Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two enzymes, protein kinase C and microsomal Ca(2+)-ATPase help regulate levels of Ca2+ in many types of cells. Since proteins that regulate Ca2+ often influence sensitivity to Pb2+, we determined the possible roles played by protein kinase C and microsomal Ca(2+)-ATPase for the Pb(2+)-evoked release of norepinephrine (NOR) in PC cells. NOR release was observed at 10 microM Pb2+ when PC 12 cells were stimulated with inhibitors of microsomal Ca(2+)-ATPase such as thapsigargin, cyclopiazonic acid, or 2,5-di-(t-butyl)-hydroquinone. At 5 microM, Pb2+ evoked the release of NOR in PC 12 cells stimulated with activators of protein kinase C such as phorbol 12-myristate 13-acetate (PMA) or (-)-7-octylindolactam. NOR release was observed at 1 microM Pb2+ in the presence of both PMA and thapsigargin. Ni2+ and Cd2+ blocked NOR release stimulated by Pb2+ in the presence of thapsigargin but not by PMA. NOR released by thapsigargin stimulation was not altered in PC 12 cells depleted of protein kinase C. Two proteins found in vesicles, chromogranin B and secretogranin-II were released with NOR. Our results indicate that in PC 12 cells, PB(2+)-evokes the release of neurotransmitters. Furthermore, thapsigargin and PMA increase the cell's sensitivity to Pb2+ by different pathways.
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PMID:Distinct mechanisms of neurotransmitter release from PC 12 cells exposed to lead. 897 2

1. Endothelin (ET) B-type (ETB) receptor-mediated signal transduction was examined after stimulation with ET-3 in cultured aortic endothelial cells (EC) from spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats (8 weeks old). 2. The EC from both rat strains expressed only ETB receptor mRNA. The receptor densities and affinities, which were non-selective for ET-1, -2, -3 and Sarafotoxin S6c, and mRNA expression were similar in WKY and SHR. 3. The cytosolic Ca2+ level in the absence of extracellular Ca2+, inositol 1,4,5-trisphosphate levels, protein kinase C and phospholipase C activities in response to ET-3 were greater in SHR EC than in WKY EC. 4. The 45Ca uptake in response to ET-3, which was blocked by Ni2+, was smaller in SHR EC than in WKY EC. 5. The 6-keto-PGF1alpha production was augmented in SHR, though nitric oxide formation after stimulation with ET-3 was similar. 6. These results suggest that ETB receptor-mediated phosphoinositide turnover signalling is augmented in SHR EC through postreceptor mechanism.
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PMID:Enhanced phosphoinositide turnover signalling stimulated by endothelin B-type receptor in endothelial cells from spontaneously hypertensive rats. 907 52

1. The aim of the present study was to identify the sources of Ca2+ contributing to acetylcholine (ACh)-induced release of endothelium-derived hyperpolarizing factor (EDHF) from endothelial cells of rat mesenteric artery and to assess the pathway involved. The changes in membrane potentials of smooth muscles by ACh measured with the microelectrode technique were evaluated as a marker for EDHF release. 2. ACh elicited membrane hyperpolarization of smooth muscle cells in an endothelium-dependent manner. The hyperpolarizing response was not affected by treatment with 10 microM indomethacin, 300 microM NG-nitro-L-arginine or 10 microM oxyhaemoglobin, thereby indicating that the hyperpolarization is not mediated by prostanoids or nitric oxide but is presumably by EDHF. 3. In the presence of extracellular Ca2+, 1 microM ACh generated a hyperpolarization composed of the transient and sustained components. By contrast, in Ca(2+)-free medium, ACh produced only transient hyperpolarization. 4. Pretreatment with 100 nM thapsigargin and 3 microM cyclopiazonic acid, endoplasmic reticulum Ca(2+)-ATPase inhibitors, completely abolished ACh-induced hyperpolarization. Pretreatment with 20 mM caffeine also markedly attenuated ACh-induced hyperpolarization. However, the overall pattern and peak amplitude of hyperpolarization were unaffected by pretreatment with 1 microM ryanodine. 5. In the presence of 5 mM Ni2+ or 3 mM Mn2+, the hyperpolarizing response to ACh was transient, and the sustained component of hyperpolarization was not observed. On the other hand, 1 microM nifedipine had no effect on ACh-induced hyperpolarization. 6. ACh-induced hyperpolarization was nearly completely eliminated by 500 nM U-73122 or 200 microM 2-nitro-4-carboxyphenyl-N, N-diphenylcarbamate, inhibitors of phospholipase C, but was unchanged by 500 nM U-73343, an inactive form of U-73122. Pretreatment with 20 nM staurosporine, an inhibitor of protein kinase C, did not modify ACh-induced hyperpolarization. 7. These results indicate that the ACh-induced release of EDHF from endothelial cells of rat mesenteric artery is possibly initiated by Ca2+ release from inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pool as a consequence of stimulation of phospholipid hydrolysis due to phospholipase C activation, and maintained by Ca2+ influx via a Ni(2+)- and Mn(2+)-sensitive pathway distinct from L-type Ca2+ channels. The Ca(2+)-influx mechanism seems to be activated following IP3-induced depletion of the pool.
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PMID:Sources of Ca2+ in relation to generation of acetylcholine-induced endothelium-dependent hyperpolarization in rat mesenteric artery. 910 9

