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

Leukotriene B4 (LTB4) induced rapid breakdown of prelabeled inositol phospholipids in rat peritoneal polymorphonuclear leukocytes (PMNs). Formation of [3H]inositol triphosphate ([3H]IP3) was rapid, with a peak of 250-300% of the control level, after 5-15 sec of exposure to LTB4. Accumulation of [3H]inositol bisphosphate was rapid, peaking after 30 sec of treatment. Accumulation of [3H]inositol monophosphate was also rapid in the presence of LiCl. The kinetics of [3H]IP3, [3H]inositol bisphosphate, and [3H]inositol monophosphate accumulation suggest that LTB4 may interact with receptors in PMNs and activate phospholipase C which in turn induces hydrolysis of inositol-phospholipids. The agonist activities of several LTB4 analogs were employed to investigate the structure-activity relationships of LTB4 receptor-mediated activation of phosphatidylinositol hydrolysis. Increases in [3H]IP3 formation were dependent upon the concentration of LTB4 and the agonist analogs. The rank order potency of these analogs was equivalent to that of the pharmacological activity of LTB4 agonists in the PMN chemotaxis assay. Furthermore, the islet activation protein isolated from Bordetella pertussis inhibited LTB4-induced [3H]IP3 formation. The tumor-promoting phorbol myristate acetate also inhibited LTB4-induced [3H]IP3 formation. The LTB4 receptors on a partially purified PMN membrane were characterized. LTB4 binding to the receptors was stereoselective and specific. The binding affinity (Kd) of [3H] LTB4 to the receptors was 1.3 +/- 0.2 nM. The maximum density of binding was 5.5 +/- 1.8 pmol/mg of protein. The rank order potency of binding affinities of several LTB4 analogs was equivalent to that of the induction of IP3 response induced by LTB4 and analogs. These results suggest that LTB4 may interact with receptors in rat PMNs, activate G protein-regulated phospholipase C, and induce [3H]IP3 formation.
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PMID:Leukotriene B4 induces formation of inositol phosphates in rat peritoneal polymorphonuclear leukocytes. 301 80

The primary action of a family of mitogens including bombesin, bradykinin, vasopressin and alpha-thrombin is to activate the hydrolysis of polyphosphoinositides. Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by phospholipase C is mediated through coupling of surface receptors to a GTP-binding protein (Gp protein) which, in some cells, is inactivated by the toxin of Bordetella pertussis. It is not known whether this signalling pathway is involved in initiating DNA replication, whereas it has been firmly established that reinitiation of DNA synthesis can be triggered without activation of PtdIns(4,5)P2 hydrolysis by, for example, EGF (epidermal growth factor), FGF (fibroblast growth factor) and insulin/IGF-I (insulin-like growth factor-I), members of a class of mitogens known to activate receptor tyrosine kinases. Taking advantage of the fact that Chinese hamster lung fibroblasts respond to either class of mitogens and that their Gp protein appears to be sensitive to pertussis toxin, we have now analysed the toxin's effect on reinitiation of DNA synthesis and find that it inhibits up to 95% of thrombin-induced mitogenicity without affecting EGF- or FGF-induced DNA synthesis and proliferation. These findings strongly suggest that activation of PtdIns(4,5)P2-phospholipase C has a determinant function in growth control, and confirm the existence of alternative growth factor-signalling pathways independent of polyphosphoinositide breakdown.
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PMID:Two growth factor signalling pathways in fibroblasts distinguished by pertussis toxin. 303 10

