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)

This study involved an investigation of the role of phospholipase C (PLC) in generating repetitive Ca2+ spikes at fertilization. Using a PCR-based strategy we have demonstrated that mouse oocytes have mRNA coding for PLC beta 1, PLC beta 3 and PLC gamma isoenzymes. Furthermore, immunodetection of PLC gamma 1 using monoclonal antibodies reveals that PLC gamma 1 protein is present in mature mouse oocytes, ruling out the possibility that mRNA was being transcribed but not expressed. We were unsuccessful at detecting the presence of PLC beta protein, but the presence of this isoform can be inferred from functional studies. The PLC inhibitor, U73122, exerted an inhibitory effect on oocytes activated by spermatozoa or acetylcholine at concentrations of 10 and 30 microM respectively, while its inactive analogue had no effect. The soluble tyrosine kinase inhibitors, genistein (100 microM), herbimycin (10 microM) and geldanamycin (0.6 microM) which could affect signalling through PLC gamma hindered but never completely inhibited Ca2+ spiking in response to fertilization. We conclude that the activation of PLC to generate InsP3 may play a critical role in fertilization.
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PMID:Phospholipase C in mouse oocytes: characterization of beta and gamma isoforms and their possible involvement in sperm-induced Ca2+ spiking. 868 4

Image analysis techniques have been used to demonstrate that progesterone induces a rapid calcium transient in the acrosomal domain of greater than 90% of human spermatozoa (n = 2354). These results are at variance with previous reports, suggesting that progesterone receptors are only expressed on a small subpopulation of these cells, by virtue of their ability to bind fluorescent probes incorporating progesterone 3- (O-carboxymethyl) oxime conjugated to BSA. In the present study, we could confirm that such probes only bound to a small proportion of human spermatozoa (3.01 +/- 0.29%; n = 7557) although 91.79 +/- 1.8% of the same sperm populations exhibited a calcium transient in response to progesterone. These results indicate that the binding of labeled progesterone conjugates to human spermatozoa does not reflect the size of the progesterone responsive population; the response elicited by this steroid is essentially ubiquitous. Progesterone action was shown to involve an influx of extracellular calcium via mechanisms that did not involve voltage sensitive- or second messenger operated-channels, phospholipase C, or G proteins. Despite previous evidence suggesting that progesterone action might involve a GABAA receptor/chloride channel, neither GABA nor the GABA agonist muscimol had any effect on intracellular calcium concentrations in human spermatozoa or influenced their functional competence. The only factor that disrupted the responses of human spermatozoa to progesterone was this steroid itself. Progesterone exposure induced a prolonged period of refractoriness to further stimulation that influenced the capacity of these cells to generate calcium transients, and their ability to exhibit a biological response to changes in intracellular calcium. There are implications in these results for our understanding of the extragenomic action of progesterone on human spermatozoa and the clinical manipulation of this system for the assessment and suppression of human sperm function.
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PMID:The extragenomic action of progesterone on human spermatozoa: evidence for a ubiquitous response that is rapidly down-regulated. 875 77

Presence and intracellular distribution of phosphoinositide-specific phospholipase C, phosphatidylinositol 4,5-bisphosphate and protein kinase C have been investigated in rat maturing germ cells and spermatozoa. The isoforms beta 1 and gamma 1 of phosphoinositide-specific phospholipase C were immunologically identified and found to be predominantly nuclear or cytoplasmic and nuclear, respectively. The two enzymes were present in the maturing cell lineage of the seminiferous tubule, except for the nucleus of late spermatids, and absent in spermatozoa, in which, however, a phosphoinositide-specific phospholipase C activity persisted, due to yet uncharacterized enzyme(s). Protein kinase C paralleled these developmental changes, and was completely down-regulated in both total cell homogenates and isolated nuclei obtained from spermatozoa. On the contrary, phosphatidylinositol 4,5-bisphosphate, present at the nuclear level in all cell types, accumulated in the nuclei of late spermatids and spermatozoa. These data support the contention that the spermatozoon nucleus stores a lipid-dependent signaling apparatus which could be reactivated either during sperm maturation or at fertilization.
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PMID:Nuclear phosphoinositide-specific phospholipase C, phosphatidylinositol 4,5-bisphosphate and protein kinase C during rat spermatogenesis. 890 92

