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)

Dioctanoylthiophosphatidylcholine, a racemic thiophosphate analog of L-alpha-dioctanoylphosphatidylcholine, has been synthesized and isolated by flash chromatography. In contrast with the didecanoylthiophosphatidylcholine synthesized previously, the analog is easily dispersed on sonication in aqueous media and is rapidly hydrolyzed to produce a free thiol group in the presence of the extracellular phospholipase C from either Bacillus cereus or Clostridium perfringens. When 5,5'-dithiobis (2-nitro-benzoic acid) was included as a thiol reactive chromogenic agent, the resultant measurement of product release, as an increase in absorbance at 412 nm, showed a linear relationship with added enzyme.
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PMID:Synthesis of a thiophosphate analog of dioctanoylphosphatidylcholine: a phospholipase C substrate. 288 86

It has previously been shown that exposure of Chlamydomonas to low pH induces the cells to shed their flagella. In the present paper we report that a 30-s treatment with 20 mM acetic, carbonic, formic, or benzoic acid at pH 4.0 will induce flagellar excision. In contrast, 20 mM concentrations of the stronger aspartic, phosphoric, citric, and tartaric acids (pH 4) do not induce excision. Further, the excision efficacy of acetate is a function of the concentration of protonated acetate. Thus, excision correlates with the presence of a protonated, membrane-permeant species of acid. Relative to acetate, the more permeant benzoate induces excision at a much lower concentration of protonated acid. We conclude that a flux of acid into the cell is the signal for excision. Previous work has shown that detergent-permeabilized cells excise their flagella in response to calcium but not in response to low pH. This suggests that the acidification of intact cells triggers excision by stimulating an increase in intracellular calcium. We have previously reported that the source of this calcium might be IP3-sensitive. In our model for the mechanism of pH-induced flagellar excision, a flux of acid into the cell activates phospholipase C, leading to IP3 production, the activation of an IP3-gated calcium channel (located on either an intracellular or surface membrane), and an increase in cytosolic calcium, which is the trigger of flagellar excision.
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PMID:Mechanisms of flagellar excision. I. The role of intracellular acidification. 835 14

Epidermal enzymes play an important role in the process of differentiation of keratinocytes. The present preliminary in vitro study was undertaken to observe if topical enzyme treatment influenced permeation of compounds across the skin. Due to the noted function and importance of phosphatidylcholine metabolism during maturation of the barrier lipids, the effects of topical application of the phosphatidylcholine dependent phospholipase C enzyme (not present in epidermis) on skin penetration of three model drugs, viz. benzoic acid, mannitol and testosterone, were studied. Similar studies were also carried out using epidermal enzymes like triacylglycerol hydrolase, acid phosphatase, and phospholipase A2 (present in epidermis). Pretreatment of skin with phospholipase C significantly enhanced permeation of benzoic acid, mannitol, and testosterone relative to untreated skin. Triacylglycerol hydrolase (neutral) increased the penetration of mannitol 3-fold and had no effect on benzoic acid penetration. Topical application of acid phosphatase did not alter the permeation of any of these compounds. Phospholipase A2 significantly enhanced permeation of benzoic acid and mannitol while it did not have any effect on the penetration of testosterone. These results for the first time demonstrate that enzymes may remarkably affect and/or regulate the permeation of topically applied compounds.
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PMID:Epidermal enzymes as penetration enhancers in transdermal drug delivery? 869 22

The effects of the phosphoinositide-mobilizing agonist bradykinin (BK) on membrane potential and intracellular calcium in monolayers of normal rat kidney (NRK) fibroblasts were investigated. BK induced a rapid transient depolarization in these cells, which was mimicked by other phosphoinositide-mobilizing factors such as prostaglandin F2alpha (PGF2alpha), lysophosphatidic acid (LPA), platelet-derived growth factor (PDGF-BB), and serum. Depolarization by BK was independent of extracellular Ca2+ or Na+. It was shown using extracellular Cl- substitutions that the depolarization was caused by an increased Cl- conductance. Depolarization was inhibited by 5-nitro-2-3-phenylpropyl(amino)benzoic acid (NPPB), niflumic acid, and flufenamic acid, inhibitors of calcium-dependent chloride channels. The depolarization provoked by BK could be mimicked by raising intracellular calcium with ionomycin or thapsigargin and could be blocked with geneticin, a blocker of phospholipase C. When intracellular calcium was buffered by loading the cells with 1,2-bis(2-aminophenoxy)ethane-NNN'N'-tetra-acetic acid (BAPTA), depolarization was prevented. We conclude that in NRK fibroblasts extracellular stimuli that increase intracellular calcium, depolarize the cells via the activation of a calcium-dependent chloride conductance. In addition to an increase in intracellular calcium, depolarization may be an important effector pathway in response to extracellular stimuli in fibroblasts. It is hypothesized that, in electrically coupled cells such as NRK fibroblasts, intercellular transmission of these depolarizations may represent a mechanism to coordinate uniform multicellular responses to Ca2+-mobilizing agonists.
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PMID:Membrane depolarization in NRK fibroblasts by bradykinin is mediated by a calcium-dependent chloride conductance. 900 45