The increased sympathetic neurotransmission in benign prostatic hyperplasia (BPH) results in a alpha 1C-adrenoceptor-mediated increase in prostatic smooth muscle tone which seems to be responsible for the dynamic infravesical obstruction occurring in BPH. The prostatic smooth muscle contractions evoked by norepinephrine can be efficiently blocked by alpha 1-adrenoceptor blockers. Moreover, an impressive number of clinical trials illustrated the beneficial results of alpha 1-adrenoceptor blockers in the treatment of BPH. However, despite knowledge of alpha 1-adrenergic neurotransmission and the clinical application of its blockade by selective alpha 1-adrenoceptor antagonists, very little is known about the intracellular pathways involved in the regulation of prostatic smooth muscle contractility. To study the intracellular mechanism of the alpha 1C-adrenoceptor-induced prostatic smooth muscle contraction, the patch-clamp technique in the whole-cell configuration mode combined with the Fura-II fluorescence technique was used in human, enzymatically isolated smooth muscle cells obtained from patients undergoing transurethral resection of the prostate because of symptomatic BPH. Furthermore changes in prostatic smooth muscle contractility were registered in organ bath experiments. Application of the selective alpha 1-adrenoceptor agonist phenylephrine (PE) increased the L-type Ca(2+)-channel current (ICa) dose dependently from 8 up to 18.5 microA/cm2, simultaneously elevating the free cytoplasmic Ca2+ concentration ([Ca2+]i) up to 1.9 microM. Pretreating the myocytes with pertussis toxin, an exotoxin of Bordetella pertussis which inactivates GTP-binding proteins (G proteins) of the Gi and G(o) family by ADP ribosylation, reduced the PE-induced ICa stimulation by 71.5 +/- 5.6% (n = 21). Dialysis of the cytosol with the second messenger inositol-1,4,5-trisphosphate (IP3), which releases Ca2+ from intracellular non-mitochondrial, IP3-sensitive Ca2+ pools, imitated the PE-evoked responses. Pretreating the myocytes with the Ca(2+)-release blockers ryanodine (10-100 microM, n = 8), thapsigargin (0.1 microM, n = 11) or low-molecular weight heparin (n = 14) largely attenuated the PE-evoked responses. The experimental results suggest a coupling of alpha 1-adrenoceptors to phospholipase C-converting phosphoinositol-4,5-bisphosphate into diacylglycerol, an endogenous activator of the protein kinase C and IP3 which releases Ca2+ from intracellular stores stimulating ICa via Ca(2+)-calmodulin-dependent protein kinase induced phosphorylation of voltage-dependent Ca2+ channels. This knowledge could be of interest for conservative treatment in symptomatic BPH.
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PMID:Regulation of prostatic smooth muscle contractility by intracellular second messengers: implications for the conservative treatment of benign prostatic hyperplasia. 753 57

The steroid hormone 1,25(OH)2-vitamin D-3 [1,25(OH)2D3] stimulated phospholipase A2 (PLA2) activity in embryonic chick myoblasts releasing [3H]arachidonic acid from the sn-2 position of phospholipids. GTP-binding protein mediation of 1,25(OH)2D3-dependent PLA2 activity was investigated in cells prelabeled with [3H]arachidonic acid. AIF4-, a G-protein activator, mimicked 1,25(OH)2D3-stimulated arachidonic acid release from myoblasts in a dose-dependent manner. Consistent with the involvement of a G-protein in the activation of PLA2 by the hormone, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), a stable GTP analogue which activates G-protein mediated signals, strongly enhanced arachidonic acid release in myoblasts. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which competitively inhibits G-protein activation by GTP and its analogues, abolished 1,25(OH)2D3-dependent arachidonic acid release. Bordetella pertussis toxin pretreatment significantly suppressed the hormone action whereas cholera toxin had minor effects on 1,25(OH)2D3 action. Hormone-induced activation of PLA2 was mimicked by the Ca2+ ionophore A23187 and blocked by nifedipine, but was unaffected by neomycin, a phospholipase C inhibitor, ruling out the contribution of phosphoinositide metabolism to arachidonic acid release. These results suggest that 1,25(OH)2D3-stimulation of PLA2 activity in embryonic chick myoblasts is mediated by a pertussis toxin-sensitive GTP-binding protein coupled to influx of extracellular calcium.
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PMID:1,25(OH)2-vitamin D-3 stimulates phospholipase A2 activity via a guanine nucleotide-binding protein in chick myoblasts. 764 3

The steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] activates in chick myoblasts the breakdown of phosphoinositides by phospholipase C and the hydrolysis of phosphatidylcholine by phospholipase D. Extracellular Ca2+ requirement and GTP-binding protein mediation of 1,25(OH)2D3-dependent activation of phospholipases C and D were investigated in cells prelabelled with [3H]glycerol or [3H]arachidonic acid. Generation of diacylglycerol by phospholipase C and phosphatidylethanol by phospholipase D were shown to be dependent on extracellular calcium, since both responses were suppressed by EGTA and the Ca(2+)-channel blockers nifedipine and verapamil, and were mimicked by the calcium ionophore A23187. The G-protein activators guanosine 5'-O-(3-thiotriphosphate) and AlF4- strongly enhanced diacylglycerol and phosphatidylethanol release in myoblasts while guanosine 5'-O-(2-thiodiphosphate), which inhibits G-protein-mediated signals, abolished 1,25(OH)2D3-dependent diacylglycerol and phosphatidylethanol release. Bordetella pertussis toxin pretreatment suppressed the hormone action. These results suggest that 1,25(OH)2D3-stimulation of phosphoinositide-specific phospholipase C and phospholipase D in chick myoblasts is mediated by a pertussis-sensitive GTP-binding protein(s) and the influx of extracellular calcium.
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PMID:1,25(OH)2-vitamin D3 stimulation of phospholipases C and D in muscle cells involves extracellular calcium and a pertussis-sensitive G protein. 890 51

We have examined the effects in vitro of calcitriol [1,25(OH)2D3], the hormonal form of vitamin D3, on the breakdown of membrane phosphoinositides in skeletal muscle from young (3 months) and aged (24 months) rats. Calcitriol (10(-9) M) induced a rapid and transient release of IP3/inositol phosphates and diacylglycerol (DAG) from muscle slices/membranes prelabeled with [3H]myo-inositol and [3H]arachidonate, respectively. Inositol phosphate release was maximal at 15 s and then declined. The effects of hormone specificity exhibited as the closely related derivatives of vitamin D3, 25OHD3, 1alphaOHD3 and 24,25(OH)2D3 did not alter muscle inositol phosphate levels. The stimulation of DAG was biphasic, the early phase (15 s) being abolished by neomycin (0.5 mM), an inhibitor of phosphoinositide hydrolysis, similar to IP3 formation and consistent with a role of phospholipase C (PLC) in intracellular signal generation. Neomycin had no effect on the second DAG peak (2 min) induced by calcitriol, suggesting that the late phase of DAG formation is independent from the hydrolysis of phosphoinositides. Higher basal inositol phosphate and DAG levels were detected in muscle from aged rats thereby reducing the effects of the hormone on second messenger generation ( -80 and -60% for IP3 and DAG, respectively). Calcitriol stimulation of PLC was mimicked, in both young and old rats, by GTPgammaS, a non-hydrolyzable analogue of GTP, while GDPbetaS, a G protein inhibitor, suppressed the effect of the hormone. The early effects of calcitriol and GTPgammaS were not additive. Bordetella pertussis toxin abolished by 85% the effects of calcitriol on inositol phosphate release in young rats but was without effect in aged animals. These results demonstrate that calcitriol activates phosphoinositide-PLC in rat skeletal muscle by a mechanism which involves a pertussis-sensitive G protein and that the effects of the hormone are altered with ageing.
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PMID:Age-related loss of calcitriol stimulation of phosphoinositide hydrolysis in rat skeletal muscle. 954 16

We compared the effects of Pasteurella multocida toxin (PMT) with Bordetella bronchiseptica dermonecrotizing toxin (DNT) at a cellular level under same conditions. Both PMT and DNT cause actin stress fiber formation in MC3T3-E1 cells which is known to be regulated by the small GTP-binding protein Rho. DNT induced mobility shifts of Rho on SDS-polyacrylamide gel electrophoresis, indicating direct modification as reported elsewhere. In contrast, no alternations in the electrophoretic mobility of Rho were found in lysates from PMT-treated cells. PMT but not DNT increased the intracellular level of inositol phosphates, indicating the elevation of phospholipase C (PLC) activity in the PMT-treated cells. These results indicate that PMT does not have Rho as a target but activates PLC. The formation of actin stress fiber by PMT seems to be stimulated through the indirect activation of Rho, which resides downstream of PLC, PMT and DNT seem to elicit similar toxic effects, at least in part, through the activation of Rho.
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PMID:Pasteurella multocida toxin and Bordetella bronchiseptica dermonecrotizing toxin elicit similar effects on cultured cells by different mechanisms. 956 Jul 76

