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. Lactotrophs are adenohypophysial cells that synthesize and secrete prolactin (PRL), a hormone principally involved in mammalian milk production. An increase in the intracellular Ca2+ concentration ([Ca2+]i) is an important signal for PRL secretion. Thyrotrophin-releasing hormone (TRH) generates Ca2+ signals derived from both the release of Ca2+ from intracellular stores and the entry of extracellular Ca2+, the latter being particularly important for PRL secretion. The identity of this TRH-sensitive Ca2+ entry pathway is unknown and therefore the subject of the present study. 2. [Ca2+]i was measured by video imaging of fura-2 loaded into single rat anterior pituitary cells. Ca2+ influx was detected by quenching of fura-2 fluorescence by external Mn2+. All data are from lactotrophs isolated from lactating female rats. Individual lactotrophs were identified by postexperimental immunofluorescent detection of PRL in fixed cells. 3. TRH induced the release of Ca2+ from intracellular stores and also stimulated Mn(2+)-permeable Ca2+ influx. U73122 (1 microM), a phospholipase C inhibitor, prevented the Ca(2+)-mobilizing actions of TRH. The chemically similar but inactive analogue, U73343 (1 microM), had no effect on TRH responses. U73122 did not act as a global G protein inhibitor because the reduction of basal [Ca2+]i by dopamine (1 microM, a G protein-mediated event) was not affected. 4. TRH-stimulated Mn2+ influx occurred either immediately after addition of TRH (early entry) or after a delay of about 130 s (late entry). There were no statistically significant differences in the magnitude or temporal characteristics of the Ca2+ signals evoked from cells showing early or late Mn2+ entry. 5. The identity of Ca2+ channels permeable to Mn2+ was investigated. Cell depolarization with 50 mM KCl stimulated Ca2+/Mn2+ influx and was prevented by nifedipine (1 microM). Bay K 8644 (1 microM) also stimulated Mn2+ influx. Thus, the presence of Mn(2+)-permeable L-type voltage-operated Ca2+ channels is likely. A second Mn(2+)-permeable pathway was present in lactotrophs. Depletion of Ca2+ stores by thapsigargin (1 microM) stimulated a Ca2+ signal and Mn2+ influx. This 'capacitative entry pathway' was insensitive to nifedipine (1 microM), indicating that putative L-type Ca2+ channels were not activated. 6. TRH-stimulated Mn2+ influx was not prevented by nifedipine (1 microM). TRH added during KCl-induced Mn2+ influx reduced the quench rate within the time frame of the TRH-induced Ca2+ spike. TRH may therefore inhibit putative L-type Ca2+ channels. 7. Addition of thapsigargin in Ca(2+)-free medium transiently increased [Ca2+]i and prevented subsequent Ca2+ responses to TRH.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Control of Ca2+ entry into rat lactotrophs by thyrotrophin-releasing hormone. 747 2

Angiotensin II (AII) evokes a biphasic increase in inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) levels in adrenal glomerulosa cells, with an extracellular Ca(2+)-independent early peak followed by a secondary sustained elevation that is highly dependent on the presence of extracellular Ca2+. The Ca(2+)-dependent sustained phase of agonist-induced Ins(1,4,5)P3 production was closely correlated with Ca2+ influx and was inhibited by inorganic Ca2+ channel blockers with the potency ratio: La3+ >> Cd2+ > Mn2+ > Co2+ > Ni2+. Of the two Ca2+ surrogates, Sr2+ and Ba2+, Sr2+ was partially active compared with Ca2+, and Ba2+ was inactive in restoring Ins(1,4,5)P3 formation in cells stimulated with AII in Ca(2+)-free medium. However, unlike Ca2+, Sr2+ only weakly supported and Ba2+ failed to affect the calmodulin-activation of Ins(1,4,5)P3 3-kinase. Also, there was an accumulation of Ins(1,4,5)P3 and diminished formation of Ins(1,3,4,5)P4 and Ins(1,3,4)P3 when intact glomerulosa cells were stimulated by AII in the presence of Sr2+. This difference between the Sr2+ sensitivity of phospholipase C and Ins(1,4,5)P3 3-kinase provides a means for the potentiation of agonist-induced elevations of Ins(1,4,5)P3 in the intact cell and for direct analysis of the role of the inositol tris-/tetrakisphosphate pathway in cellular signaling.
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PMID:Cation sensitivity of inositol 1,4,5-trisphosphate production and metabolism in agonist-stimulated adrenal glomerulosa cells. 751 76