Mammalian circadian rhythms originate in the hypothalamic suprachiasmatic nuclei (SCN), from which rhythmic neural activity can be recorded in vitro. Application of neurochemicals can reset this rhythm. Here we determine cellular correlates of the phase-shifting properties of neuropeptide Y (NPY) on the hamster circadian clock in vitro. Drug or control treatments were applied to hypothalamic slices containing the SCN on the first day in vitro. The firing rates of individual cells were sampled on the second day in vitro. Control slices exhibited a peak in firing rate in the middle of the day. Microdrop application of NPY to the SCN phase advanced the time of peak firing rate. This phase-shifting effect of NPY was not altered by block of sodium channels with tetrodotoxin or block of calcium channels with cadmium and nickel, consistent with a direct postsynaptic site of action. Pretreatment with the glutamate receptor antagonists (DL-2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione disodium) also did not alter phase shifts to NPY. Blocking GABAA receptors with bicuculline (Bic) had effects only at very high (millimolar) doses of Bic, whereas blocking GABAB receptors did not alter effects of NPY. Phase shifts to NPY were blocked by pretreatment with inhibitors of protein kinase C (PKC), suggesting that PKC activation may be necessary for these effects. Bathing the slice in low Ca2+/high Mg2+ can block phase shifts to NPY, possibly via a depolarizing action. A depolarizing high K+ bath can also block NPY phase shifts. The results are consistent with direct action of NPY on pacemaker neurons, mediated through a signal transduction pathway that depends on activation of PKC.
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PMID:Circadian phase shifts to neuropeptide Y In vitro: cellular communication and signal transduction. 933 19

