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)

1. Spontaneous inhibitory postsynaptic currents (IPSCs) and evoked IPSCs were recorded by a whole-cell patch-recording technique from cultured Purkinje cells of the rat. The size of spontaneous IPSCs, after a train of depolarizing pulses was applied to the Purkinje cells, increased to 163 +/- 6% (mean +/- S.E.M., n = 7 cells) of the control levels measured before the stimulus train. 2. The GABAergic postsynaptic currents were recorded under voltage clamp from the synapse formed between two Purkinje cells. These IPSCs increased to 218 +/- 31% (n = 4) of control levels after depolarizing stimulation was applied to the postsynaptic Purkinje cells. Size-increased IPSCs were observed as long as recording continued and the phenomena will be called potentiation in this paper. 3. Intracellular application of Ruthenium Red (20 microM) did not block the potentiation of spontaneous IPSCs induced by the depolarizing stimulus (165 +/- 9%, n =6), but heparin (2 mg ml-1) partially blocked the potentiation (123 +/- 10%, n = 6). Heparin applied together with Ruthenium Red (20 microM) blocked potentiation completely (96 +/- 5%, n = 8) at concentrations higher than 1 mg ml-1. 4. Intracellular free calcium concentrations ([Ca2+]i) was monitored as the ratio of fura-2 fluorescences excited at 340 and 380 nm. In control cells, [Ca2+]i was increased by each depolarizing pulse. When Purkinje cells were dialysed with heparin or heparin with Ruthenium Red, the rise in [Ca2+]i was suppressed. 5. Bath application of thapsigargin (1 microM) blocked the potentiation (99 +/- 2%, n = 4) and suppressed the rise in [Ca2+]i. 6. When 30 mM BAPTA was applied intracellularly, a train of depolarizing pulses failed to induce potentiation of IPSCs and failed to raise [Ca2+]i. The results from points 3-6 suggest that the increase in [Ca2+]i, most probably coupled with the release from intracellular stores especially from the inositol trisphosphate (IP3)-sensitive stores, is crucial for the potentiation of IPSCs. 7. Bath application of a metabotropic glutamate receptor activator (t-ACPD, 200 microM) increased both the amplitude and frequency of spontaneous IPSCs and increased the [Ca2+]i slightly in dendrites. The inward current induced by the puff-applied GABA (2 microM) was increased, after t-ACPD application, to 186 +/- 36% of the control level (n = 3). Bath application of quisqualate (2 microM) caused a rapid increase in [Ca2+]i in dendrites and in the cell body and increased both the amplitude and frequency of spontaneous IPSCs. 8. The bath application of an inhibitor of phospholipase C (PLC), U73122 (1 microM), suppressed a rise in [Ca2+]i and blocked the potentiation (106 +/- 3%, n = 5). The inactive form, U73343 (1 microM), did not affect the potentiation (151 +/- 11%, n = 7) or the rise in [Ca2+]i. These observations suggest a possible involvement of the mechanism of Ca2+ activation of PLC and the IP3-induced Ca2+ release in the induction of IPSC potentiation in Purkinje cells.
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PMID:Release of Ca2+ is the crucial step for the potentiation of IPSCs in the cultured cerebellar Purkinje cells of the rat. 900 48

In U373 MG cells, a line derived from a human astrocytoma, histamine stimulated the release of [3H]gamma-aminobutyric acid ([3H]GABA) in a concentration-dependent manner (286 +/- 23% of basal release at 1 mM histamine). Neither Ca2+ removal nor Cd2+ (100 microM) affected [3H]GABA release evoked by 100 microM histamine but the response was significantly reduced by 10 microM U-73122 ({1-[6-((17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl)-amino)-hexyl]-1 H-pyrrole-2,5-dione}), an inhibitor of phospholipase C activation (79 +/- 8% inhibition) and by 10 microM dimethylbenzamil, a selective blocker of plasma membrane Na+/Ca2+ exchange (58 +/- 6% inhibition). In [3H]inositol-labelled cells histamine stimulated [3H]inositol phosphate accumulation (EC50, 17 +/- 2 microM; maximum effect, 203 +/- 4% of basal). Histamine-evoked Ca2+ mobilisation yielded an EC50 of 12 +/- 2 microM and maximum delta[Ca2+]i of 337 +/- 23 nM. Thapsigargin (1 nM) increased [Ca2+]i (delta[Ca2+]i 164 +/- 12 nM) and prevented any further increase by histamine (100 microM). The effects of histamine on [3H]GABA release, [3H]inositol phosphate accumulation and Ca2+ mobilisation were blocked by the selective histamine H1 receptor antagonist mepyramine. Taken together, these results indicate that histamine stimulates [3H]GABA release by increasing [Ca2+]i. The mechanism of release may be related to changes in transmembranal Na+ gradients and reversal of GABA carrier transport due to stimulation of plasma membrane Na+/Ca2+ exchange.
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PMID:Histamine H1 receptor activation stimulates [3H]GABA release from human astrocytoma U373 MG cells. 900 31