When single rat hepatocytes were stimulated with the phospholipase C-activating hormone, vasopressin (from 300 pM to 1 microM), the [Ca2+]i signals were always "all-or-none" responses. At low concentrations of vasopressin, Ca2+ release was maximal because liberation of additional inositol 1,4,5-trisphosphate (IP3) by photolysis of its caged precursor at the top of the [Ca2+]i spike failed to increase [Ca2+]i further. However, if IP3 was generated by photolysis of caged IP3 in previously unstimulated cells, [Ca2+]i increased immediately, and the magnitude of the response was a graded function of the quantity of IP3 released. We also analyzed the kinetics of activation of intracellular IP3 receptor/Ca2+ channels by monitoring the quench of sequestered dye by the entry of cytoplasmic Mn2+ into fura-2-loaded intracellular IP3-sensitive organelles. This Mn(2+)-induced quench was precipitous and always preceded by a delay inversely related to the vasopressin concentration. In hepatocytes stimulated with 10 nM vasopressin, IP3 increased slowly, and the half-time of the IP3 rise was comparable with the latency for the release of intracellular calcium. The slow rise in IP3 would be predicted to produce accelerating Ca2+ release. This is consistent with the results of the Mn2+ quench experiments, which revealed accelerating activation of intracellular IP3-regulated calcium channels. We conclude that this accelerating release of Ca2+, which does not occur with instantaneous increases in IP3 due to flash photolysis, is likely to be important for generating the all-or-none Ca2+ mobilization that initiates the processes of intracellular [Ca2+]i oscillations.
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PMID:Delayed "all-or-none" activation of inositol 1,4,5-trisphosphate-dependent calcium signaling in single rat hepatocytes. 752 88

It is well recognized that JMV-180, a cholecystokinin (CCK) analogue, acts as an agonist on the high-affinity CCK receptor in pancreatic acinar cells. It caused Ca2+ oscillations and amylase secretion in a manner independent of the phospholipase C-inositol 1,4,5-trisphosphate (IP3) pathway. We investigated the mechanism by which the high-affinity CCK receptor utilizes IP3-independent Ca2+ signal transduction to mediate amylase secretion. JMV-180 (1-1,000 nM)-stimulated Ca2+ oscillations and amylase secretion were significantly inhibited by the phospholipase A2 (PLA2) inhibitor, ONO-RS-082 (10 microM). Using streptolysin O-permeabilized cells, we showed that a porcine pancreatic anti-PLA2 antibody from rabbit serum (250 ng/ml) inhibited JMV-180-stimulated amylase secretion. In contrast to CCK octapeptide, JMV-180 (1 nM-10 microM) had no effect on intracellular IP3 levels. These concentrations of JMV-180 did, however, increase intracellular levels of arachidonic acid (AA) metabolite by 2.5-fold in a biphasic manner. Application of exogenous AA (10 microM) released 60% of ATP-incorporated 45Ca2+ from permeabilized pancreatic acini within 3 min in a transient manner. We also showed that active phorbol ester (100 nM) inhibited Ca2+ oscillations and amylase secretion stimulated by JMV-180 (10 nM) or CCK-OPE (100 nM). Application of Mn2+ (2 mM) to superfused acini resulted in a rapid quench of fura 2 fluorescence during 10 nM JMV-180 stimulation, suggesting an involvement of extracellular Ca2+ influx. However, the major source of Ca2+ utilized for oscillations during high-affinity CCK receptor activation was intracellular. In conclusion, we have demonstrated that the high-affinity CCK receptors are coupled to PLA2 pathways to produce AA, which mediates cytosolic Ca2+ oscillation and monophasic amylase secretion, in rat pancreatic acinar cells.
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PMID:High-affinity CCK receptors are coupled to phospholipase A2 pathways to mediate pancreatic amylase secretion. 757 55