1. The effects of endothelin-1 (ET-1) were studied in bovine oviductal arteries and compared to those of noradrenaline (NA) and high K+ (K+). The influence of endothelium, the receptor subtypes involved, and the mechanisms of Ca2+ mobilization were assessed. 2. ET-1 (0.1-300 nM) induced concentration-dependent contractions with a potency of 10(3) and 10(2) times higher than NA (0.1 microM-0.1 mM) and K+ (9.5-119 mM), respectively. Removal of endothelium or NG-nitro-L-arginine (L-NOARG, 0.1 mM) pretreatment did not affect responses to either ET-1 or K+, whereas the NA response was significantly increased. Indomethacin (1 microM) had no effect on either of these agonists. 3. The rank order of potency for the ET isopeptides was: ET-1 = ET-2 > ET-3. The ETA receptor-selective agonist, sarafotoxin 6c (S6c), had no effect. The ETA receptor-selective antagonist, BQ-123, showed a competitive antagonism on the ET-1 response (pA2 value of 6.58 +/- 0.01), whereas contractions to ET-3 were completely abolished by BQ-123 at 0.1 microM. 4. Concentration-response curves to both ET-1 and NA were shifted to the right and their maximum response reduced to approximately 56% and 65% of controls, respectively, under 30 min of incubation in Ca(2+)-free solution, whereas responses to K+ were almost abolished by this treatment. Contractions to both NA (30 microM) and ET-1 (30 nM) were maximally inhibited after 10 min of extracellular Ca2+ deprivation. 5. Contractions to ET-1 were more potently inhibited by nickel (Ni2+, 0.3 mM), whereas nifedipine (1 microM) and cadmium (Cd2+, 0.1 mM) induced only a slight effect. In contrast, opposite effects were found for both NA and K+. 6. Treatment with ryanodine (100 microM) and caffeine (10 mM) in Ca(2+)-free solution reduced the tension measured 5 min after NA (30 microM) and ET-1 (30 nM) addition, but the sustained response (tension at 25 min) remained unaffected. 7. Calphostin C (1 microM), a specific protein kinase C (PKC) inhibitor, reduced the maximum contractile response to ET-1 by about 50% without significantly affecting its pD2 value. 8. These results suggest that ET-1 acts in bovine oviductal arteries by directly activating a homogenous population of ETA receptors in smooth muscle, without endothelial modulation. Several Ca2+ activation mechanisms seem to be involved in the contractile action of the peptide, including: (1) extracellular Ca2+ entrance through Ni(2+)-sensitive and L-type Ca2+ channels; (2) intracellular Ca2+ release from a ryanodine-sensitive Ca2+ store; and (3) sensitization of the contractile machinery to Ca2+ via PKC.
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PMID:Endothelin receptor-mediated Ca2+ mobilization and contraction in bovine oviductal arteries: comparison with noradrenaline and potassium. 935 11

The involvement of phosphatidylcholine-specific phospholipase C (PC-PLC) and D (PC-PLD) in the regulation of the thapsigargin-induced Ca2+ increase was investigated. Pretreatment of human lymphocytes with the PC-PLC inhibitors D609 or U73122 enhanced the thapsigargin-induced Ca2+ influx. By contrast, no effect was observed in the presence of phospholipase D inhibitor butanol. Addition of exogenous PC-PLC but not PC-PLD to lymphocytes prestimulated with thapsigargin led to a decrease of intracellular Ca2+. In addition, thapsigargin was shown to release diacylglycerol (DAG) from cellular phosphatidylcholine pools. The thapsigargin-induced DAG formation was inhibited by U73122 and D609 but not by butanol. Moreover, no formation of the PC-PLD activity marker phosphatidylbutanol was detected. Thapsigargin-induced DAG formation was dependent on the Ca2+ entry, as it was abolished in the absence of extracellular Ca2+ or in the presence of Ni2+. Further investigations demonstrated that the inhibition of the cellular DAG target, protein kinase C (PKC), enhanced thapsigargin-induced Ca2+ increase, whereas direct PKC activation had an inhibitory effect. Taken together, our results reveal the involvement of PC-PLC in the regulation of the thapsigargin-induced Ca2+ increase and point to the existence of a physiologic feedback mechanism activated by Ca2+ influx and acting via consecutive activation of PC-PLC and PKC to limit the rise of intracellular Ca2+.
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PMID:Phosphatidylcholine-specific phospholipase C regulates thapsigargin-induced calcium influx in human lymphocytes. 940 64