Testicular myoid cells surrounding the seminiferous tubule are contractile cells responsible for peritubular contractility and for the propulsion of tubular fluid and spermatozoa. We have investigated the contractile response of rat myoid cells to endothelins (ETs) in cell and organ culture and analyzed the cell signaling involved. ET-2, ET-3, and IRL 1620, a highly selective agonist of ETB receptor, elicit [Ca2+]i increases, though with dissimilar potencies and kinetics. Competition binding assays using [125I]ET-1, [125I]ET-3 and [125I]IRL 1620 show that myoid cells express both ETA and ETB receptors with high affinity for ET-1 and ET-1/ ET-3, respectively. All endothelin isoforms activate phosphoinositide (PI) turnover, but only stimulation of the ETA receptor mediates both PI turnover and mobilization of [Ca2+]i. Although stimulation of the ETB receptor with IRL 1620 fails to produce a significant effect on inositol phosphate (IP) production, it induces mobilization of a thapsigargin-sensitive intracellular Ca2+ pool in the absence of any measurable IP production. We also studied the effect of U-73122 [1-(6-[17-beta-3-methoxyestra-1,3,5 (10)-trien-17-yl] amino/hexyl)-1H-pirrole-2,5-dione] and its inactive analog, U-73343, on Ca2+ mobilization and IP production after selective stimulation of ET receptors. U-73122 (1 microM) completely inhibited the effect of ETA-mediated ET-1 stimulation of IP production, whereas U-73343 was inactive. However, in the presence of U-73122, the selective stimulation of ETB receptors induced the mobilization of a thapsigargin-sensitive and inositol phosphate-independent intracellular Ca2+ pool. The ETB receptor-dependent mobilization of [Ca2+]i resulted mainly from Ca2+ release from intracellular Ca2+ stores. This paper illustrates contraction of myoid cells in the seminiferous tubule in response to selective activation of either ET receptor. Scanning electron microscopy of the peritubular tissue demonstrates that the contractile response to ET was inhibited by a combination of BQ-123 and BQ-788, but not by either antagonist alone. Moreover, the observation that selective stimulation of ETB receptor with IRL 1620 also resulted in cell contraction strongly suggests that stimulation of either ETA or ETB receptors alone may be sufficient to elicit seminiferous tubule contractility. Two types of receptors account for the actions of endothelin on contractile activity of seminiferous tubule: 1) an ETA receptor that is positively coupled to phospholipase C (PLC) and Ca2+ mobilization; and 2) an ETB receptor that induces the mobilization of a thapsigargin-sensitive intracellular Ca2+ pool in a manner independent of the formation of inositol phosphates. ET may play a complex role in regulating the flux of spermatozoa along the seminiferous tubule through its contractile effect on peritubular myoid cells.
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PMID:Dual control of seminiferous tubule contractility mediated by ETA and ETB endothelin receptor subtypes. 906 17

The binding of the spermatozoon to the oocyte zona pellucida (ZP) occurs via specific receptors localized over the anterior head region of the spermatozoon. Zona pellucida binding stimulates the spermatozoa to undergo the acrosome reaction resulting in the release of hydrolytic enzymes and in the exposure of new membrane domains, both of which are essential for fertilization. We suggest that ZP binds to at least two different receptors in the plasma membrane. One (R) is a Gi-coupled receptor that activates phospholipase C (PLC) beta 1. The other (TK) is a tyrosine kinase receptor coupled to PLC gamma. Binding to R would regulate adenylyl cyclase (AC) leading to elevation of cAMP and protein kinase (PKA) activation. The PKA activates a voltage-dependent Ca2+ channel in the outer acrosomal membrane which releases Ca2+ from the interior of the acrosome to the cytosol. This is the first, relatively small, rise in [Ca2+]i (I) which leads to activation of the PLC gamma. The products of phosphatidyl-inositol bisphosphate (PIP2) hydrolysis by PLC diacylglycerol (DAG) and inositol-trisphosphate (IP3) will lead to PKC translocation to the plasma membrane and its activation. PKC opens a voltage-dependent Ca2+ channel (L) in the plasma membrane, leading to the second (II) higher increase in [Ca2+]i. The Gi or TK can also activate an Na+/H+ exchanger leading to alkalization of the cytosol. PKC also activates phospholipase A2 (PLA2) to generate arachidonic acid (AA) from membrane phospholipids. AA will be converted to prostaglandins (PG) and leukotriens (LT) by the enzymes cyclooxygenase (COX) and lipoxygenase (LOX) respectively. The increase in [Ca2+]i and pH leads to membrane fusion and acrosomal exocytosis.
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PMID:The biochemistry of the acrosome reaction. 923 45