The regulation of intracellular Ca2+ concentration ([Ca2+]i) by glutamate metabotropic receptors (mGluR) was studied in 8-day-old rat forebrain synaptoneurosomes using spectrofluorimetric methods. Here we demonstrate that metabotropic glutamate agonists induce in rat brain synaptoneurosomes a Ca2+ influx largely dependent upon the presence of Ca2+ in the external medium. The pharmacological profile of this influx is strongly correlated with the pharmacological profile of the activation of phosphoinositide hydrolysis, i.e. quisqualic acid >> 1S,3R-amino-1-dicarboxylate-1,3 cyclopentane approximately equal to glutamate. This metabotropic glutamate receptor-induced Ca2+ influx is insensitive to voltage-dependent Ca2+ channel antagonists and occurs through a Mn2+ impermeant pathway. The study of the rapid kinetics shows that this influx is triggered after a 300 ms delay compared with that elicited by depolarizing agents and Ca2+ ionophore A23187. In order to assess further if mGluR stimulate this influx through the recruitment of inositol triphosphate (IP3)-sensitive intracellular Ca2+ stores, we have tested the effect of thapsigargin on membrane potential and intracellular Ca2+ simultaneously. Thapsigargin induces a depolarization of the synaptoneurosomal membrane followed by a massive Ca2+ influx, occurring via a Mn2+ nonpermeant route. This depolarizing effect is sensitive to the presence of the intracellular Ca2+ chelator [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetoxymethyl ester], and partially sensitive to extracellular Na+, but insensitive to the presence of extracellular Ca2+. Taken together, our data suggest that mGluR stimulate self-maintained increases of [Ca2+]i in rat forebrain synaptoneurosomes via the activation of a multistep mechanism, sequenced in the following steps: (i) mGluR-induced IP3 synthesis; (ii) IP3-stimulated intracellular Ca2+ release; (iii) Ca(2+)-activated non-specific cation channel, leading to local depolarization and a Ca2+ influx; and (iv) activation of Ca(2+)-sensitive phospholipase C.
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PMID:Stimulation of Ca(2+)-activated non-specific cationic channels by phospholipase C-linked glutamate receptors in synaptoneurosomes? 758 31

In this study, we showed that cross-linking CD3 molecules on the T cell surface resulted in Ca2+ release from the intracellular stores followed by a sustained Ca2+ influx. Inhibition of release with TMB-8 did not block the influx. However, inhibition of phospholipase C activity suppressed both Ca2+ release and influx. Once activated, the influx pathway remained open in the absence of further hydrolysis of PIP2. Thapsigargin, a microsomal Ca(2+)-ATPase inhibitor, stimulated Ca2+ entry into the cells by a mechanism other than emptying Ca2+ stores. In addition, Ca2+ entry into the Ca(2+)-depleted cells was stimulated by low basal level of cytosolic Ca2+, not by the emptying of intracellular Ca2+ stores. Both the Ca2+ release and influx were dependent on high and low concentrations of extracellular Ca2+. At low concentrations, Mn2+ entered the cell through the Ca2+ influx pathway and quenched the sustained phase of fluorescence; whereas, at higher Mn2+ concentration both the transient and the sustained phases of fluorescence were quenched. Moreover, Ca2+ release was inhibited by low concentrations of Ni2+, La3+, and EGTA, while Ca2+ influx was inhibited by high concentrations. Thus, in T cells Ca2+ influx occurs independently of IP3-dependent Ca2+ release. However, some other PIP2 hydrolysis-dependent event was involved in prolonged activation of Ca2+ influx. Extracellular Ca2+ influenced Ca2+ release and influx through the action of two plasma membrane Ca2+ entry pathways with different pharmacological and biochemical properties.
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PMID:T cell receptor-mediated Ca2+ signaling: release and influx are independent events linked to different Ca2+ entry pathways in the plasma membrane. 759 56