Muscarinic receptor stimulation elicits a redistribution of calmodulin (CaM) from the membrane fraction to cytosol in the human neuroblastoma cell line SK-N-SH. Increasing the intracellular Ca2+ concentration with ionomycin also elevates cytosolic CaM. The aim of this study was to investigate the roles of extracellular and intracellular Ca2+ pools in the muscarinic receptor-mediated increases in cytosolic CaM in SK-N-SH cells. Stimulus-mediated changes in intracellular Ca2+ were monitored in fura-2-loaded cells, and CaM was measured by radioimmunoassay in the 100,000-g cytosol and membrane fractions. The influx of extracellular Ca2+ normally seen with carbachol treatment in SK-N-SH cells was eliminated by pretreatment with the nonspecific Ca2+ channel blocker Ni2+. Blocking the influx of extracellular Ca2+ had no effect on carbachol-mediated increases in cytosolic CaM (168 +/- 18% of control values for carbachol treatment alone vs. 163 +/- 28% for Ni2+ and carbachol) or decreases in membrane CaM. Similarly, removal of extracellular Ca2+ from the medium did not affect carbachol-mediated increases in cytosolic CaM (168 +/- 26% of control). On the other hand, prevention of the carbachol-mediated increase of intracellular free Ca2+ by pretreatment with the cell-permeant Ca2+ chelator BAPTA/AM did attenuate the carbachol-mediated increase in cytosolic CaM (221 +/- 37% of control without BAPTA/AM vs. 136 +/- 13% with BAPTA/AM). The effect of direct entry of extracellular Ca2+ into the cell by K+ depolarization was assessed. Incubation of SK-N-SH cells with 60 mM K+ elicited an immediate and persistent increase in intracellular free Ca2+ concentration, but there was no corresponding alteration in CaM localization. On the contrary, in cells where intracellular Ca2+ was directly elevated by thapsigargin treatment, cytosolic CaM was elevated for at least 30 min while particulate CaM was decreased. In addition, treatment with ionomycin in the absence of extracellular Ca2+, which releases Ca2+ from intracellular stores, induced an increase in cytosolic CaM (203 +/- 30% of control). The mechanism for the CaM release may involve activation of the alpha isozyme of protein kinase C, which was translocated from cytosol to membranes much more profoundly by thapsigargin than by K+ depolarization. These data demonstrate that release of Ca2+ from the intracellular store is important for the carbachol-mediated redistribution of CaM in human neuroblastoma SK-N-SH cells.
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PMID:Cytosolic calmodulin is increased in SK-N-SH human neuroblastoma cells due to release of calcium from intracellular stores. 942 56

cDNA encoding the full-length hKv1.3 lymphocyte channel and a C-terminal truncated (delta 459-523) form that lacks the putative PKA Ser468 phosphorylation site were stably transfected in human embryonic kidney (HEK) 293 cells. Immunostaining of the transfected cells revealed a distribution at the plasma membrane that was uniform in the case of the full-length channel whereas clustering was observed in the case of the truncated channel. Some straining within the cell cytoplasm was found in both instances, suggesting an active process of biosynthesis. Analyses of the K+ current by the patch-clamp technique in the whole cell configuration showed that depolarizing steps to 40 mV from a holding potential (HP) of -80 mV elicited an outward current of 2 to 10 nA. The current threshold was positive to -40 mV and the current amplitude increased in a voltage-dependent manner. The parameters of activation were -5.7 and -9.9 mV (slope factor) and -35 mV (half activation, V0.5) in the case of the full-length and truncated channels, respectively. The characteristics of the inactivation were 14.2 and 24.6 mV (slope factor) and -17.3 and -39.0 mV (V0.5) for the full-length and truncated channels, respectively. The activation time constant of the full-length channel for potentials ranging from -30 to 40 mV decreased from 18 to 12 msec whereas the inactivation time constant decreased from 6600 msec at -30 mV to 1800 msec at 40 mV. The unit current amplitude measured in cells bathing in 140 mM KCl was 1.3 +/- 0.1 pA at 40 mV, the unit conductance, 34.5 pS and the zero current voltage, 0 mV. Both forms of the channels were inhibited by TEA, 4-AP, Ni2+ and charybdotoxin. In contrast to the native (Jurkat) lymphocyte Kv1.3 channel that is fully inhibited by PKA and PKC, the addition of TPA resulted in 34.6 +/- 7.3% and 38.7 +/- 9.4% inhibition of the full-length and the truncated channels, respectively, 8-BrcAMP induced a 39.4 +/- 5.4% inhibition of the full-length channel but had no effect (8.6 +/- 8.3%) on the truncated channel. Cell dialysis with alkaline phosphatase had no effects, suggesting that the decreased sensitivity of the transfected channels to PKA and PKC was not due to an already phosphorylated channel. Patch extract experiments suggested that the hKv1.3 channel was partially sensitive to PKA and PKC. Cotransfecting the Kv beta 1.2 subunit resulted in a decrease in the value of the time constant of inactivation of the full-length channel but did not modify its sensitivity to PKA and PKC. The cotransfected Kv beta 2 subunit had no effects. Our results indicate that the hKv1.3 lymphocyte channel retains its electrophysiological characteristics when transfected in the Kv beta-negative HEK 293 cell line but its sensitivity to modulation by PKA and PKC is significantly reduced.
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PMID:The sensitivity of the human Kv1.3 (hKv1.3) lymphocyte K+ channel to regulation by PKA and PKC is partially lost in HEK 293 host cells. 943 74