The mechanism through which kainate receptors downregulate the release of GABA in the hippocampus is not known. We have found that the action of kainate on the hippocampal inhibitory postsynaptic current (IPSC) is mediated by a metabotropic process that is sensitive to Pertussis toxin (PTx) and independent of ion channel current. The downregulation of GABA IPSCs by kainate was also prevented in a dose-dependent manner by calphostin C, a specific inhibitor of PKC, and the inhibition of phospholipase C (PLC) drastically reduced the action of kainate. The effect of kainate was completely occluded by phorbol esters and by increasing extracellular Ca2+ but remained unaltered after inhibition or activation of protein kinase A (PKA). These results demonstrate that the activation of kainate receptors triggers a second messenger cascade, which results in the stimulation of PKC, and therefore document a metabotropic action of kainate receptors, which results in the inhibition of GABA release.
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PMID:Kainate receptor modulation of GABA release involves a metabotropic function. 965 8

alpha-Latrotoxin stimulates neurotransmitter release probably by binding to two receptors, CIRL/latrophilin 1 (CL1) and neurexin Ialpha. We have now produced recombinant alpha-latrotoxin (LtxWT) that is as active as native alpha-latrotoxin in triggering synaptic release of glutamate, GABA and norepinephrine. We have also generated three alpha-latrotoxin mutants with substitutions in conserved cysteine residues, and a fourth mutant with a four-residue insertion. All four alpha-latrotoxin mutants were found to be unable to trigger release. Interestingly, the insertion mutant LtxN4C exhibited receptor-binding affinities identical to wild-type LtxWT, bound to CL1 and neurexin Ialpha as well as LtxWT, and similarly stimulated synaptic hydrolysis of phosphatidylinositolphosphates. Therefore, receptor binding by alpha-latrotoxin and stimulation of phospholipase C are insufficient to trigger exocytosis. This conclusion was confirmed in experiments with La3+ and Cd2+. La3+ blocked release triggered by LtxWT, whereas Cd2+ enhanced it. Both cations, however, had no effect on the stimulation by LtxWT of phosphatidylinositolphosphate hydrolysis. Our data show that receptor binding by alpha-latrotoxin and activation of phospholipase C do not by themselves trigger exocytosis. Thus receptors recruit alpha-latrotoxin to its point of action without activating exocytosis. Exocytosis probably requires an additional receptor-independent activity of alpha-latrotoxin that is selectively inhibited by the LtxN4C mutation and by La3+.
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PMID:alpha-latrotoxin action probed with recombinant toxin: receptors recruit alpha-latrotoxin but do not transduce an exocytotic signal. 979 28

The suprachiasmatic nucleus (SCN) harbors an endogenous oscillator generating circadian rhythms that are synchronized to the external light/dark cycle by photic information transmitted via the retinohypothalamic tract (RHT). The RHT has recently been shown to contain pituitary adenylate cyclase-activating polypeptide (PACAP) as neurotransmitter/neuromodulator. PACAPergic effects on cAMP-mediated signaling events in the SCN are restricted to distinct time windows and sensitive to melatonin. In neurons isolated from the SCN of neonatal rats we investigated by means of the fura-2 technique whether PACAP and melatonin also influence the intracellular calcium concentration ([Ca2+]i). PACAP elicited increases of [Ca2+]i in 27% of the analyzed neurons, many of which were also responsive to the RHT neurotransmitters glutamate and/or substance P. PACAP-induced changes of [Ca2+]i were independent of cAMP, because they were not mimicked by forskolin or 8-bromo-cAMP. PACAP caused G-protein- and phospholipase C-mediated calcium release from inositol-trisphosphate-sensitive stores and subsequent protein kinase C-mediated calcium influx, demonstrated by treatment with GDP-beta-S, neomycin, U-73122, calcium-free saline, thapsigargin, bisindolylmaleimide, and chelerythrine. The calcium influx was insensitive to antagonists of voltage-gated calcium channels of the L-, N-, P-, Q- and T-type (diltiazem, nifedipine, verapamil, omega-conotoxin, omega-agatoxin, amiloride). Immunocytochemical characterization of the analyzed cells revealed that >50% of the PACAP-sensitive neurons were GABA-immunopositive. Our data demonstrate that in the SCN PACAP affects the [Ca2+]i, suggesting that different signaling pathways (calcium as well as cAMP) are involved in PACAPergic neurotransmission or neuromodulation. Melatonin did not interfere with calcium signaling, indicating that in SCN neurons the hormone primarily affects the cAMP signaling pathway.
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PMID:Pituitary adenylate cyclase-activating polypeptide and melatonin in the suprachiasmatic nucleus: effects on the calcium signal transduction cascade. 987 Sep 51

