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

Thrombin is thought to stimulate responsive cells by cleaving cell-surface receptors coupled to intracellular second-messenger-generating enzymes via G-proteins. In order to understand this process better, we have examined the regulation of adenylate cyclase by thrombin in the megakaryoblastic HEL cell line and compared it with platelets. A notable difference was found. In HEL-cell membrane preparations, thrombin inhibited cyclic AMP (cAMP) formation by a pertussis-toxin-sensitive mechanism comparable with that observed in platelets. In contrast, when added to intact HEL cells, thrombin activated adenylate cyclase and caused an increase in cAMP formation synergistic with that produced by forskolin and prostaglandin I2. This increase, which was not seen with platelets, was accompanied by an increase in cAMP metabolism by phosphodiesterase. Like other responses to thrombin, the increase in cAMP formation required proteolytically active thrombin and was subject to homologous desensitization. An equivalent response could be evoked by the addition of a polypeptide, derived from the N-terminus of the thrombin receptor, that has been shown to activate the receptor. The effects of thrombin could not, however, be reproduced by the addition of phorbol ester and the Ca2+ ionophore, A23187, nor be prevented with inhibitors of arachidonate metabolism. Preincubation of the cells with adrenaline, which inhibited Gs-mediated activation of adenylate cyclase, or pertussis toxin, which inhibited phospholipase C activation, had no effect on thrombin-induced cAMP formation. These results suggest that thrombin can regulate cAMP formation by two different mechanisms. First, thrombin can inhibit adenylate cyclase in a Gi-dependent manner. This effect predominates in HEL-cell membrane preparations, as it does in platelets, but is not detectable when thrombin is added to intact HEL cells. Instead, in intact HEL cells thrombin activates adenylate cyclase. Although clearly receptor-mediated, this response does not appear to involve Gi, Gs, protein kinase C, eicosanoid formation or changes in the cytosolic Ca2+ concentration.
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PMID:Dual regulation of cyclic AMP formation by thrombin in HEL cells, a leukaemic cell line with megakaryocytic properties. 131 10

The activation of membrane-bound phospholipase D (PLD) resulting in the generation of phosphatidic acid (PA) is increasingly recognized as an integral event in the initiation of a variety of cellular responses. We explored whether alpha-thrombin is a physiologic agonist for PLD activation in human umbilical vein endothelial cells (HUVEC). HUVEC monolayers were labeled with [32Pi] and PLD activity determined by formation of the PLD metabolite [32P] phosphatidylethanol (PEt) in the presence of 5 g/L ethanol by thin-layer chromatography. alpha-Thrombin rapidly (1 minute) increased PA and PEt formation in a dose-dependent manner (10(-6) to 10(-10)) with maximal PLD stimulation achieved with 10 nmol/L alpha-thrombin producing a threefold to fourfold increase in PA and a sixfold to eightfold increase in PEt over controls at 15 minutes. Esterolytically active zeta-thrombin (10 nmol/L) and gamma-thrombin (1 mumol/L), but not inactive DIP-alpha-thrombin (1 mumol/L) also increased PLD activity. The role of Ca2+ flux in human endothelial cell PLD activation was investigated and PEt formation was significantly enhanced by Ca2+ ionophores A23187 and ionomycin (1 mumol/L, three-fold to fourfold increase in PEt). Alpha-Thrombin-stimulated PEt formation was abolished (greater than 90% inhibition) with chelation of intracellular calcium (Ca2+i) by pretreatment with BAPTA-AM (25 mumol/L, 30 minutes) but only mildly attenuated (30% inhibition) by removal of extracellular calcium (Ca2+E) with EGTA (5 mmol/L). The protein kinase C (PKC) inhibitor staurosporine reduced alpha-thrombin-induced PEt formation in a dose-dependent manner (10 mumol/L, 78% inhibition) and PKC downregulation with chronic PMA treatment (18 hours) also resulted in marked inhibition of alpha-thrombin-induced PEt formation. Neither pertussis nor botulinum C bacterial toxins significantly altered alpha-thrombin-induced PLD responses. In contrast, similar pretreatment with cholera toxin (1 microgram/mL, 60 minutes) consistently augmented alpha-thrombin-stimulated PLD activity by 50% to 90%. Comparable results were observed with agents which increased cAMP such as forskolin, 8-bromo cAMP, or dibutyryl cAMP and cholera toxin augmentation was abolished by 2-dideoxyadenosine, a competitive inhibitor of adenylyl cyclase activity. These studies demonstrate that alpha-thrombin is a potent stimulus for human PLD-mediated PA formation and that cyclic adenosine nucleotides modulate agonist-induced cellular PLD activity. In this model of PLD activation, alpha-thrombin receptor occupancy leads to the breakdown of phosphatidylinositol 4,5-bisphosphate catalyzed by phospholipase C producing the Ca2+ secretagogue IP3 and DAG.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Thrombin stimulation of human endothelial cell phospholipase D activity. Regulation by phospholipase C, protein kinase C, and cyclic adenosine 3'5'-monophosphate. 131 12

