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)

Previous findings have shown that osteoblasts respond to parathyroid hormone (PTH) with an increase in extracellular acidification rate (ECAR) in addition to the known effect of PTH to increase local acidification by osteoclasts. We, therefore, investigated use of the Cytosensor to measure the ECAR response of whole intact bone to PTH employing microphysiometry. The Cytosensor measures a generic metabolic increase of cells to various agents. Using neonatal mouse calvaria, we found that the area surrounding the sagittal suture was particularly responsive to PTH. In this bone, the increase in ECAR was slower to develop (6 minutes) and more persistent than in cultured human osteoblast-like SaOS-2 cells and was preceded by a brief decrease in ECAR. Salmon calcitonin also produced an increase in ECAR in this tissue but with a different pattern than that elicited by PTH. Because PTH stimulates osteoclastic bone resorption in mouse calvaria via a cyclic adenosine monophosphate (cAMP)-mediated mechanism, we showed that the adenylyl cyclase activator forskolin also stimulated ECAR in this tissue. When the protein kinase A (PKA) pathway was activated by maintaining a high intracellular concentration of cAMP using N6-2'-0-dibutyryladenosine-cAMP (db-cAMP), there was a reduction of PTH-induced acidification, while isobutylmethylxanthine pretreatment potentiated the PTH-induced acidification, consistent with a PKA-mediated pathway. Thapsigargin and the protein kinase C (PKC) activator phorbol myristate acetate had no effect on the PTH-induced increase in ECAR in calvaria, indicating that PKC does not play a major role in the ECAR response in intact bone. These results indicate the utility of using microphysiometry to study ECAR responses in intact tissue and should enable elucidation of the relative importance of extracellular acidification by osteoblasts and osteoclasts to the anabolic and catabolic activities of PTH, respectively.
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PMID:Direct measurement of hormone-induced acidification in intact bone. 1075 May 70

Experiments on isolated superior cervical ganglia from rats were used to study the effects of substances affecting intracellular second messengers on membrane currents evoked by iontophoretic application of acetylcholine (ACh currents) and on excitatory postsynaptic currents (EPSC) induced by single discharges of preganglionic nerve fibers. These studies showed that the adenylate cyclase activator forskolin, the phosphodiesterase inhibitor isobutylmethylxanthine (IMBX), and the protein kinase C activator phorbol ester decreased the amplitude of the ACh current. Neither IMBX nor phorbol ester had any effect on the amplitude or decay time constant of EPSC, while forskolin increased the amplitude of EPSC without altering its decay time constant. Thapsigargin, which liberates intracellular calcium, not only decreased the amplitude of the ACh current, but also decreased EPSC amplitude without affecting its decay time constant. These results suggest that intracellular signaling via protein kinases A and C may affect neuronal nicotinic cholinoceptors (nAChR) only by altering receptor desensitization and not affecting receptor sensitivity to transmitters released from nerves or the kinetics of receptor ion channels. At the same time, neuronal nAChR are influenced by intracellular calcium, which decreases their ability to be activated by exogenous (perhaps acting via desensitization) and nerve-released acetylcholine without affecting the kinetics of ion channel function.
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PMID:Intracellular regulation of neuronal nicotinic cholinoreceptors. 1076 68

Isolated segments of porcine tracheal epithelium were mounted in Ussing chambers, current required to maintain transepithelial potential difference at 0 mV (short circuit current, I(SC)) was monitored and effects of nucleotides upon I(SC) were studied. Mucosal UTP (100 microM) evoked a transient rise in I(SC) that was followed by a sustained fall below basal I(SC) maintained for 30 min. Mucosal ATP (100 microM) also stimulated a transient rise in I(SC) but in contrast to UTP did not inhibit basal I(SC). Submucosal UTP and ATP both transiently increased I(SC). UTP-prestimulated epithelia were refractory to ATP but prestimulation with ATP did not abolish the response to UTP. The epithelia thus appear to express two populations of apical receptors allowing nucleotides to modulate I(SC). The UTP-induced rise was reduced by pretreatment with either bumetanide (100 microM), diphenylamin-2-carboxylic acid (DPC, 1 mM), or Cl(-) and HCO(3)(-)-free solution whilst the fall was abolished by amiloride pretreatment. Thapsigargin (0.3 microM) abolished the UTP-induced increase in I(SC) but not the subsequent decrease. Staurosporine (0.1 microM) inhibited basal I(SC) and blocked UTP-induced inhibition of I(SC). Inhibitors of either protein kinase C (PKC) (D-erythro sphingosine) or PKA (H89) had no effect. This study suggests that UTP stimulates Cl(-) secretion and inhibits basal Na(+) absorption. ATP has a similar stimulatory effect, which may be mediated by activation of P2Y(2) receptors and an increase in [Ca(2+)](in), but no inhibitory effect, which is likely mediated by activation of a pyrimidine receptor and possible inhibition of a protein kinase other than PKC or PKA.
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PMID:Differential effects of UTP and ATP on ion transport in porcine tracheal epithelium. 1080 75

