolysis, Ins(1,4,5)P3 receptor availability, or Ca2+ gradients lengthens the duration of the cell cycle in Xenopus laevis blastomeres. 130 10

Decreases in D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] content and changes in inositol phospholipid contents occurred during the time of atropine-induced relaxation of swine tracheal smooth muscle contracted with 55 microM carbachol. Decrease in Ins(1,4,5)P3 occurred in a pool which makes up 40% of the total content of this inositol phosphate and which has access to Ins(1,4,5)P3 5-phosphatase and/or 3-kinase. A 50% decrease in this pool occurred at 16 s after addition of atropine and within 6-10 s after inhibition of phospholipase C (PLC). The maximal fall in Ins(1,4,5)P3 occurred at a time when force had only decreased 30% of the maximal response. A phosphatidylinositol 4-phosphate (PIP) pool linked to muscarinic receptor-activation increased 160% after addition of atropine, the maximal response occurring at a time when relaxation was 80% complete. The mechanisms for this increase were the maintained formation of PIP and phosphatidylinositol 4,5-bisphosphate (PIP2) even though PIP2 hydrolysis was inhibited and the apparent chemical equilibrium between PIP and PIP2.
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PMID:Inositol 1,4,5-trisphosphate, inositide flux rates and pool sizes during smooth muscle relaxation. 131 Feb 25

In previous works, we synthesized a series of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) analogs, with a substituent on the second carbon of the inositol ring. Using these analogs, the Ins(1,4,5)P3 affinity media were also synthesized (Hirata, M., Watanabe, Y., Ishimatsu, T., Yanaga, F., Koga, T., and Ozaki, S. (1990) Biochem. Biophys. Res. Commun. 168, 379-386). When the cytosol fraction from the rat brain was applied to an Ins(1,4,5)P3 affinity column, an eluate with a 2 M NaCl solution was found to have remarkable Ins(1,4,5)P3-binding activity. The active fraction was further fractionated with gel filtration chromatography, and two proteins with an apparent molecular mass of 130 or 85 kDa were found to be Ins(1,4,5)P3-binding proteins but with no Ins(1,4,5)P3 metabolizing activities. Partial amino acid sequences determined after proteolysis and reversed-phase chromatography revealed that the protein with an apparent molecular mass of 85 kDa is the delta-isozyme of phospholipase C and that of 130 kDa has no sequence the same as the Ins(1,4,5)P3-recognizing proteins hitherto examined. Ins(1,4,5)P3 at concentrations greater than 1 microM strongly inhibited 85-kDa phospholipase C delta activity, without changing its dependence on the concentrations of free Ca2+ and H+. Among inositol phosphates examined, Ins(3,4,5,6)P4 inhibited the binding of [3H]Ins(1,4,5)P3 to the 130-kDa protein at much the same concentrations as seen with Ins(1,4,5)P3. This report seems to be the first evidence for the presence of soluble Ins(1,4,5)P3-binding proteins in the rat brain, one of which is the delta isozyme of phospholipase C.
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PMID:Putative inositol 1,4,5-trisphosphate binding proteins in rat brain cytosol. 131 9

We have identified a Ca(2+)-dependent polyphosphoinositide-specific phospholipase C activity in Dictyostelium discoideum. Addition of Ca2+ (20 microM) results in the rapid formation of Ins(1,4,5)P3 within 5 s and leads to sustained inositol phosphate production for up to 40 min in membranes prepared from [3H]inositol-labelled cells. The phospholipase C activity is primarily membrane-bound under the conditions used to lyse the cells. In addition to this activity we also identified a family of Ca(2+)-regulated phospholipase activities active on a range of phospholipid substrates, using [3H]palmitate labelling. Inositol-specific phospholipase C activity is highest in vegetatively growing cells and in starved cells during the first 6 h in development, during which time Ca2+ elicited a 5-fold stimulation of inositol phosphate formation. After this time, total activity decreased progressively until 15 h, after which the activity remained constant up until 24 h. During this period, Ca2+ was able to stimulate a 2-fold increase in inositol phosphates.
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PMID:Characterization of phospholipase activity in Dictyostelium discoideum. Identification of a Ca(2+)-dependent polyphosphoinositide-specific phospholipase C. 131 14