Fructose-1,6-diphosphate (FDP) is a physiological product which exhibits pharmacological properties. This study shows that FDP (1-3 mM) inhibits platelet aggregation induced by the agonists thrombin, vasopressin, platelet activating factor, ADP, adrenaline, arachidonate and the stable thromboxane analogue U 44069. Thrombin-promoted ATP secretion and cytosolic Ca2+ rise are also drastically inhibited by FDP, which decreases, although to a lesser extent, the protein kinase C-dependent phosphorylation of the 47 kDa protein. The inhibition on thrombin-induced aggregation is shared, albeit less efficiently, by glucose-1,6-diphosphate and fructose-2,6-diphosphate but not by other phosphorylated monosaccharides (fructose-1:2 cyclic,6-diphosphate, glucose-1- and glucose-6-phosphate, fructose-1- and fructose-6-phosphate, mannose-6-phosphate and 5-phosphoryl ribose-1-pyrophosphate). FDP does not affect platelet activation induced by the protein kinase C activators dioctanoylglycerol or phorbol 12-myristate 13-acetate. No increase of cAMP concentration is observed in FDP-treated platelets. Altogether, these results indicate that FDP inhibits platelet activation at a level preceding phospholipase C. The data are consistent with a general inhibitory action of FDP on signal transmission.
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PMID:Fructose-1,6-diphosphate inhibits platelet activation. 131 5

The protease thrombin is a potent activator of various cell types. Thrombin cleaves and thereby activates its own seven-transmembrane-domain receptor which couples to G proteins. Thrombin also can inhibit neuronal differentiation, supposedly by degrading components of the extracellular matrix. Here we report that active thrombin induces immediate cell rounding and neurite retraction in differentiating N1E-115 and NG108-15 neural cells in serum-free culture. Serum (0.5-5% vol/vol) evokes similar responses, but the cell-rounding and neurite-retracting activity of serum is not attributable to thrombin. Neural cell rounding is transient, subsiding after 10-15 min, and subject to homologous desensitization, whereas retracted neurites rapidly degenerate. Thrombin action is inhibited by cytochalasin, but not colchicine. A novel 14-amino acid peptide agonist of the thrombin receptor fully mimics thrombin's morphoregulatory activity, indicating that thrombin-induced shape changes are receptor-mediated and not secondary to extracellular matrix degradation. Although thrombin receptors couple to phosphoinositide hydrolysis and Ca2+ mobilization, thrombin-induced shape changes appear to depend neither on the Ca2+/protein kinase C- nor the cyclic nucleotide-mediated signal transduction pathways; however, the morphological response to thrombin is blocked by pervanadate, an inhibitor of tyrosine phosphatases, and by broad-specificity kinase inhibitors. Our results suggest that the thrombin receptor communicates to an as-yet-uncharacterized effector to reorganize the actin cytoskeleton and to reverse the differentiated phenotype of neural cells.
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PMID:Thrombin receptor activation causes rapid neural cell rounding and neurite retraction independent of classic second messengers. 132 Nov 60

Platelets from stroke-prone spontaneously hypertensive rats (SHRSP) show severe hypofunctions accompanied by defective protein (P47) phosphorylation. To examine the mechanism of platelet hypofunctions, phospholipid metabolism in SHRSP was compared with that in Wistar Kyoto rats (WKY). Phosphatidylinositol (PI) content was 20% less in SHRSP than in WKY, but no changes were observed in other phospholipids. Incorporation of [3H]-arachidonic acid (AA) into PI and phosphatidylethanolamine (PE) was 12% and 11% lower, and that into phosphatidylcholine (PC) was 6% higher in SHRSP than in WKY. Thrombin-induced diacylglycerol and phosphatidic acid formation were similar in both groups of platelets. Thrombin-induced release of [14C]-AA from the labeled platelets and its metabolism to eicosanoids occurred at similar rates. These results suggest that reduced formation of diacylglycerol, an activator of protein kinase C (PKC), does not cause defective phosphorylation of P47, a substrate of PKC, in SHRSP. However it remains unclear how the lower PI content and the altered distribution of AA in PC and PE is related to SHRSP platelet hypofunctions.
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PMID:Phospholipid metabolism in platelets from stroke-prone spontaneously hypertensive rats and Wistar Kyoto rats. 132 96