The whole cell recording technique was used to examine an outwardly rectifying chloride current activated by hypotonic shock in bovine pigmented ciliary epithelial (PCE) cells. Removal of internal and external Ca2+ did not affect the activation of these currents, but they were abolished by the phospholipase C inhibitor neomycin. The current was blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, and 4,4'-disothiocyanostilbene-2,2'-disulfonic acid (DIDS) in a voltage-dependent manner, but tamoxifen, dideoxyforskolin, and quinidine did not affect it. This blocking profile differs from that of the volume-sensitive chloride channel in neighboring nonpigmented ciliary epithelial cells (Wu, J., J. J. Zhang, H. Koppel, and T. J. C. Jacob, J. Physiol, Lond. 491: 743-755, 1996), and this difference implies that the volume responses of the two cell types are mediated by different chloride channels (Jacob, T. J. C., and J. J. Zhang. J. Physiol. Lond. In press). Intracellular administration of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to PCE cells induced a transient, time-independent, outwardly rectifying chloride current that closely resembled the current activated by hypotonic shock. DIDS produced a voltage-dependent block of the GTP gamma S-activated current similar to the block of the hypotonically activated current. Intracellular neomycin completely prevented activation of this current as did incubation of the cells in calphostin C. and inhibitor of protein kinase C (PKC). Removal of Ca2+ did not affect activation of the current by GTP gamma S but extended the duration of the response. Inhibition of phospholipase A2 (PLA2) with p-bromophenacyl bromide prevented the activation of the hypotonically induced current and also inhibited the current once activated by hypotonic solution. The findings imply that the hypotonic response in PCE cells is mediated by both phospholipase C (PLC) and PLA2. Both phospholipases generate arachidonic acid, and, in addition, the PLC pathway regulates the PLA2 pathway via a PKC-dependent phosphorylation of PLA2.
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PMID:Volume-sensitive chloride current in pigmented ciliary epithelial cells: role of phospholipases. 935 90

In previous studies, we have shown that mouse RAW 264.7 macrophages possess pyrimidinoceptors, coupled to a phosphoinositide-specific phospholipase C, with a higher specificity for UTP than for ATP. In the current study, we explored the mechanism involved in the UTP-induced intracellular acidification seen in this cell line. UTP (30 microM) caused a reversible pHi decrease of 0.16 +/- 0.01 unit; this effect was not influenced by the removal of extracellular Cl- or Na+ ions or by pretreatment with 5-(N-ethyl-N-isopropyl)-amiloride (10 microM), 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 microM), staurosporine (1 microM), or Ro 31-8220 (1 microM) but was completely abolished by the removal of extracellular Ca2+. UTP (30 microM), thapsigargin (1 microM), and ionomycin (1 microM) each induced a similar extent of external Ca2+-dependent acidification with a similar time-dependency, but the effects were nonadditive. To further investigate the Ca2+-dependent mechanism, we studied the involvement of arachidonic acid (AA) and eicosanoid metabolites. The addition of AA (10 microM) but not arachidic acid (100 microM) produced a reduction in pHi. UTP, thapsigargin, and ionomycin induced Ca2+-dependent AA release. Furthermore, 4-bromo-phenacyl bromide [30 microM, a phospholipase A2 (PLA2) inhibitor-, nordihydroguaiaretic acid (50 microM, a lipoxygenase inhibitor), and MK-886 (10 microM, a 5-lipoxygenase-activating protein inhibitor) abolished the UTP- or ionomycin-induced responses, whereas indomethacin (30 microM, a cyclooxygenase inhibitor) and baicalein (10 microM, a selective 12-lipoxygenase inhibitor) had no effect. MAFP (a cPLA2 inhibitor) and REV 5901 (a 5-lipoxygenase inhibitor as well as a competitive antagonist of peptide leukotrienes), but not RHC 80267 (a diacylglycerol lipase inhibitor), also inhibited the UTP-induced response. In contrast, the pHi response to AA was unaffected by the presence of 4-bromo-phenacyl bromide or the removal of extracellular Ca2+ ions but abolished by addition of NDGA. Exogenous 5-hydroperoxyeicosatetraenoic acid (2 microM) also produced marked acidification, and UTP and ionomycin both induced peptide leukotriene formation. In conclusion, this is the first report indicating that lipoxygenase metabolites act as mediators of the Ca2+-dependent acidification seen in macrophages in response to UTP or ionomycin via activation of cPLA2 and AA release.
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PMID:Lipoxygenase metabolites as mediators of UTP-induced intracellular acidification in mouse RAW 264.7 macrophages. 946 90