The membrane-bound form of aminopeptidase P (aminoacylprolyl-peptide hydrolase) (EC 3.4.11.9) was purified 670-fold to apparent homogeneity from rat lung microsomes. The enzyme was solubilized from the membranes using a phosphatidylinositol-specific phospholipase C. The purification scheme also resulted in homogeneous preparations of dipeptidylpeptidase IV (EC 3.4.14.5) and membrane dipeptidase (EC 3.4.13.19). Aminopeptidase P had a subunit molecular weight of 90,000, which included at least 17% N-linked carbohydrate. The molecular weight by gel permeation chromatography varied from 220,000 to 340,000, depending on the conditions used. The amino acid composition was determined and the N-terminal sequence was found to be X1-Gly2-Pro3-Glu4-Ser5-Leu6-Gly7-Arg8-Glu9-As p10-Val11-Arg12-Asp13-X14-Ser15- Thr16-Asn17-Pro18-Pro19-Arg20-Leu21- X22-Val23-Thr24-Ala25-. Aminopeptidase P cleaved the Arg1-Pro2 bond of bradykinin with a kcat/Km of 5.7 x 10(5) s-1 M-1. N-Terminal fragments of bradykinin including Arg-Pro-Pro, but not Arg-Pro, were also cleaved. The enzyme was shown to have four binding subsites (S1, S1', S2'. S3'), the first three of which must be occupied for hydrolysis to occur. Neuropeptide Y and allatostatin I were hydrolyzed at the Tyr1-Pro2 bond and Ala1-Pro2 bond, respectively. The pH optimum for Arg-Pro-Pro cleavage was 6.8-7.5 in most buffers. The enzyme was most stable in the range of pH 7.0-10.5 in the presence of poly(ethylene glycol). NaCl inhibited activity completely at 2 M. Mn2+ had variable effects on activity, depending on its concentration and the substrate used. Various peptides having an N-terminal Pro-Pro sequence were inhibitory. The enzyme was also inhibited by EDTA, o-phenanthroline, 2-mercaptoethanol, dithiothreitol, p-(chloromercuri)benzenesulfonic acid, apstatin, and captopril. The carboxyalkyl angiotensin-converting enzyme inhibitors, ramiprilat and enalaprilat, inhibited activity in the micromolar range only in the presence of Mn2+.
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PMID:Purification and properties of membrane-bound aminopeptidase P from rat lung. 766 81

Serpulina hyodysenteriae produces an oxygen-stable heat-labile hemolysin that may be an important virulence factor in the pathogenesis of swine dysentery. We examined the effect of Ca2+, Co2+, Cu2+, Fe2+, Mg2+, Mn2+, Ni2+, and Zn2+ on the hemolytic activity of cell-free supernatant (CFS) from S. hyodysenteriae, isolate B204. Cells harvested from late logarithmic phase cultures were incubated in phosphate-buffered saline containing glucose and RNA-core (PBS-GR) with or without cations and the hemolytic activity of CFS obtained after successive 30 min incubation and washing cycles was determined. The addition of either ZnSO4 or CuSO4 to the PBS-GR caused complete inhibition of hemolytic activity after 3 cycles; other cations gave results similar to control extracts. Reduction in the concentration of Zn2+ in CFS by 60 to 80% after each incubation cycle and binding of Zn2+ by EDTA indicated that Zn2+ was associated with the cell fraction, and inhibition of hemolysin activity was specifically mediated by Zn2+. When the spirochetes were washed after incubation in the presence of ZnSO4 for 2 cycles and incubated in fresh PBS-GR without Zn2+, inhibition of hemolysin activity remained unchanged, indicating that the inhibitory effect of ZnSO4 was due to a direct action of ZnSO4 on the spirochetes. Since neither the viability of the spirochetes nor the activity of pre-formed hemolysin were affected by the presence of ZnSO4, the inhibitory effect of Zn2+ cations was attributed to reduced biosynthesis by viable S. hyodysenteriae cells rather than interference of Zn2+ cations with lysis of erythrocytes by the hemolysin. Transmission electron microscopic examination of spirochetes after incubation in PBS-GR containing ZnSO4 revealed clumping of ribosomes and clearing of cell cytoplasm.
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PMID:Effect of divalent cations on hemolysin synthesis by Serpulina (Treponema) hyodysenteriae: inhibition induced by zinc and copper. 780 26