A possible role for altered signal transduction mechanisms in impaired alpha 1-adrenergic-stimulated secretory function during aging was investigated in parotid cells prepared from adult (6 mo) and old (24 mo) rats. Compared with adults, epinephrine-stimulated 45Ca2+ efflux and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] production were reduced 31 and 36% in cells of old rats, respectively. There was a highly significant correlation between 45Ca2+ efflux and Ins(1,4,5)P3 production. In saponin-permeabilized cells, no significant differences in Ins(1,4,5)P3-stimulated 45Ca2+ efflux in adult and old preparations were observed. When G proteins were stimulated by guanosine 5'-O-(3-thiotriphosphate) or NaF, no age differences in Ins(1,4,5)P3 production were detected. Stimulation of phosphoinositide-specific phospholipase C (PLC) by CaCl2 in adult and old cells was also comparable. Moreover, no differences in immunolabeled common alpha (GTP binding site), Gi alpha, PLC-gamma, or PLC-delta could be detected in either cytosol or membranes of adult and old preparations. In the absence of 5'-guanylylimidodiphosphate [Gpp(NH)p], no age-related changes in epinephrine competition for [3H]prazosin binding sites were observed. Approximately 30% of the agonist binding sites existed in a high-affinity form at both ages. Gpp(NH)p caused large rightward shifts of epinephrine displacement curves in adult membranes (converting all binding sites to the low-affinity form), but not old. Moreover, epinephrine was much more effective in stimulating G protein low-Km GTPase in parotid membranes from adult than old rats. These data suggest that age-related impairments in alpha 1-adrenergic responsiveness are mediated, at least in part, by the functional alterations in the coupling of G proteins with alpha 1-adrenergic receptors.
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PMID:Altered coupling of alpha 1-adrenergic receptor-G protein in rat parotid during aging. 131 99

Recent studies have indicated two major mechanisms for the release of arachidonic acid (20:4) from membrane phospholipids: 1) activation of phospholipase A2 and 2) stimulated hydrolysis of poly-phosphoinositides (PI) and diacylglycerols (DG) through phospholipase C and diacylglycerol lipase, respectively. In mammalian brain both mechanisms seem to be operable, although the relative contributions by these two pathways have not been carefully assessed. In this study three experimental protocols were used to examine 20:4 release in brain due to ischemia and agonist stimulation, as well as the metabolic relationship between this release and the increase in diacylglycerols, lysophospholipids, and inositol phosphates. The preferential release of arachidonic acid during the initial phase after decapitation was attributed mainly to the sequential hydrolysis of poly-PI to DG. During the second phase, the release of 20:4 along with other free fatty acids (FFA) correlated well with the increase in labeled lysophospholipids, suggesting the involvement of phospholipase A2. Diacylglycerols in brain are enriched in 18:0 and 20:4. Decapitation induced a rapid increase in the level of DG, which remained elevated during the 30 min period under study. Between 5 sec and 5 min, the increase in FFA lagged behind that of DG. The parallel increases in 18:0 and 20:4 in the FFA pool further support the notion that, during the early phase, 20:4 could be derived from the sequential hydrolysis of poly-PI and DG. Decapitation also induced a sequential appearance of Ins(1,4,5)P3, Ins(1,4)P2, and Ins(4)P, which peaked at 30 sec, 1 min, and 2 min, respectively. The level of 20:4 in brain was also examined with respect to poly-PI turnover due to stimulation by cholinergic agonists. Administration of pilocarpine to lithium-treated mice resulted in increased accumulation of labeled inositol monophosphate (IP1) compared to the amount in controls receiving lithium alone, as well as a less obvious increase in 20:4. Both pilocarpine-mediated increases (IP1 and 20:4) could be blocked by atropine. These results point to the presence of an active mechanism for poly-PI turnover and for the recycling of 20:4 in brain.
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PMID:Contributions to arachidonic acid release in mouse cerebrum by the phosphoinositide-phospholipase C and phospholipase A2 pathways. 132 24