Using human spermatozoa stimulated with either progesterone or the Ca2+ ionophore A23187 to undergo acrosomal exocytosis, we have investigated potential pathways for generation of diacylglycerol (DAG) and have examined the possibility that DAG plays an important role in the exocytotic response. Both treatments resulted in rapid and considerable generation of DAG, followed by a limited rise in phosphatidic acid (PA). Further experiments indicated that phospholipase C (PLC) activity is important in this generation of DAG, but phospholipase D activity probably is not. In addition, polyphosphoinositide-specific phosphoinositidase C activation and hydrolysis, of phosphatidylinositol 4,5-bisphosphate appears to be a necessary prerequisite for activation of the PLC pathway. Finally the DAG formed appears to be important in acrosomal exocytosis: (i) blocking DAG metabolism with a DAG kinase inhibitor resulted in both increased endogenous levels of DAG and a significantly increased exocytotic response in stimulated cells and (ii) exogenous DAG induced exocytosis in capacitated spermatozoa whereas PA did not. Taken together, these results suggest that DAG plays a key role in events leading to membrane fusion during human sperm acrosomal exocytosis stimulated by natural agonists.
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PMID:A role for diacylglycerol in human sperm acrosomal exocytosis. 923 98

We have investigated pathways of lipid metabolism in spermatozoa and generation of various metabolites with potential messenger functions during exocytosis stimulated with A23187/Ca2+. Stimulation of boar spermatozoa resulted in a considerable rapid increase in saturated/unsaturated 1,2-diacylglycerol (1,2-SU-DAG) and, concomitantly, a substantial reduction in disaturated 1,2-diacylglycerol (1,2-DS-DAG), and in phosphatidylcholine (PC). These changes preceded the onset of exocytosis. Phosphatidic acid was sometimes generated in parallel, but usually rose later, suggesting that 1,2-SU-DAG may be formed directly by phospholipase C action. Lipid changes observed in stimulated spermatozoa that have been prelabelled with several lipid precursors ([14C]palmitic acid, [14C]glycerol, [14C]choline, or [14C]arachidonic acid) suggested the existence of a unique process involving the utilization of the high basal levels of 1,2-DS-DAG to form 1,2-SU-DAG, with the latter being subsequently employed to replenish the PC pool. An ensuing generation of lysoPC and arachidonic acid, which paralleled the occurrence of exocytosis, revealed that the newly synthesized PC was hydrolyzed by phospholipase A2. The highest levels of 1,2-SU-DAG, minimum levels of 1,2-DS-DAG, and the regeneration of the PC pool were tightly coupled to the beginning of visible exocytosis. These results suggest that changes in these lipid metabolites may be fundamental processes during acrosomal exocytosis occurring in response to physiological agonists.
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PMID:Diacylglycerol species as messengers and substrates for phosphatidylcholine re-synthesis during Ca2+-dependent exocytosis in boar spermatozoa. 926 66

Acrosomal exocytosis occurs after the binding of the spermatozoon to the zona pellucida of the oocyte via specific receptors. We suggest that the zona pellucida binds to at least two different receptors in the plasma membrane. One (R) is a Gi-coupled receptor that activates phospholipase C beta 1. The other (TK) is a tyrosine kinase receptor coupled to phospholipase C gamma. Binding to R would regulate adenylyl cyclase leading to an increase in cyclic adenosine monophosphate and protein kinase A activation. The protein kinase A activates a voltage-dependent Ca2+ channel in the outer acrosomal membrane that releases Ca2+ from the interior of the acrosome to the cytosol. This is the first (I), relatively small, rise in intracellular Ca2+ which leads to activation of the phospholipase C gamma. The products of phosphatidyl-inositol bisphosphate hydrolysis by phospholipase C, diacylglycerol and inositol-trisphosphate lead to protein kinase C translocation to the plasma membrane and its activation. Protein kinase C opens a voltage-dependent Ca2+ channel (L) in the plasma membrane, leading to the second (II), higher, increase in intracellular Ca2+ leading to acrosomal exocytosis. Spermine, a physiological constituent of the seminal plasma regulates sperm acrosomal exocytosis by modulating intracellular Ca2+ binding sites and phospholipase C activity. Spermine is rapidly incorporated into the sperm cells during ejaculation and temporarily inhibits premature capacitation and acrosome reaction. During the passage of the spermatozoon through the female genital tract, there is a progressive depletion of spermine from spermatozoa, so that capacitation and consequently the acrosomal exocytosis take place at the appropriate time, when the spermatozoon reaches the vicinity of the egg.
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PMID:Regulatory mechanisms in acrosomal exocytosis. 941 80