Activation of either muscarinic cholinergic or thrombin receptors increases phosphoinositide turnover, Ca2+ mobilization, and redistribution of protein kinase C and induces rapid transient increases in c-fos mRNA and c-jun mRNA in 1321N1 cells. To determine whether the increases in c-fos and c-jun mRNA induced by carbachol and thrombin are sufficient to stimulate AP-1-mediated transactivation, 1321N1 cells were transfected with a reporter carrying two copies of the tetradecanoyl phorbol acetate response element and the firefly luciferase gene. Thrombin was significantly more effective than carbachol at stimulating AP-1-mediated transactivation. To identify the factors underlying the difference in AP-1 activity induced by carbachol and thrombin, members of the fos and jun families which encode components of AP-1 were examined. Carbachol and thrombin have similar effects on expression of c-fos, fosB, fra-2, junB, and junD, both acutely and over a 24-h time course. However, whereas carbachol leads only to transient induction of c-jun (maximal at 0.5 h), thrombin induces a biphasic increase in c-jun mRNA--an initial peak at 0.5 h and a second, more-prolonged increase at 12 h. Thrombin but not carbachol also induces a late increase in fra-1 mRNA, which peaks at 12 h. The secondary increase in c-jun mRNA is associated with marked increases in c-Jun protein levels and AP-1 DNA-binding activity. The late induction of c-jun and fra-1 mRNA can be prevented by adding the antagonist hirudin 30 min after thrombin, which results in loss of thrombin-stimulated increases in c-Jun protein, AP-1 DNA-binding activity, and AP-1-mediated transactivation. These findings suggest that rapid and transient conduction of c-fos and c-jun mRNA is insufficient to induce prominent changes in gene transcription, while the sustained increase in c-jun mRNA and perhaps the late induction of fra-1 mRNA are required for generation of AP-1 DNA-binding activity and transactivation through AP-1.
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PMID:Biphasic increase in c-jun mRNA is required for induction of AP-1-mediated gene transcription: differential effects of muscarinic and thrombin receptor activation. 132 61

The human erythroleukemia cell line (HEL) has been used as a model system for studying signal transduction processes as they might relate to platelet/megakaryocyte function. We were interested in examining the role of thrombin in the regulation of adenylyl cyclase in this cell line. As opposed to its predominantly inhibitory effects on cyclic AMP production in platelets or in membranes from HEL cells, our initial experiments in intact HEL cells revealed that thrombin markedly potentiated the cyclic AMP response to prostaglandin E1 (2.9 +/- 0.2-fold), prostacyclin (1.9 +/- 0.2-fold) and carbacyclin (2.5 +/- 0.5-fold), measured either by radioimmunoassay or by the [3H]adenine preloading procedure. Thrombin, although ineffective alone, also potentiated cyclic AMP production stimulated by vasoactive intestinal peptide (1.6 +/- 0.2-fold), cholera toxin (3.0 +/- 0.6-fold) and AIF4- (2.3 +/- 0.6-fold), but not by forskolin (0.9 +/- 0.1-fold). The thrombin effect 1) produced an increase in the efficacy of the prostaglandins with no change in potency; 2) was long-lived; 3) required the proteolytic activity of thrombin; 4) was insensitive to pertussis toxin; and 5) was at least partially mimicked by trypsin, extracellular ATP and UTP, platelet activating factor and activators of protein kinase C. Down-regulation of protein kinase C or pre-exposure to the protein kinase inhibitor staurosporine blocked the potentiating effect. Together, these results suggest that in HEL cells, the mechanism of thrombin potentiation of cyclic AMP production may involve alterations in the interaction between stimulatory guanine nucleotide binding protein and the catalytic subunit of adenylyl cyclase, possibly involving protein kinase C-mediated phosphorylation.
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PMID:Potentiation of cyclic adenosine monophosphate production by thrombin in the human erythroleukemia cell line, HEL. 133 12