UTP activates P2Y, receptors in both 1321N1 cell transfectants expressing the P2Y2 receptor and human HT-29 epithelial cells expressing endogenous P2Y, receptors with an EC50 of 0.2-1.0 microM. Pretreatment of these cells with UTP diminished the effectiveness of a second dose of UTP (the IC50 for UTP-induced receptor desensitization was 0.3-1.0 microM for both systems). Desensitization and down-regulation of the P2Y2 nucleotide receptor may limit the effectiveness of UTP as a therapeutic agent. The present studies investigated the phenomenon of P2Y2 receptor desensitization in human 1321N1 astrocytoma cells expressing recombinant wild type and C-terminal truncation mutants of the P2Y2 receptor. In these cells, potent P2Y2 receptor desensitization was observed after a 5 min exposure to UTP. Full receptor responsiveness returned 5-10 min after removal of UTP. Thapsigargin, an inhibitor of Ca2+-ATPase in the endoplasmic reticulum, induced an increase in the intracellular free calcium concentration, [Ca2+]i, after addition of desensitizing concentrations of UTP, indicating that P2Y2 receptor desensitization is not due to depletion of calcium from intracellular stores. Single cell measurements of increases in [Ca2+]i induced by UTP in 1321N1 cell transfectants expressing the P2Y2 receptor indicate that time- and UTP concentration-dependent desensitization occurred uniformly across a cell population. Other results suggest that P2Y2 receptor phosphorylation/dephosphorylation regulate receptor desensitization/resensitization. A 5 min preincubation of 1321N1 cell transfectants with the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), reduced the subsequent response to UTP by about 50%, whereas co-incubation of PMA with UTP caused a greater inhibition in the response. The protein phosphatases-1 and -2A inhibitor, okadaic acid, partially blocked resensitization of the receptor. Furthermore, C-terminal truncation mutants of the P2Y2 receptor that eliminated several potential phosphorylation sites including two for PKC were resistant to UTP-, but not phorbol ester-induced desensitization. Down regulation of protein kinase C isoforms prevented phorbol ester-induced desensitization but had no effect on agonist-induced desensitization of wild type or truncation mutant receptors. These results suggest that phosphorylation of the C-terminus of the P2Y2 receptor by protein kinases other than protein kinase C mediates agonist-induced receptor desensitization. A better understanding of the molecular mechanisms of P2Y2 nucleotide receptor desensitization may help optimize a promising cystic fibrosis pharmacotherapy based on the activation of anion secretion in airway epithelial cells by P2Y, receptor agonists.
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PMID:Mechanisms of agonist-dependent and -independent desensitization of a recombinant P2Y2 nucleotide receptor. 1082 29

Thapsigargin, which elevates cytosolic calcium levels by inhibiting the sarcoplasmic/endoplasmic reticulum calcium-dependent ATPase, was tested for its ability to degranulate bone marrow-derived mast cells (BMMCs) from src homology 2-containing inositol phosphatase +/+ (SHIP+/+) and SHIP-/- mice. As was found previously with steel factor, thapsigargin stimulated far more degranulation in SHIP-/- than in SHIP+/+ BMMCs, and this was blocked with the phosphatidylinositol-3 (PI-3) kinase inhibitors, LY294002 and wortmannin. In contrast to steel factor, however, this heightened degranulation of SHIP-/- BMMCs was not due to a greater calcium influx into these cells, nor was the thapsigargin-induced calcium influx inhibited by LY294002, suggesting that the heightened thapsigargin-induced degranulation of SHIP-/- BMMCs was due to a PI-3 kinase-regulated step distinct from that regulating calcium entry. An investigation of thapsigargin-stimulated pathways in both cell types revealed that MAPK was heavily but equally phosphorylated. Interestingly, the protein kinase C inhibitor, bisindolylmaleimide (compound 3), totally blocked thapsigargin-induced degranulation in both SHIP+/+ and SHIP-/- BMMCs. As well, thapsigargin stimulated a PI-3 kinase-dependent, transient activation of protein kinase B, and this activation was far greater in SHIP-/- than in SHIP+/+ BMMCs. Consistent with this, thapsigargin was found to be a potent survival factor, following cytokine withdrawal, for both cell types and was more potent with SHIP-/- cells. These studies have both identified an additional PI-3 kinase-dependent step within the mast cell degranulation process, possibly involving 3-phosphoinositide-dependent protein kinase-1 and a diacylglycerol-independent protein kinase C isoform, and shown that the tumor-promoting activity of thapsigargin may be due to its activation of protein kinase B and subsequent promotion of cell survival.
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PMID:Thapsigargin-induced degranulation of mast cells is dependent on transient activation of phosphatidylinositol-3 kinase. 1086 Oct 44