The molecular mechanisms underlying the cerebral symptoms of ethanol withdrawal syndrome are poorly understood. In addition to ethanol's effect on GABA and NMDA receptors, ethanol affects muscarinic acetylcholine signaling. This interaction has attracted attention because of the importance of muscarinic signaling in consciousness. Chronic ethanol exposure increases muscarinic receptor binding. Increased transcription of receptor message has been suggested as the underlying mechanism, but this hypothesis has not been tested directly. Therefore, we studied the effects of ethanol on muscarinic signaling in a model that bypasses transcription of muscarinic receptor genes. We expressed rat m1 muscarinic receptors by cRNA microinjection in Xenopus oocytes. Cells were voltage-clamped at -70 mV and effects of prolonged (24, 48, and 72 hr) exposure to ethanol (25, 50, and 100 mM) on methylcholine-induced calcium-activated Cl- currents were determined. Effects of prolonged ethanol exposure on currents induced by stimulation of lysophosphatidate receptors, direct G protein activation, or inositol trisphosphate receptor activation were studied as well. Prolonged ethanol exposure enhanced methylcholine (or lysophosphatidate-)-induced currents in a time- and concentration-dependent manner. Thus, enhanced muscarinic gene transcription is not required for ethanol enhancement of muscarinic signaling. Lack of ethanol effect on inositol trisphosphate-induced signaling suggests that intracellular signaling systems downstream of phospholipase C are not involved. In contrast, currents induced by direct G protein stimulation were enhanced significantly. Therefore, one potential site of ethanol's action on muscarinic signaling is upregulation of the associated G protein or enhancement of its functioning.
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PMID:Chronic ethanol exposure enhances signaling through muscarinic receptors expressed by cRNA injection in Xenopus oocytes: implications for mechanism of action. 1037 97

Glutamate is the major excitatory neurotransmitter in the brain and plays a unique role in a variety of central nervous system (CNS) functions. The discovery of the metabotropic receptors (mGluRs), a family of G-protein coupled receptors than can be activated by glutamate, has led to an impressive number of studies in recent years aimed at understanding their biochemical, physiological and pharmacological characteristics. The eight mGluRs now known are divided into three groups according to their sequence homology, signal transduction mechanisms, and agonist selectivity. Group I mGluRs include mGluR1 and mGluR5, which are linked to the activation of phospholipase C; Groups II and III include all others and are negatively coupled to adenylyl cyclases. The availability in recent years of agents selective for Group I mGluRs has made possible the study of the physiological roles of these receptors in the CNS. In addition to mediating glutamatergic neurotransmission, Group I mGluRs can modulate other neurotransmitter receptors, including GABA and the ionotropic glutamate receptors. Group I mGluRs are involved in many CNS functions and may participate in a variety of disorders such as pain, epilepsy, ischemia, and chronic neurodegenerative diseases. This class of receptor may provide important pharmacological therapeutic targets and elucidating its functions will be relevant to develop new treatments for neurological and psychiatric disorders in which glutamatergic neurotransmission is abnormally regulated. In this review anatomical, physiological and pharmacological results are presented with a special emphasis on the role of Group I mGluRs in functional and pathological processes.
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PMID:Group I metabotropic glutamate receptors: implications for brain diseases. 1041 61