Oxidative stress plays an important role in the induction of T lymphocyte hyporesponsiveness observed in several human pathologies including cancer, rheumatoid arthritis, leprosy, and AIDS. To investigate the molecular basis of oxidative stress-induced T cell hyporesponsiveness, we have developed an in vitro system in which T lymphocytes are rendered hyporesponsive by co-culture with oxygen radical-producing activated neutrophils. We have observed a direct correlation between the level of T cell hyporesponsiveness induced and the concentration of reactive oxygen species produced. Moreover, induction of T cell hyporesponsiveness is blocked by addition of N-acetyl cysteine, Mn(III)tetrakis(4-benzoic acid)porphyrin chloride, and catalase, confirming the critical role of oxidative stress in this system. The pattern of tyrosine-phosphorylated proteins was profoundly altered in hyporesponsive as compared with normal T cells. In hyporesponsive T cells, T cell receptor (TCR) ligation no longer induced phospholipase C-gamma1 activation and caused reduced Ca(2+) flux. In contrast, despite increased levels of ERK1/2 phosphorylation, TCR-dependent activation of mitogen-activated protein kinase ERK1/2 was unaltered in hyporesponsive T lymphocytes. A late TCR-signaling event such as caspase 3 activation was as well unaffected in hyporesponsive T lymphocytes. Our data indicate that TCR-signaling pathways are differentially affected by physiological levels of oxidative stress and would suggest that although "hyporesponsive" T cells have lost certain effector functions, they may have maintained or gained others.
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PMID:Reactive oxygen species differentially affect T cell receptor-signaling pathways. 1191 64

Characteristics of pituitary adenylate cyclase-activating polypeptide (PACAP)-induced increase of Ca(2+) entry and catecholamine (CA) release were studied in bovine adrenal medullary chromaffin cells. PACAP induced intracellular free Ca(2+) concentration ([Ca(2+)](i)), showing an initial transient [Ca(2+)](i) rise followed by a sustained rise and CA release, which were not blocked by the blocking agents for nicotinic acetylcholine receptor (nAChR) channel, the voltage-dependent Ca(2+) channel (VOC), or the Na(+) channel. The sarcoendoplasmic Ca(2+)-ATPase inhibitors thapsigargin and cyclopiazonic acid did not affect the PACAP-induced sustained rise of [Ca(2+)](i), but did inhibit the initial [Ca(2+)](i) rise. In cells pretreated with cyclopiazonic acid or membrane-permeable, low-affinity Ca(2+) chelator N',N',N',N'-tetrakis(2-pyridylmethyl)ethylenediamine, PACAP further stimulated the entry of Ca(2+) or Mn(2+), whereas these treatments masked [Ca(2+)](i) dynamics induced by bradykinin. PACAP-induced sustained [Ca(2+)](i) rise and Mn(2+) entry were enhanced by acidic extracellular solution and reduced by alkalinization, whereas thapsigargin-induced Mn(2+) entry was regulated by the opposite. PACAP-induced [Ca(2+)](i) rise and Mn(2+) entry were not affected by blockers of cAMP-dependent protein kinase, phospholipase C, or protein kinase C. All store-operated Ca(2+) channel (SOC) blocking agents tested inhibited thapsigargin-induced Mn(2+) entry. 1(beta-[3-(4-Methoxyphenyl)-propoxy]-4-methoxyphenylethyl)-1H-imidazole hydrochloride (SK&F 96365), (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-[2-(2,3,4-trimethoxyphenyl)ethyl]-acetamide, and econazole inhibited PACAP-induced Ca(2+) or Mn(2+) entry, whereas GdCl(3), 7,8-benzoflavone, nor-dihydroguaiaretic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, fulfenamic acid, and niflumic acid did not. SK&F 96365 and econazole but not GdCl(3) inhibited PACAP-induced CA release. These results suggest that PACAP activates a novel Ca(2+) entry pathway associated with sustained CA release independent of the nAChR channel, VOC and SOC, activated by acid pH, with different sensitivity to blockers of SOC. This pathway may provide a useful model for the study of receptor-operated Ca(2+) entry.
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PMID:Pituitary adenylate cyclase-activating polypeptide induces a sustained increase in intracellular free Ca(2+) concentration and catechol amine release by activating Ca(2+) influx via receptor-stimulated Ca(2+) entry, independent of store-operated Ca(2+) channels, and voltage-dependent Ca(2+) channels in bovine adrenal medullary chromaffin cells. 1218 54