I investigated the role of protein kinase C (PKC) in regulation of the capacitative Ca2+ entry and steroidogenesis in bovine adrenocortical (BA) cells. Thapsigargin (TG)-treatment depleted intracellular Ca2+ stores followed by induction of Ca2+ influx from the extracellular pool and also increasing of Mn2+ influx as an indicator of divalent cation influx in BA cells. Calphostin C, a PKC inhibitor, inhibited the TG-induced [Ca2+]i elevation dose-dependently (0.1-1 microM) and attenuated Mn2+ entry. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, potentiated the elevation of [Ca2+]i and enhanced Mn2+ entry by TG treatment. These results suggest that PKC may modulate capacitative Ca2+ entry in BA cells. In the presence of extracellular Ca2+, TG enhanced cortisol production in BA cells. Calphostin C attenuated the TG-induced steroidogenesis dose-dependently (0.25-1 microM). PMA enhanced the steroidogenesis dose-dependently (1-100 nM). These results suggested that PKC may have a modulatory effect on the capacitative Ca2+ entry that links to steroidogenesis in BA cells.
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PMID:Protein kinase C potentiates capacitative Ca2+ entry that links to steroidogenesis in bovine adrenocortical cells. 1088 51

The release of [(3)H] arachidonic acid (AA) and its connection with the triggering of the MAP kinase cascade were studied in the human A549 epithelial cell line upon stimulation with thapsigargin. Thapsigargin can increase AA release along with the increase of intracellular calcium concentration, phosphorylation, and activation of extracellular regulated kinase (ERK) and cytosolic phospholipase A(2) (cPLA(2)). Both ERK and cPLA(2) phosphorylation in response to thapsigargin were inhibited by PD 98059, a specific inhibitor of MAP kinase kinase of the ERK group (MEK), and EGTA. cPLA(2) phosphorylation was not affected by Ro 31-8220 (an inhibitor of all PKC isoforms) or LY 379196 (a PKCbeta selective inhibitor), while both of them indeed attenuated ERK activation. On the other hand, rottlerin (the selective PKCdelta inhibitor), SB 203580 (the selective p38 MAPK inhibitor), and wortmannin (the PI 3-kinase inhibitor) can affect neither cPLA(2) nor ERK phosphorylation. In A549 cells, PKC activator PMA cannot increase either the basal or thapsigargin-induced (3)H-AA release, while it can induce the phosphorylation of ERK and cPLA(2.) The PMA-induced ERK phosphorylation was inhibited by Ro 31-8220, LY 379196, rottlerin, and PD 98059, but unaffected by SB 203580 and wortmannin. Moreover, the phosphorylation by PMA was non-additive with that of thapsigargin. This implies that intracellular Ca(2+) level is the key factor for induction of cPLA(2) activity and thapsigargin-elicited ERK activation itself is substantially sufficient for cPLA(2) activation upon intracellular Ca(2+) increase.
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PMID:Basal cPLA(2) phosphorylation is sufficient for Ca(2+)-induced full activation of cPLA(2) in A549 epithelial cells. 1099 51

At the synapse, neurotransmitter release is triggered physiologically by Ca(2+) influx through voltage-gated Ca(2+) channels. Non-physiologically, release can be evoked by a potent neurotoxin, alpha-latrotoxin, and by hypertonic sucrose. Controversy has arisen on whether release evoked by alpha-latrotoxin and hypertonic sucrose requires extracellular Ca(2+) or Ca(2+) from intracellular stores. Using synaptosomes, we have studied the Ca(2+) dependence of alpha-latrotoxin and sucrose action in different neurotransmitter systems. In agreement with previous data, no requirement for extracellular Ca(2+) in sucrose-induced secretion of norepinephrine, dopamine, glutamate or GABA was detected. Unexpectedly, we observed large differences between these neurotransmitters in the Ca(2+) dependence of alpha-latrotoxin-stimulated release: norepinephrine release required Ca(2+), dopamine release was only partially Ca(2+) dependent, and glutamate and GABA release did not require Ca(2+). To test if Ca(2+) derived from intracellular Ca(2+) stores participates in neurotransmitter release triggered by alpha-latrotoxin or hypertonic sucrose, we employed thapsigargin, a Ca(2+)-ATPase inhibitor that empties Ca(2+) stores. Thapsigargin did not induce neurotransmitter release, nor did it inhibit subsequent release stimulated by KCl depolarization, hypertonic sucrose or alpha-latrotoxin. However, intracellular Ca(2+) performs an important regulatory function, since thapsigargin increased the size of the readily releasable pool as measured by stimulation with hypertonic sucrose. This effect required extracellular Ca(2+) and protein kinase C, suggesting that depletion of internal Ca(2+) stores leads to store-operated Ca(2+) entry. The resulting Ca(2+) influx does not trigger release by itself, but activates protein kinase C that increases the readily releasable pool of neurotransmitters. Our data show that internal and external Ca(2+) is not acutely involved in hypertonic sucrose-evoked neurotransmitter release, while alpha-latrotoxin-triggered release requires external Ca(2+) for a subset of neurotransmitters. Although internal Ca(2+) is not essential for release, it modulates its extent, implying that the emptying of intracellular stores by activation of presynaptic receptors plays an important regulatory role in neurotransmitter release.
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PMID:Role of calcium in neurotransmitter release evoked by alpha-latrotoxin or hypertonic sucrose. 1111 28