In an evaluation of the contribution of swelling-induced amino acid release, through the regulatory volume decrease (RVD) process, to cerebral ischemic injury, studies of the role of phospholipases and protein kinases in the response to hyposmotic stress were undertaken using an in vivo rat cortical cup model. Hyposmotic stress induced significant releases of aspartate, glutamate, glycine, phosphoethanolamine, taurine and GABA from the rat cerebral cortex. Taurine release was most affected, exhibiting a greater than 9-fold increase during the hyposmotic stimulus. The phospholipase A2 (PLA2) inhibitors 4-bromophenacyl bromide (1 microM) and 7,7-dimethyleicosadienoic acid (5 microM) had no significant effects on hyposmotically induced amino acid release. AACOCF3 (50 microM), an inhibitor of cytosolic PLA2 decreased taurine release to 84% of DMSO controls. The release of the other amino acids was not affected. The phospholipase C inhibitor U73122 (5 microM) had no significant effects on amino acid release. The protein kinase C (PKC) inhibitor chelerythrine (5 microM) significantly reduced hyposmotically induced taurine release to 72% of saline controls but had no significant effects on the other amino acids. Stimulation of PKC with phorbol 12-myristate, 13-acetate (10 microM) did not significantly change taurine, glutamate, glycine or phosphethanolamine release. The releases of aspartate and GABA were enhanced 2 to 3 fold. Phorbol 12,13-didecanoate (10 microM), another potent stimulator of PKC, significantly increased taurine release to 122% of DMSO controls. The releases of aspartate, glutamate and glycine were enhanced 2.5 to 3.5 fold. Similarly, stimulation of protein kinase A with forskolin (100 microM) significantly increased taurine, aspartate, and glycine release 1.5- to 2-fold compared to DMSO controls. In summary, phospholipases may play a minor role in volume regulation. These studies also support the hypothesis that protein kinases play a modulatory role in the RVD response. The results show that although RVD may play a role, additional mechanisms, including phospholipase activation, must be involved in the ischemia-evoked release of excitotoxic amino acids.
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PMID:Hyposmotically induced amino acid release from the rat cerebral cortex: role of phospholipases and protein kinases. 1053 55

We developed a protocol for quantification of relative gene expression using reverse transcription-polymerase chain reaction (RT-PCR) without the use of radioisotopes, special equipment or extra nucleotide fragments, such as competitors. The relative gene expression of GABA(A) receptor beta(1) subunit (GABA(A)Rbeta(1)) and phospholipase C beta(4) subtype (PLCbeta(4)) in rat cerebrum and cerebellum were determined by comparing the ratio of PCR products generated by linear amplification of the target cDNA segments and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA segment as a reference. The density of PCR products was measured from digitized images of photographs of ethidium-bromide-stained agarose gels. The linear region of PCR amplification was within the linear range (from 0.3 to 12 ng DNA in a single band) of the detection system. The accuracy of the present method was <2-fold difference in gene expression in a single determination and a 1.5-fold difference was statistically significant after repeated measurements. The estimated relative expression of PLCbeta(4) was significantly higher in cerebellum than cerebrum, and that of GABA(A)Rbeta(1) was the same in these two regions. Using the present method, it is possible to quantify several different subunits and subtypes of known ion channel, neurotransmitter receptor and intracellular signaling enzyme gene families.
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PMID:Quantification of relative mRNA expression in the rat brain using simple RT-PCR and ethidium bromide staining. 1093 41

During gamete interaction, sperm acrosome reaction (AR) induced by oocyte investment is a prerequisite event for the spermatozoa to pass through the zona pellucida (ZP), fuse with and penetrate the oocyte. Progesterone (P4), secreted by cumulus cells, is an important cofactor for the occurrence of this exocytosis event. The AR results from the fusion between outer acrosomal and plasma membranes, leading to inner acrosomal membrane exposure. Binding of agonists, P4 or ZP3 glycoprotein, to plasma membrane sperm receptors activates intraspermatic signals and enzymatic pathways involved in the AR. Among the proteins or glycoproteins described as potential sperm receptors for ZP, Gi/Go protein-coupled and tyrosine kinase receptors have been described. Sperm receptors for P4 are poorly characterized, except a putative GABA(A)-like receptor. ZP- and P4-promoted AR is mediated by an obligatory intracellular calcium increase, appearing first at the acrosome equatorial segment and spreading throughout the head. The plasma membrane channels involved in calcium entry are operated by a plasma membrane depolarization and protein phosphorylations mediated by protein kinase C and tyrosine kinase protein. Part of the calcium increase could also be due to intracellular store release through IP3- and nucleotide (cAMP)-gated channels. Besides adenylate cyclase and phospholipase C activations, intracellular calcium increase also stimulates PLA2 activity and actin depolymerization, leading to membrane fusion. Evaluation of AR by staining or fluorescent probes can be useful to predict fertilization success and to direct the therapeutic strategy in male infertility.
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PMID:The acrosome reaction in human spermatozoa. 1104 13


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