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)

This study examines the calcium store-regulated (capacitative) calcium influx pathway in the endocrine pancreatic cell line RINm5F, utilizing thapsigargin. After preincubation of the cells with the phorbol ester TPA, thapsigargin induced a sustained elevation of cytosolic calcium as well as a sustained stimulation of manganese entry, the latter being used to assess calcium influx. Thapsigargin given alone provoked a smaller and only transient elevation of cytosolic calcium and stimulation of manganese entry. The protein kinase C inhibitor staurosporine antagonized the effect of the phorbol ester. Verapamil, nifedipine, or measures to hyperpolarize the cells exerted no inhibitory action against this effect, which excludes an involvement of voltage-dependent calcium channels. In conclusion, our data shows for the first time that protein kinase C stimulation activates the capacitative calcium influx pathway of endocrine pancreatic insulin-producing cells.
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PMID:Protein kinase C activates capacitative calcium entry in the insulin secreting cell line RINm5F. 811 72

Remodeling of the pulmonary vascular tree in pulmonary hypertension is associated with hypertrophy and proliferation of smooth muscle cells. Since the stimuli and signaling pathways for these processes are not well understood, we used a rat pulmonary arterial smooth muscle cell line (PAC1) to examine the effects of thrombin and platelet-derived growth factor (PDGF) on cellular growth and immediate-early gene expression. Over 72 h, thrombin (1 unit/ml) caused hypertrophy as reflected by a 102 +/- 12% increase in protein synthesis and a 49 +/- 11% increase in protein content per cell, but no change in cell number. PDGF (2.5 ng/ml) stimulated proliferation as evidenced by an increase in cell number (doubling in 5 days), but no significant change in protein content per cell. Immediate-early gene expression was examined by Northern blotting: both thrombin and PDGF induced egr-1, c-fos, c-jun, junB, and fra-1 mRNAs within 15 min; the response was maximal at 30-60 min (increases ranging from 2.9- to 9.3-fold over control serum-deprived cells) and returned to base-line levels within 2-4 h. Neither agent affected junD mRNA levels. However, thrombin but not PDGF, caused an increase in fosB mRNA levels (7.7 +/- 4.0-fold higher than control, n = 12, p < 0.0005). The immediate-early gene response to both agonists was generally dependent on extracellular Ca2+, Na2+/H+ exchange, and protein kinase C activation, but not on cAMP. The exception was c-jun mRNA, the levels of which were not affected by inhibition of protein kinase C, but decreased significantly by prevention of cAMP formation. Thapsigargin-sensitive intracellular Ca2+ stores were necessary for the response to thrombin, but not to PDGF. These results demonstrate that thrombin is a hypertrophic agent and that PDGF is a proliferative agent in PAC1 cells. These two agonists stimulate increases in a variety of immediate-early gene mRNAs, but only thrombin induces fosB mRNA.
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PMID:Immediate-early gene expression in response to hypertrophic and proliferative stimuli in pulmonary arterial smooth muscle cells. 811 89

Thapsigargin-activated Ca2+ entry into platelets was examined in the presence of S-145, a thromboxane A2 receptor antagonist, to inhibit indirect effects by endogenously formed prostaglandin H2/thromboxane A2. With external Ca2+ present, 0.2 microM thapsigargin caused a prompt increase in intracellular Ca2+ concentration ([Ca2+]i) followed by a gradual increase. Pretreatment with 6 microM wortmannin, a specific inhibitor of myosin light chain kinase, partly inhibited the increase in [Ca2+]i. In Ca(2+)-free EGTA buffer, thapsigargin induced a smaller increase in [Ca2+]i, and subsequent addition of Ca2+ to the buffer caused a further prompt increase in [Ca2+]i, demonstrating external Ca2+ entry. Wortmannin only partly inhibited this entry of external Ca2+. The wortmannin-insensitive Ca2+ entry pathway remained open for more than 6 min in Ca(2+)-free buffer. On the other hand, when receptor agonists such as thrombin and U46619 were substituted for thapsigargin, activation of the wortmannin-insensitive Ca2+ entry was transient (Hashimoto et al., J. Biol. Chem (1992) 267, 17078-17081). In the presence of S-145 and wortmannin, thapsigargin stimulated phosphorylation of neither the 20-kDa myosin light chain nor the 47-kDa protein, a substrate of protein kinase C. These results suggest that thapsigargin induces external Ca2+ entry by two mechanisms: (1) a mechanism involving myosin light chain kinase; (2) a mechanism, not activated by receptor agonists, that is independent of the major protein kinases of platelets.
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PMID:Ca2+ entry pathways activated by the tumor promoter thapsigargin in human platelets. 826 42