The increase of intracellular free calcium concentration ([Ca(2+)](i)) and protein kinase C (PKC) activity are two major early mitogenic signals to initiate proliferation of human T cells. However, a rapid change in intracellular pH (pH(i)), acidification or alkalinization during the activation, is also associated after these two signals. The aim of this study was to define whether the change in pH(i) is affected by calcium and protein kinase C (PKC), in phytohemagglutinin (PHA)-stimulated T cells. T cells were isolated from human peripheral blood. The [Ca(2+)](i) and the pH(i) were measured using, respectively, the fluorescent dyes, Fura-2, and BCECF. In addition, down-regulation of PKC activity by PMA (1 microM, 18 h) was confirmed in these cells using a protein kinase assay. The results indicated that, (1) alkalinization was induced by PHA or PMA in T cells; the results of alkalinization was PKC-dependent and Ca(2+)-independent, (2) in PKC down-regulated T cells, PHA induced acidification; this effect was enhanced by pre-treating the cells with the Na(+)/H(+) exchange inhibitor, 5-(N,N-dimethyl)-amiloride, (DMA, 10 microM, 20 min), (3) the acidification was dependent on the Ca(2+) influx and blocked by removal of extracellular calcium or the addition of the inorganic channel blocker, Ni(2+), and (4) Thapsigargin (TG), a Ca(2+)-ATPase inhibitor, confirmed that acidification by the Ca(2+) influx occurred in T cells in which PKC was not down-regulated. These findings indicate two mechanisms, alkalinization by PKC and acidification by Ca(2+) influx, exist in regulating pH(i) in T cells. This is the first report that PHA stimulates the acidification by Ca(2+) influx but not alkalinization in T cells after down-regulation of PKC. In conclusion, the activity of PKC in T cells determines the response in alkalinization or acidification by PHA.
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PMID:Response of alkalinization or acidification by phytohemagglutinin is dependent on the activity of protein kinase C in human peripheral T Cells. 1132 15

1. The role of intracellular Ca(2+) mobilization in the mechanism of increased endothelial permeability was studied. Human umbilical vein endothelial cells (HUVECs) were exposed to thapsigargin or thrombin at concentrations that resulted in similar increases in intracellular Ca(2+) concentration ([Ca(2+)](i)). The rise in [Ca(2+)](i) in both cases was due to release of Ca(2+) from intracellular stores and influx of extracellular Ca(2+). 2. Both agents decreased endothelial cell monolayer electrical resistance (a measure of endothelial cell shape change) and increased transendothelial (125)I-albumin permeability. Thapsigargin induced activation of PKCalpha and discontinuities in VE-cadherin junctions without formation of actin stress fibres. Thrombin also induced PKCalpha activation and similar alterations in VE-cadherin junctions, but in association with actin stress fibre formation. 3. Thapsigargin failed to promote phosphorylation of the 20 kDa myosin light chain (MLC(20)), whereas thrombin induced MLC(20) phosphorylation consistent with formation of actin stress fibres. 4. Calphostin C pretreatment prevented the disruption of VE-cadherin junctions and the decrease in transendothelial electrical resistance caused by both agents. Thus, the increased [Ca(2+)](i) elicited by thapsigargin and thrombin may activate a calphostin C-sensitive PKC pathway that signals VE-cadherin junctional disassembly and increased endothelial permeability. 5. Results suggest a critical role for Ca(2+) signalling and activation of PKCalpha in mediating the disruption of VE-cadherin junctions, and thereby in the mechanism of increased endothelial permeability.
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PMID:Ca(2+) signalling and PKCalpha activate increased endothelial permeability by disassembly of VE-cadherin junctions. 1138 3


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