1. Barrier function and cytosolic free calcium content [Ca2+]i was measured in monolayers of bovine pulmonary artery endothelial cells (BPAEC) and bovine aortic endothelial cells (BAEC). 2. Thrombin (1 u ml-1) increased albumin transfer across monolayers of BPAEC but not BAEC, yet induced biphasic increases in [Ca2+]i in both endothelial cell types, consisting of a rapid, initial phasic component which decayed to a lower, more sustained plateau phase. 3. 4 beta-Phorbol 12-myristate 13-acetate (PMA; 0.3-3000 nM) increased albumin transfer across monolayers of BPAEC and BAEC, but had no effect on basal levels of [Ca2+]i in either endothelial cell type. 4. Treatment of BPAEC and BAEC with forskolin (30 microM), an activator of adenylate cyclase, had no effect on resting transfer of albumin, but inhibited that stimulated by PMA (600 nM). It also inhibited the thrombin (1 u ml-1)-induced increase in albumin transfer across monolayers of BPAEC, but enhanced the plateau phase of the associated increase in [Ca2+]i. 5. Treatment of BPAEC and BAEC with either atriopeptin II (100 nM), an activator of particulate guanylate cyclase, or 8 bromo cyclic GMP (30 microM) had no effect on resting or PMA (600 nM)-stimulated transfer of albumin. Both agents did, however, inhibit the thrombin (1 u ml-1)-induced increase in albumin transfer across monolayers of BPAEC, but had no effect on the associated increase in [Ca2+]i. 6. These data suggest a dissociation between the ability of agents that increase or decrease albumin transfer and their effects on [Ca2+]i. Consequently, activation of protein kinase C may be the major stimulus for trans-endothelial transfer of macromolecular solutes. Endothelial barrier function is enhanced by elevation of either cyclic AMP or cyclic GMP content. Cyclic AMP appears to act by inhibiting the actions of protein kinase C, while cyclic GMP may act to inhibit a key step proximal to activation of this enzyme.
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PMID:Modulation of barrier function of bovine aortic and pulmonary artery endothelial cells: dissociation from cytosolic calcium content. 133 54

Cell surface aminopeptidases N (APN) and A (APA) have been characterized on cultured human glomerular epithelial cells and a SV40-transformed cell line derived from them. APN had a wide substrate specificity whereas APA only attacked peptides with an acidic N terminal amino acid. Both enzymes also differed by their sensitivity to divalent cations and to aminopeptidase inhibitors. Phorbolmyristate acetate (PMA) stimulated APN but not APA expression after a lag time of 12 hours. An increase of twice the basal value was observed with 10 ng.ml-1 PMA. This effect was confirmed by immunofluorescence staining using a specific anti-APN monoclonal antibody. Both ecto- and total enzyme activities were stimulated by PMA. The effect of PMA was suppressed by H7, a PKC inhibitor, and cycloheximide, an inhibitor of protein synthesis. Thrombin (1 to 2.5 U.ml-1) and interferon (IFN)-gamma (100 U.ml-1) also stimulated APN activity, the latter after longer exposure of the cells. APA activity was increased by 8-bromo-cAMP and two cAMP-stimulating agents, forskolin and isobutylmethylxanthine (IBMX). A twofold increase above basal value was obtained with 100 microM forskolin after 72 hours of treatment. cAMP-stimulated APA activity was suppressed by cycloheximide. Dexamethasone also stimulated APA activity. The effects of forskolin and dexamethasone were additive. These results demonstrate that APN and APA in glomerular epithelial cells are under different regulations: mitogens and IFN-gamma for APN, cAMP and glucocorticoids for APA. This selective expression may imply possible functional consequences in glomerular diseases.
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PMID:Cell surface aminopeptidase A and N activities in human glomerular epithelial cells. 135 70

Thrombin, the key regulatory protein of hemostasis, is a potent stimulus for endothelial cell activation, a process implicated in a variety of ischemic, thrombotic, and inflammatory vascular disorders. Activation of the thrombin receptor requires a novel mechanism of receptor proteolysis generating a tethered receptor ligand. Synthetic peptides whose sequences are identical to this newly exposed receptor NH2-terminus reproduce thrombin effects on human and bovine endothelial cell activation. Receptor cleavage by catalytically active alpha-thrombin is tightly coupled to a PI-PLC, with resultant generation of IP3 and DAG, increases in [Ca2+]i, and translocation of PKC (Fig. 3). Both the increase in [Ca2+]i and PKC activation are required for thrombin-stimulated PLA2 and PLD activity, PGI2 synthesis, and barrier dysfunction, the latter occurring as the result of Ca2+ and PKC effects on specific cytoskeletal protein elements and other contractile proteins (Fig. 3). Further investigations are ongoing to identify more clearly not only the precise biochemical intermediates involved in the endothelial cell response to thrombin but also the specific protein kinase systems involved in thrombin-mediated signal transduction in vascular endothelium.
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PMID:Molecular mechanisms of thrombin-induced human and bovine endothelial cell activation. 140 26


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