Microinjection of a truncated form of the c-kit tyrosine kinase present in mouse spermatozoa (tr-kit) activates mouse eggs parthenogenetically, and tr-kit- induced egg activation is inhibited by preincubation with an inhibitor of phospholipase C (PLC) (Sette, C., A. Bevilacqua, A. Bianchini, F. Mangia, R. Geremia, and P. Rossi. 1997. Development [Camb.]. 124:2267-2274). Co-injection of glutathione-S-transferase (GST) fusion proteins containing the src-homology (SH) domains of the gamma1 isoform of PLC (PLCgamma1) competitively inhibits tr-kit- induced egg activation. A GST fusion protein containing the SH3 domain of PLCgamma1 inhibits egg activation as efficiently as the whole SH region, while a GST fusion protein containing the two SH2 domains is much less effective. A GST fusion protein containing the SH3 domain of the Grb2 adaptor protein does not inhibit tr-kit-induced egg activation, showing that the effect of the SH3 domain of PLCgamma1 is specific. Tr-kit-induced egg activation is also suppressed by co-injection of antibodies raised against the PLCgamma1 SH domains, but not against the PLCgamma1 COOH-terminal region. In transfected COS cells, coexpression of PLCgamma1 and tr-kit increases diacylglycerol and inositol phosphate production, and the phosphotyrosine content of PLCgamma1 with respect to cells expressing PLCgamma1 alone. These data indicate that tr-kit activates PLCgamma1, and that the SH3 domain of PLCgamma1 is essential for tr-kit-induced egg activation.
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PMID:Involvement of phospholipase Cgamma1 in mouse egg activation induced by a truncated form of the C-kit tyrosine kinase present in spermatozoa. 972 17

Epididymal mouse spermatozoa have a surface-associated decapacitation factor (DF) that can be removed precociously by centrifugation, resulting in acceleration of capacitation and increased fertilizing ability. Addition of exogenous DF to capacitated suspensions inhibits fertilizing ability and reverses capacitation in acrosome-intact cells. DF appears to regulate a Ca2+-ATPase, located primarily in the post-acrosomal region. The present investigations of DF<-->spermatozoon interaction indicate that DF can be removed from uncapacitated cells by treatment with phosphatidylinositol-specific phospholipase C (PIC), suggesting the involvement of a glycosylphosphatidylinositol (GPI) moiety. However, exogenous DF cannot reassociate with PIC-treated spermatozoa, suggesting that DF may bind to spermatozoa via a GPI-anchored receptor. DF binding appears to involve fucose residues, since depletion of endogenous DF followed by brief exposure to fucose (0.1-10 mM) prevented DF reassociation with cells. Furthermore, 5 mM fucose could displace DF from uncapacitated cells, accelerating capacitation and resulting in a higher proportion of fertilized oocytes, with increased polyspermy, than obtained with untreated controls. FITC-labelled fucosylated BSA bound specifically to the postacrosomal region, binding being inhibited by both excess fucose and crude DF. UEA I, a lectin with specificity for fucose residues, bound to the postacrosomal region of cells preincubated in fucose but not crude DF, and blocked DF binding to DF-depleted cells. These results are consistent with the DF binding, via fucose residues, to a GPI-anchored receptor. Fucose binding sites are in the same region where Ca2+-ATPase, the enzyme regulated by DF, has been localized; these results support the hypothesis that DF modulates capacitation by regulating enzyme activity and hence the intracellular Ca2+ concentration.
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PMID:Interactions between a decapacitation factor and mouse spermatozoa appear to involve fucose residues and a GPI-anchored receptor. 974 Mar 27


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