Thapsigargin raises intracellular free calcium ([Ca2+]i) by potently inhibiting the endoplasmic reticulum Ca-ATPase, which sequesters calcium from the cytosol. In human keratinocytes a rise in [Ca2+]i has been associated with differentiation and therefore we investigated the action of thapsigargin on this process. At concentrations above 3 nM thapsigargin inhibited keratinocyte proliferation. Thapsigargin induced an immediate transient [Ca2+]i rise in calcium-free or 70 microM calcium medium but a more prolonged rise in 2 mM calcium. For keratinocytes cultured in 70 microM calcium medium a late [Ca2+]i rise was also observed, after 6 h, similar to the effect of known differentiation stimuli. However, immunohistochemical techniques did not show any expression of the differentiation-specific protein involucrin, a component of the cornified envelope. When keratinocyte differentiation was induced by an increase in the extracellular calcium from 70 microM to 2 mM abundant involucrin and desmoplakin, a component of desmosomes, were synthesised. Both proteins gave staining patterns which suggested incorporation into structural proteins, but thapsigargin disrupted the calcium-induced pattern of involucrin and desmoplakin synthesis. Thapsigargin did not induce differentiation, possibly due to its inability to activate protein kinase C and raise inositol trisphosphate levels. We conclude that a rise in [Ca2+]i does not alone induce keratinocyte differentiation but may act with other intracellular signals to promote differentiation.
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PMID:Thapsigargin raises intracellular free calcium levels in human keratinocytes and inhibits the coordinated expression of differentiation markers. 826 99

In C6-2B rat glioma cells, agonist-stimulated cAMP accumulation is potently inhibited after the stimulation of endogenous bradykinin receptors or stably transfected substance K receptors, coupled to phosphatidylinositol hydrolysis. In the present report, pharmacological tools were used to selectively stimulate either protein kinase C or Ca2+, the two final effectors activated upon phosphatidylinositol hydrolysis, and their role in the inhibition of the C6-2B cell cAMP signaling pathway was investigated. Activation of protein kinase C by an acute treatment with phorbol 12-myristate 13-acetate or L-alpha-1-oleoyl-2-acetyl-sn-3-glycerol did not reduce, but rather enhanced, the cAMP accumulation elicited by forskolin, a direct activator of adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. This effect was antagonized by the protein kinase inhibitor H-7 and mimicked by the protein phosphatase inhibitor okadaic acid. Thapsigargin, a selective microsomal Ca(2+)-ATPase inhibitor, evoked a sustained increase in the intracellular free Ca2+ concentration, with an EC50 of 24.8 +/- 4.3 nM, and inhibited the cAMP accumulation induced by the beta-adrenergic receptor agonist isoproterenol with comparable potency (IC50 = 19.3 +/- 0.2 nM), strongly suggesting a causal relationship between the two phenomena. The inhibition by thapsigargin of isoproterenol- or forskolin-stimulated cAMP accumulation was not affected by pertussis toxin or down-regulation or inhibition of protein kinase C. Dantrolene, a blocker of Ca2+ release from intracellular stores, antagonized 1) the Ca2+ transient in response to thapsigargin and substance K and 2) the inhibitory effect of these compounds on isoproterenol- or forskolin-induced cAMP accumulation. Moreover, sequestration of intracellular Ca2+ with the cell-permeable Ca2+ chelator ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid acetoxymethyl ester abolished the cAMP inhibition mediated by thapsigargin. Finally, isoproterenol- or forskolin-stimulated adenylyl cyclase activity in digitonin-permeabilized cells was not affected by either thapsigargin or substance K. These data provide compelling evidence that increases in intracellular free Ca2+ concentration without activation of protein kinase C suffice and are responsible for the inhibition of cAMP accumulation in C6-2B cells.
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PMID:Ca2+ inhibition of beta-adrenergic receptor- and forskolin-stimulated cAMP accumulation in C6-2B rat glioma cells is independent of protein kinase C. 838 3