The regulation of Ca2+ influx in rat hepatocytes by glucagon and cyclic AMP (cAMP) was investigated. Exposing hepatocytes to glucagon resulted in an increase in the initial rate of Ca2+ entry. The concentrations of glucagon producing half-maximal and maximal stimulation of Ca2+ entry were 10(-10) and 10(-8) M, respectively. A similar stimulation of Ca2+ influx was obtained in cells exposed to cAMP analogues or to forskolin. Exposing hepatocytes suspended in nominally Ca(2+)-free medium to glucagon for 3 min produced a 9% decrease in the size of the vasopressin-sensitive Ca2+ pool; in contrast, N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (Bt2cAMP) slightly augmented the size of this pool. Glucagon and Bt2cAMP synergized the initial vasopressin-stimulated Ca2+ and Mn2+ influx rates, but only moderately increased the initial rate of Ca2+ entry after thapsigargin addition. The glucagon- and Bt2cAMP-stimulated Ca2+ influx was inhibited by the same antagonists of the plasma membrane Ca2+ carriers that mediate Ca2+ entry during stimulation by vasopressin. Thus, cAMP does not stimulate Ca2+ entry through either a capacitative type of mechanism or inositol phosphate turnover. The authors' findings instead suggest that cAMP acts directly, or through protein kinase A on the same Ca2+ carriers that are activated by phospholipase C-linked receptor agonists.
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PMID:Cyclic AMP stimulates Ca2+ entry in rat hepatocytes by interacting with the plasma membrane carriers involved in receptor-mediated Ca2+ influx. 781 85

Calcium signalling was examined in CHO-k1 cells that stably express the m3 subtype of the muscarinic receptor. The calcium indicator Fura-2 was retained in these cells only in the presence of probenecid (1 mM), suggesting that Fura-2 efflux was mediated by an organic anion transporter. The addition of carbachol (CCh) to Fura-2 loaded cells in suspension caused a rapid transient increase in intracellular calcium [Ca]i followed by a smaller sustained plateau phase. The transient rise in [Ca]i was dose-dependent with a threshold response of 89 +/- 18 nM above baseline with 10 nM CCh and a maximum stimulation of 734 +/- 46 nM with 10 microM CCh. This phase was accompanied by a similar dose-dependent stimulation of total inositol phosphate production and was assumed to be generated by release from intracellular stores of the endoplasmic reticulum (ER). The sustained increase in [Ca]i was generated by entry from the extracellular bath since it was blocked by pretreatment with La3+ (1 microM) and was absent when bath calcium was chelated with EGTA. This phase was not dependent on CCh dose, and a stimulation of [Ca]i of approximately 90 nM above baseline was observed with CCh concentrations between 50 nM and 10 microM. With this dose range, the rate of Mn2+ quenching of Fura-2 at the Ca-insensitive excitation wavelength of 360 nm was likewise maximally stimulated. At lower CCh concentrations (10-50 nM), it was clear that the activation of Ca entry could not be dissociated from a threshold release of Ca from intracellular stores. The phorbol ester PMA, which uncouples the muscarinic receptor from phospholipase C, reduced the transient rise in [Ca]i by approximately 50% with little or no effect on Ca entry at higher CCh levels (> or = 1 microM). At lower CCh concentrations (< or = 100 nM) however, pretreatment with PMA completely blocked all Ca mobilization and supports the contention that Ca entry is coupled to Ca release from stores or to store depletion. The emptying of inositol trisphosphate-sensitive stores with thapsigargin (10 nM) stimulated Ca entry and also the rate of Mn2+ quenching. Store depletion by incubation in Ca-free media likewise stimulated Mn2+ uptake without a rise in [Ca]i. Our data are therefore consistent with a 'capacitative' coupling model, whereby the activation of the plasma membrane receptor leads to an InsP3-induced change in the degree of filling of the ER Ca pool.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Differential effects of carbachol on calcium entry and release in CHO cells expressing the m3 muscarinic receptor. 782 72


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