Interactions between ATP and adenosine on the formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and mobilization of intracellular calcium were investigated in the smooth muscle cell line DDT1 MF-2. Activation of adenosine A1 receptors with adenosine or cyclopentyladenosine (CPA) or of nucleotide receptors with ATP increased both Ins(1,4,5)P3 formation and intracellular calcium concentrations. The A1 receptor-induced Ins(1,4,5)P3 formation (EC50 10 nM) was antagonized by the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and by pretreatment of the cells with pertussis toxin (PTX). ATP-stimulated Ins(1,4,5)P3 formation (EC50 21 microM) was attenuated, but still present, after PTX treatment. ATP and CPA had supraadditive effects on Ins(1,4,5)P3 accumulation and CPA increased ATP-induced Ins(1,4,5)P3 accumulation in a concentration-dependent manner with an EC50 of 3 nM, a concentration which per se had little or no effect on Ins(1,4,5)P3 accumulation. ATP (EC50 4 microM) and CPA (EC50 4 nM) both increased intracellular calcium levels. The effect of ATP was partially sensitive to PTX treatment, whereas the effect of CPA was blocked both by PTX and by DPCPX. Concentrations of ATP and CPA that by themselves were insufficient to raise intracellular calcium were able to do so when combined. The synergy between ATP and CPA on the mobilization of intracellular calcium was abolished after treatment of cells with PTX or when DPCPX was included in the experiment. Since ATP was metabolized by ecto-enzymes to ADP, AMP, and adenosine, we also examined whether adenosine formed from ATP could enhance the ATP effects on Ins(1,4,5)P3 accumulation. Indeed, the addition of the A1 receptor antagonist DPCPX or removal of endogenous adenosine by inclusion of adenosine deaminase in the experimental medium significantly attenuated the ATP response, and the two treatments did not have additive effects. The present study thus demonstrates that in a clonal cell line two types of receptors increase phospholipase C activity, but via different pathways; nucleotide receptors appeared to act via partially PTX-insensitive, and A1 receptors via PTX-sensitive G-proteins. ATP and CPA are not only able per se to induce formation of Ins(1,4,5)P3 and mobilize intracellular calcium, but they also act synergistically. Finally, it is demonstrated that endogenous adenosine, possibly formed from the rapid breakdown of ATP, can significantly enhance some ATP effects.
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PMID:ATP and its metabolite adenosine act synergistically to mobilize intracellular calcium via the formation of inositol 1,4,5-trisphosphate in a smooth muscle cell line. 132 90

Inositol 1,4,5-trisphosphate (InsP3) is an intracellular messenger generated from the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C in response to Ca(2+)-mobilizing stimuli. InsP3 interacts with a specific receptor responsible for the release of sequestered Ca2+ from an intracellular store. The purpose of the present study was to evaluate the relative affinities of the naturally occurring D-isomer of InsP3 and that of its L-stereoisomer for the InsP3 receptor and the InsP3 metabolizing enzymes from bovine adrenal cortex. The InsP3 receptor recognized D- and L-isomers with respective affinities of 4.8 nM and 7.3 microM. This high degree of selectivity was also reflected in the capacity of both isomers to mobilize Ca2+ from the microsomal preparation. The partially purified InsP3 kinase preparation was also able to discriminate between the two stereoisomers. The activity of the kinase was half-maximally inhibited in the presence of 11 microM L-InsP3, a value much higher than the Km of the kinase for D-InsP3 (0.4 microM). Both stereoisomers exhibited equipotent affinities (around 17 microM) for the particulate preparation of InsP3 phosphatase. The enzyme, however, appeared to hydrolyze L-InsP3 at a much slower rate. These results demonstrated that the different recognition sites for InsP3 were expressing distinct levels of stereoselectivity. This property, which is an important aspect of ligand-receptor interaction, could be exploited for the design of new selective drugs interfering with InsP3 action and metabolism.
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PMID:Study on the stereoselectivity of inositol 1,4,5-trisphosphate recognition sites of bovine adrenal cortex. 132 74