The biochemical transductional events involved in NO synthesis are not fully understood. These studies, therefore, were undertaken to elucidate the role of intracellular calcium and protein kinase C (PKC) in the induction of nitric oxide (NO) synthesis in murine peritoneal macrophages. Thapsigargin (TG), Ca(2+)-ATPase inhibitor of endoplasmic reticulum, had modest activity on NO synthesis by itself, whereas phorbol ester, PKC activator, alone had no effect. When TG was used in combination with phorbol ester, there was a marked cooperative induction of NO synthesis in a dose-dependent manner. The optimal effect of phorbol ester was shown in the first 6 h after TG treatment. In addition, the ability of TG with phorbol ester on NO synthesis could be mimicked by another chemically unrelated inhibitor of Ca(2+)-ATPase, 2,5-di-(t-butyl)-1, 4-benzohydroquinone, and Ca2+ ionophore, A23187. This increase of NO synthesis was reflected as increased amount of NO synthase (NOS) mRNA, as determined by Northern blotting. Intracellular Ca2+ transient by TG was not affected in the presence or absence of extracellular Ca2+, indicating that TG must be effective on cytosolic Ca2+ pool. In addition, chelation of intracellular Ca2+ by acetoxymethyl ester of 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM), an intracellular Ca2+ chelating agent, blocked TG- or TG + PMA-induced NO production. PKC inhibitors such as staurosporine or polymyxin B reduced only the synergistic cooperative effect of TG with phorbol ester without affecting TG-induced NO production. In addition, when the cells were pretreated with phorbol ester before TG treatment, there was no synergy between TG and phorbol ester, indicating that PKC is not directly involved in the expression of NOS but involved in "triggering" signal. Secretion of NO corresponded with tumor cell killing, but TG plus phorbol ester-activated macrophages failed to kill tumor cell targets in the presence of Ng-monomethyl-L-arginine. Collectively, these data illustrate that mobilization of intracellular Ca2+ provides a "priming" signal for induction of NOS gene expression by itself and it also requires PKC as a "triggering" signal for macrophage tumoricidal activity.
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PMID:Synergistic cooperation between thapsigargin and phorbol ester for induction of nitric oxide synthesis in murine peritoneal macrophages. 872 23

Although the sesquiterpene lactone thapsigargin has been shown to possess hyperplastic and tumor-promoting activities when applied topically to mouse skin in vivo, the cellular mechanism(s) which underlie these effects are unclear. We show here that thapsigargin treatment of Primary mouse epidermal keratinocytes increased intracellular free Ca2+ concentration (Cai) in a concentration-dependent manner. Thapsigargin induced a rapid, transient elevation in keratinocyte Cai, in part due to the release of Ca2+ from intracellular stores. This response was followed by a sustained elevation in Ca2+, resulting entirely from calcium influx. Thapsigargin elicited a biphasic effect on keratinocyte DNA synthesis: a rapid inhibitory effect (50-60% inhibition at 4-8 h), followed by a very marked and sustained elevation. Prolonged treatment of keratinocytes with thapsigargin at relatively high concentrations resulted in cytotoxicity (inhibition of neutral red uptake). The rapid antiproliferative effect of thapsigargin was not associated with cytotoxicity, as determined by either neutral red uptake or by trypan blue exclusion, and was not blocked by pretreatment with Ro 31-7349, a selective inhibitor of protein kinase C. The rapid antiproliferative effect of thapsigargin was associated with rapid, transient activation of keratinocyte c-fos expression and rapid inhibition of total protein synthesis. Taken together, these findings raise the possibility that the hyperplastic and tumor-promoting activities of thapsigargin on epidermis in vivo result from direct keratinocyte growth stimulation as a consequence of a prolonged elevation in levels of Cai.
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PMID:Thapsigargin induces rapid, transient growth inhibition and c-fos expression followed by sustained growth stimulation in mouse keratinocyte cultures. 875 61