In vitro differentiation of mouse embryonic stem cells within three-dimensional cell aggregates called embryoid bodies parallels the development of postimplantation embryos at the egg cylinder stage, where visceral and parietal endoderm diverge from the primitive endoderm. We have investigated spontaneous [Ca2+]i oscillations by means of confocal laser-scanning microscopy in primitive endodermal cell layers of embryoid bodies during their differentiation to parietal and visceral endoderm. The frequency of [Ca2+]i oscillations increased from day 4 to day 19 of development, whereas their duration decreased from day 3 to days 16-17. Oscillations depended on both extracellular Ca2+ and Ca2+ release from intracellular stores as they were abolished in Ca(2+)-free solution and in the prescence of Ni2+ and thapsigargin. Signal transduction operated via the phospholipase C (PLC)-mediated inositol 1,4,5-triphosphate (InsP3) pathway with a negative feedback loop via protein kinase C (PKC) as U73,122, a blocker of PLC; bisindolylmaleimide 1, staurosporine, and H-7, blockers of PKC; and 10 mM caffeine totally inhibited [Ca2+]i spiking. Thimerosal, which hypersensitizes the InsP3 receptor, as well as vasopressin and bradykinin, which act via the InsP3 pathway, increased the frequency of [Ca2+]i spikes. In the prescence of brefeldin A (50 microM) or monensin (20 microM), which both inhibit endo/exocytotic vesicle pathways, an immediate transient increase in spiking activity was followed by a decline within 1 to 2 h. In the presence of brefeldin A or thapsigargin or in the absence of extracellular Ca2+, endocytotic vesicles were absent, suggesting that oscillating [Ca2+]i transients are involved in the exo/endocytotic vesicle shuttle.
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PMID:Spontaneous calcium oscillations in embryonic stem cell-derived primitive endodermal cells. 945 52

Moesin, a member of the ezrin-radixin-moesin (ERM) family of membrane/cytoskeletal linkage proteins, is known to be threonine-phosphorylated at Thr558 in activated platelets within its conserved putative actin-binding domain. The pathway leading to this phosphorylation step and its control have not been previously elucidated. We have detected and characterized reactions leading to moesin phosphorylation in human leukocyte extracts. In vitro phosphorylation of endogenous moesin, which was identified by peptide microsequencing, was dependent on phosphatidylglycerol (PG) or to a lesser extent, phosphatidylinositol (PI), but not phosphatidylserine (PS) and diacylglycerol (DAG). Analysis of charge shifts, phosphoamino acid analysis, and stoichiometry was consistent with a single phosphorylation site. By using mass spectroscopy and direct microsequencing of CNBr fragments of phospho-moesin, the phosphorylation site was identified as KYKT*LRQIR (where * indicates the phosphorylation site) (Thr558), which is conserved in the ERM family. Recombinant moesin demonstrated similar in vitro phospholipid-dependent phosphorylation compared with the endogenous protein. The phosphorylation site sequence of moesin displays a high degree of conservation with the pseudosubstrate sequences of the protein kinase C (PKC) family. We identified the kinase activity as PKC-theta on the basis of immunodepletion of the moesin kinase activity and copurification of PKC-theta with the enzymic activity. We further demonstrate that PKC-theta displays a preference for PG vesicles over PI or PS/DAG, with minimal activation by DAG, as well as specificity for moesin compared with myelin basic protein, histone H1, or other cellular proteins. Expression of a human His6-tagged PKC-theta in Jurkat cells and purification by Ni2+ chelate chromatography yield an active enzyme that phosphorylates moesin. PG vesicle binding experiments with expressed PKC-theta and moesin demonstrate that both bind to vesicles independently of one another. Thus, PKC-theta is identified as a major kinase within cells with specificity for moesin and with activation under non-classical PKC conditions. It appears likely that this activity corresponds to a specific intracellular pathway controlling the function of moesin as well as other ERM proteins.
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PMID:Protein kinase C-theta phosphorylation of moesin in the actin-binding sequence. 951 63


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