We examined the effects of heparin, guanosine nucleotides, protein kinase C (PKC) modulators, such as phorbol 12,13-dibutyrate (PDBu) and H-7 on Ca(2+)-dependent K+ currents in smooth muscle cells of the rabbit portal vein using the whole-cell patch-clamp technique, to explore the effects of PKC on the oscillatory outward current (Ioo). Neomycin (30 microM), an inhibitor of phospholipase C, and intracellular applications of heparin (10 micrograms/ml) and guanosine 5'-O-(2-thiodiphosphate) (GDP[beta S]; 1 mM) partly but consistently inhibited the generation of Ioo, whereas a higher concentration of heparin (100 micrograms/ml) transiently enhanced then suppressed the generation of Ioo. Inhibition of Ioo generation by heparin was more powerful at the holding potential of +20 mV than at -20 mV. Inositol 1,4,5-trisphosphate (InsP3; 30 microM) continuously generated Ioo at holding potentials more positive than -60 mV. Noradrenaline (10 microM) and caffeine (3-20 mM) transiently augmented, then reduced the generation of Ioo. Heparin (10 micrograms/ml) completely inhibited responses induced by InsP3 and noradrenaline, but not those induced by caffeine. Intracellular application of guanosine 5'-triphosphate (GTP; 200 microM) or low concentrations of guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma S]; < or = 3 microM) continuously augmented the generation of Ioo. High concentrations of GTP[gamma S] (> or = 10 microM) transiently augmented, then inhibited Ioo. Neither GTP[gamma S] nor noradrenaline induced the transient augmentation or the subsequent inhibition of Ioo when applied in the presence of GDP[beta S] (1 mM), neomycin (30 microM) or heparin (10 micrograms/ml). PDBu (0.1 microM) reduced the generation of Ioo but failed to produce an outward current following application of caffeine (3-5 mM). This action of PDBu was inhibited by pretreatment with H-7 (20 microM). In the presence of H-7, GTP[gamma S] continuously enhanced the generation of Ioo. The suppression of the generation of Ioo during application of noradrenaline (10 microM) was reduced by pretreatment with H-7. Thus both InsP3 and protein kinase C contribute to the generation of Ioo in smooth muscle cells of the rabbit portal vein and heparin is not a specific InsP3 antagonist on the InsP3-induced Ca(2+)-release channel (PIRC). InsP3 opens PIRC and protein kinase C may deplete the stored Ca2+ by either inhibiting the reuptake of Ca2+ or by enhancement of the releasing actions of InsP3.
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PMID:Roles of inositol trisphosphate and protein kinase C in the spontaneous outward current modulated by calcium release in rabbit portal vein. 133 73

The mechanism of inhibitory effects of azelastine, an antiallergic and antiasthmatic agent, on depolarization- and alpha-1 adrenergic agonist-induced contractions of intact smooth muscle was studied. The effects of azelastine on membrane currents were determined in isolated guinea pig ileum smooth muscle cells with the whole-cell clamp technique; the effects on contraction were evaluated in receptor- and G-protein-coupled, alpha-toxin-permeabilized rabbit femoral artery and portal vein smooth muscle strips. Azelastine (1-20 microM), like dihydropyridines, inhibited spontaneous rhythmic and high K(+)-induced contractions, mainly through inhibition of the voltage-dependent (L-type) Ca++ current. The tonic component of high K+ contractions was inhibited more than the phasic component, correlating to voltage-dependent inhibition of Ca++ current by the drug. Azelastine (IC50 of 0.25 microM), a known histamine blocker, also reversibly inhibited alpha-1 agonist-induced contractions in the presence and absence of extracellular Ca++. Both major pathways of pharmacomechanical coupling, agonist-induced Ca++ release from the sarcoplasmic reticulum and Ca++ sensitization of the regulatory/contractile apparatus were blocked by the same concentration of drug in permeabilized as in intact muscle. Inositol 1,4,5-trisphosphate-induced Ca++ release and guanosine 5'-O-(tau-thiotriphosphate)-induced Ca++ sensitization, however, were not inhibited. Azelastine at high (greater than 10 microM) concentrations reversibly inhibited Ca(++)-activated contraction, more potently at lower Ca++ concentration and in phasic smooth muscle, but inhibited neither adenosine 5'-O-(tau-thiotriphosphate)-induced, Ca(++)-independent nor phorbol ester-induced contractions. These results indicate that azelastine is a genuine Ca++ antagonist that inhibits voltage-gated Ca++ inward current and agonist-induced Ca++ release and Ca++ sensitization.
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PMID:Mechanism of inhibitory effects of azelastine on smooth muscle contraction. 134 67


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