1. In this study we have investigated delta and mu opioid receptor-mediated elevation of intracellular Ca2+ concentration ([Ca2+]i) in the human neuroblastoma cell line, SH-SY5Y. 2. The Ca(2+)-sensitive dye, fura-2, was used to measure [Ca2+]i in confluent monolayers of SH-SY5Y cells. Neither the delta-opioid agonist, DPDPE ([D-Pen2,5]-enkephalin) nor the mu-opioid agonist, DAMGO (Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol enkephalin) elevated [Ca2+]i when applied alone. However, when either DPDPE or DAMGO was applied in the presence of the cholinoceptor agonist, carbachol (100 nM-1 mM) they evoked an elevation of [Ca2+]i above that caused by carbachol alone. 3. In the presence of 1 microM or 100 microM carbachol, DPDPE elevated [Ca2+]i with an EC50 of 10 nM. The elevation of [Ca2+]i was independent of the concentration of carbachol. The EC50 for DAMGO elevating [Ca2+]i in the presence of 1 microM and 100 microM carbachol was 270 nM and 145 nM respectively. 4. The delta-receptor antagonist, naltrindole (30 nM), blocked the elevations of [Ca2+]i by DPDPE (100 nM) without affecting those caused by DAMGO while the mu-receptor antagonist, CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Pen-Thr-NH2) (100 nM-1 microM) blocked the elevations of [Ca2+]i caused by DAMGO (1 microM) without affecting those caused by DPDPE. 5. Block of carbachol activation of muscarinic receptors with atropine (10 microM) abolished the elevation of [Ca2+]i by the opioids. The nicotinic receptor antagonist, mecamylamine (10 microM), did not affect the elevations of [Ca2+]i caused by opioids in the presence of carbachol. 6. Muscarinic receptor activation, not a rise in [Ca2+]i, was required to reveal the opioid response. The Ca2+ channel activator, maitotoxin (3 ng ml-1), also elevated [Ca2+]i but subsequent application of opioid in the presence of maitotoxin caused no further changes in [Ca2+]i. 7. The elevations of [Ca2+]i by DPDPE and DAMGO were abolished by pretreatment of the cells with pertussis toxin (200 ng ml-1, 16 h). This treatment did not significantly affect the response of the cells to carbachol. 8. The opioids appeared to elevate [Ca2+]i by mobilizing Ca2+ from intracellular stores. Both DPDPE and DAMGO continued to elevate [Ca2+]i when applied in nominally Ca(2+)-free external buffer or when applied in a buffer containing a cocktail of Ca2+ entry inhibitors. Thapsigargin (100 nM), an agent which discharges intracellular Ca2+ stores, also blocked the opioid elevations of [Ca2+]i. 9. delta and mu Opioids did not appear to mobilize intracellular Ca2+ by modulating the activity of protein kinases. The application of H-89 (10 microM), an inhibitor of protein kinase A, H-7 (100 microM), an inhibitor of protein kinase C, protein kinase A and cyclic GMP-dependent protein kinase, or Bis I, an inhibitor of protein kinase C, did not alter the opioid mobilization of [Ca2+]i. 10. Thus, in SH-SY5Y cells, opioids can mobilize Ca2+ from intracellular stores but they require ongoing muscarinic receptor activation. Opioids do not elevate [Ca2+]i when applied alone.
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PMID:delta- and mu-opioid receptor mobilization of intracellular calcium in SH-SY5Y human neuroblastoma cells. 878 87

