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Prostacyclin inhibits platelet aggregation induced by phorbol ester or Ca2+ ionophore at steps distal to activation of protein kinase C and Ca2+-dependent protein kinases.

ABSTRACT: Suspensions of aspirin-treated, 32P-prelabelled, washed platelets containing ADP scavengers in the buffer were activated with either phorbol 12,13-dibutyrate (PdBu) or the Ca2+ ionophore A23187. High concentrations of PdBu (greater than or equal to 50 nM) induced platelet aggregation and the protein kinase C (PKC)-dependent phosphorylation of proteins with molecular masses of 20 (myosin light chain), 38 and 47 kDa. No increase in cytosolic Ca2+ was observed. Preincubation of platelets with prostacyclin (PGI2) stimulated the phosphorylation of a 50 kDa protein [EC50 (concn. giving half-maximal effect) 0.6 ng of PGI2/ml] and completely abolished platelet aggregation [ID50 (concn. giving 50% inhibition) 0.5 ng of PGI2/ml] induced by PdBu, but had no effect on phosphorylation of the 20, 38 and 47 kDa proteins elicited by PdBu. The Ca2+ ionophore A23187 induced shape change, aggregation, mobilization of Ca2+, rapid phosphorylation of the 20 and 47 kDa proteins and the formation of phosphatidic acid. Preincubation of platelets with PGI2 (500 ng/ml) inhibited platelet aggregation, but not shape change, Ca2+ mobilization or the phosphorylation of the 20 and 47 kDa proteins induced by Ca2+ ionophore A23187. The results indicate that PGI2, through activation of cyclic AMP-dependent kinases, inhibits platelet aggregation at steps distal to protein phosphorylation evoked by protein kinase C and Ca2+-dependent protein kinases.

SUBMITTER: Siess W

PROVIDER: S-EPMC1138323 | biostudies-other | 1989 Feb

REPOSITORIES: biostudies-other

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The effects of phorbol ester, diacylglycerol, phospholipase C and Ca2+ ionophore on protein phosphorylation in human and sheep erythrocytes.

Project description:