The vacuolating cytotoxin VacA is an important virulence factor of Helicobacter pylori. Removing glycosylphosphatidylinositol-anchored proteins (GPI-Ps) from the cell surface by phosphatidylinositol-phospholipase C or disrupting the cell actin cytoskeleton by cytochalasin D reduced VacA-induced vacuolation of cells. Using the fluorescent dye 6-methoxy-N-ethylquinolinium chloride, an indicator for cytosolic chloride, we have investigated the role of either GPI-Ps or actin cytoskeleton in the activity of the selective anionic channel formed by VacA at the plasma membrane level. Removal of GPI-Ps from HeLa cell surfaces did not impair VacA localization into lipid rafts but strongly reduced VacA channel-mediated cell influx and efflux of chloride. Disruption of the actin cytoskeleton of HeLa cells by cytochalasin D did not affect VacA localization in lipid rafts but blocked VacA cell internalization and inhibited cell vacuolation while increasing the overall chloride transport by the toxin channel at the cell surface. Specific enlargement of Rab7-positive compartments induced by VacA could be mimicked by the weak base chloroquine alone, and the vacuolating activities of either chloroquine alone or VacA were blocked with the same potency by the anion channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid shown to inhibit VacA channel activity. We suggest that formation of functional VacA channels at the cell surface required GPI-Ps and that endocytosis of these channels by an actin-dependent process increases the chloride content of late endosomes that accumulate weak bases, provoking their enlargement by osmotic swelling.
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PMID:Glycosylphosphatidylinositol-anchored proteins and actin cytoskeleton modulate chloride transport by channels formed by the Helicobacter pylori vacuolating cytotoxin VacA in HeLa cells. 1467 90

Caloporoside is a natural active fungal metabolite, which was isolated from Caloporous dichrous and was described to exhibit antibacterial, antifungal and phospholipase C inhibitory activity. We have previously reported evidence that related beta-linked compounds, lactose and octyl-beta-d-mannoside, bind and functionally modulate rodent GABA(A) receptors, respectively. We have characterized the binding pharmacology of synthetic caloporoside and two further congeners, 2-hydroxy-6-([(16R)-(beta-d-mannopyranosyloxy)heptadecyl]) benzoic acid and octyl-beta-d-glucoside on GABA(A) receptors using a [35S]-t-butylbicyclophosphoorothionate (TBPS) radioligand binding assay. Caloporoside and 2-hydroxy-6-([(16R)-(beta-d-mannopyranosyloxy)heptadecyl]) benzoic acid produced concentration-dependent complete inhibition of specific [35S] TBPS binding with overall apparent IC50 values of 14.7+/-0.1 and 14.2+/-0.1 microM, respectively. In contrast, octyl-beta-d-glucoside elicited a concentration-dependent stimulation of specific [35S] TBPS binding (E(max)=144+/-4%; EC50=39.2+/-22.7 nM). The level of stimulation was similar to that elicited by diazepam (E(max)=147+/-6%; EC50=0.8+/-0.1 nM), and was occluded by GABA (0.3 microM). However, the three test compounds failed to elicit any significant effect (positive or negative) upon [3H] flunitrazepam or [3H] muscimol binding, indicating that they did not bind directly, or allosterically couple, to the benzodiazepine or agonist binding site of the GABA(A) receptor, respectively. The constituent monosaccharide, glucose, and both the closely related congeners octyl-beta-d-glucoside or hexyl-beta-d-glucoside have no significant effect upon [35S] TBPS binding. These data, together, provide strong evidence that a beta-glycosidic linkage and chain length are crucial for the positive modulation of [35S] TBPS binding to the GABA(A) receptor by this novel chemical class.
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PMID:Radioligand binding studies of caloporoside and novel congeners with contrasting effects upon [35S] TBPS binding to the mammalian GABA(A) receptor. 1616 65


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