The actions of TSH, ATP, the ionophore A23187, the endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin, and phorbol dibutyrate (PDBu) on 3H-cytidine-monophosphate phosphatidic acid (3H-CMP-PA) accumulation were studied in human thyroid slices to evaluate PA generation and inositol recycling towards phosphatidyl-inositol synthesis. The effects of the same agonists also were measured on phosphatidylbutanol (PtdBut) generation in 3H-palmitate or 3H-myristate prelabeled slices to assess the activity of phospholipase D (PLD). The phospholipid target of this PLD was determined on 3H-choline prelabeled human thyroid slices by measuring 3H-choline release in incubation medium and slices and 3H-choline incorporation in phospholipids. TSH (10 U/L) stimulated 3H-CMP-PA accumulation in an LiCl-and propranolol-insensitive way, as well as 2H-fatty acids incorporation into PA, diacylglycerol, and phosphatidylcholine (PtdCho) with on evidence of dose-dependent effects and had no detectable action on PLD activity. The effects of TSH were not reproduced by Bu2cAMP or forskolin. Thapsigargin and A23187 both increased CMP-PA accumulation and PtdBut generation, whereas ATP only stimulated PLD activity. The phorbol ester PDBu (5 x 10(-7) mol/L) increased PtdBut formation and 3-H-fatty acid incorporation into PtdCho, but had no effect on CMP-PA generation. Staurosporine (STSP) (5 x 10(-6) mol/L), a nonspecific inhibitor of protein kinase C, unexpectedly reproduced the effects of PDBu. The increase of 3H-choline in slices' supernatant and the decrease of 3H-choline-labeled PtdCho induced by PDBu, ATP, thapsigargin, and STSP indicate that the activated PLD hydrolyzed PtdCho. We suggest that the PA generation induced by PLD stimulation could contribute to the stimulated H2O2 formation and iodide organification observed with the agonists inducing PtdBut accumulation. Indeed, Bu2cAMP and forskolin, known to decrease iodide organification in human thyroid, inhibited the PLD stimulation induced by ATP and PDBu. In cultured dog thyrocytes, phorbol esters, and STSP induced DNA synthesis and dedifferentiation, whereas thapsigargin inhibited TSH-induced growth and killed phorbol esters stimulated cells, suggesting a positive role of PLD stimulation towards dedifferentiated growth and of simultaneously raised [Ca2+)i and stimulated protein kinase C-PLD towards growth arrest and cellular death.
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PMID:Regulation and metabolic role of phospholipase D activity in human thyroid and cultured dog thyrocytes. 885 96

In rat liver epithelial cells (GN4), angiotensin II (Ang II) and thapsigargin stimulate a novel calcium-dependent tyrosine kinase (CADTK) also known as PYK2, CAKbeta, or RAFTK. Activation of CADTK by a thapsigargin-dependent increase in intracellular calcium failed to stimulate the extracellular signal-regulated protein kinase pathway but was well correlated with a 30-50-fold activation of c-Jun N-terminal kinase (JNK). In contrast, Ang II, which increased both protein kinase C (PKC) activity and intracellular calcium, stimulated extracellular signal-regulated protein kinase but produced a smaller, less sustained, JNK activation than thapsigargin. 12-O-Tetradecanoylphorbol 13-acetate (TPA), which slowly activated CADTK, did not stimulate JNK. These findings suggest either that CADTK is not involved in JNK activation or PKC activation inhibits the CADTK to JNK pathway. A 1-min TPA pretreatment of GN4 cells inhibited thapsigargin-dependent JNK activation by 80-90%. In contrast, TPA did not inhibit the >50-fold JNK activation effected by anisomycin or UV. The consequence of PKC-dependent JNK inhibition was reflected in c-Jun and c-Fos mRNA induction following treatment with thapsigargin and Ang II. Thapsigargin, which only minimally induced c-Fos, produced a much greater and more prolonged c-Jun response than Ang II. Elevation of another intracellular second messenger, cAMP, for 5-15 min also inhibited calcium-dependent JNK activation by approximately 80-90% but likewise had no effect on the stress-dependent JNK pathway. In summary, two pathways stimulate JNK in cells expressing CADTK, a calcium-dependent pathway modifiable by PKC and cAMP-dependent protein kinase and a stress-activated pathway independent of CADTK, PKC, and cAMP-dependent protein kinase; the inhibition by PKC can ultimately alter gene expression initiated by a calcium signal.
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PMID:Protein kinase C and protein kinase A inhibit calcium-dependent but not stress-dependent c-Jun N-terminal kinase activation in rat liver epithelial cells. 916 74


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