The gene coding for the G-protein alphaq subunit was interrupted by homologous recombination in murine embryonic stem cells (alphaq-null ES cells) as detected by Southern analysis and reverse-transcriptase PCR. The bradykinin (BK) B2 receptor was stably transfected into wild-type (WT) alphai-2-null and alphaq-null ES cells. The B2 receptor bound BK with high affinity and mobilized Ca2+. BK also activated phospholipase C (PLC), as determined by total inositol phosphate (IP) accumulation in a Bordetella pertussis toxin- and genistein-insensitive manner. In WT and alphai-2-null ES cells, BK increased IP levels approx. 4-fold above baseline. Most interestingly, in alphaq-null ES cells, BK increased IP accumulation approx. 9-fold above baseline. Re-expression of alphaq in alphaq-null ES cells resulted in normalization of the BK-stimulated IP accumulation (4-fold above baseline). These results suggest that the B2 receptor activates PLC through more than one member of the Gq family. Additionally, the absence of alphaq alters the kinetics of IP generation, which may reflect intrinsic characteristics of individual members of the Gq family or a decreased susceptibility to heterologous regulation in the alphaq-null ES cells, thus allowing for a more sustained generation of IP.
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PMID:Enhanced bradykinin-stimulated phospholipase C activity in murine embryonic stem cells lacking the G-protein alphaq-subunit. 958 59

We previously reported that calcitriol [1,25(OH)2-vitamin D3] in rat skeletal muscle and duodenum stimulates the hydrolysis of polyphosphoinositides by phospholipase C (PLC), generating the second messengers inositol trisphosphate (IP3) and diacylglycerol (DAG), and that this mechanism is altered in old animals. As previously reported in muscle, we show in the present study that GTPgammaS (100 microM, 15 s), the non-hydrolyzable analogue of GTP, increased IP3 release from young rats duodenum to the same extent as 1 nM calcitriol (+ 100%), while GDPbetaS (100 microM) suppressed hormone-dependent IP3 production. Similarly to calcitriol, GTPgammaS response was diminished in old rats. Contrary to muscle, pretreatment with Bordetella pertussis toxin did not modify calcitriol-dependent IP3 in duodenum. The antibody, anti-G alpha q/11 (1:200) and anti-G alpha i (1:200) blocked calcitriol-dependent IP3 release in muscle from young rats, indicating that the hormone activates an isoform of PLC coupled to the alpha subunit of Gq/11 and possibly the betagamma subunits of Gi. The aged muscle was insensitive to anti G alpha i. In rat duodenum the hormone effects were suppressed by anti-Gq/11 both in young and aged animals. In 24-month-old rats, Gq/11 and Gi protein levels were greatly reduced both in muscle and duodenum, suggesting that a deficiency in G protein expression with aging may have important consequences for correct receptor/effector coupling and could explain age-related declines in the function of second messenger systems linked to G-proteins.
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PMID:Aging and calcitriol regulation of IP3 production in rat skeletal muscle and intestine. 1128 Jul 8

In this report we describe the 1,500-fold purification and characterization of the haemolytic phospholipase C (PLC) of Pseudomonas aeruginosa, the paradigm member of a novel PLC/phosphatase superfamily. Members include proteins from Mycobacterium tuberculosis, Bordetella spp., Francisella tularensis and Burkholderia pseudomallei. Purification involved overexpression of the plcHR1,2 operon, ion exchange chromatography and native preparative polyacrylamide gel electrophoresis. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry confirmed the presence of two proteins in the purified sample with sizes of 17,117.2 Da (PlcR2) and 78,417 Da (PlcH). Additionally, liquid chromatography electrospray mass spectrometry (LCMS) revealed that PlcH and PlcR2 are at a stoichiometry of 1 : 1. Western blot analysis demonstrated that the enzyme purifies as a heterodimeric complex, PlcHR2. PlcHR2 is only active on choline-containing phospholipids. It is equally active on phosphatidylcholine (PC) and sphingomyelin (SM) and is able to hydrolyse plasmenylcholine phospholipids (plasmalogens). Neither PlcHR2 nor the M. tuberculosis homologues are inhibited by D609 a widely used, competitive inhibitor of the Bacillus cereus PLC. PlcH, PlcR2, and the PlcHR2 complex bind calcium. While calcium has no detectable effect on enzymatic activity, it inhibits the haemolytic activity of PlcHR2. In addition to being required for the secretion of PlcH, the chaperone PlcR2 affects both the enzymatic and haemolytic properties of PlcH. Inclusive in these data is the conclusion that the members of this PC-PLC and phosphatase family possess a novel mechanism for the recognition and hydrolysis of their respective substrates.
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PMID:A novel class of microbial phosphocholine-specific phospholipases C. 1241 Aug 24


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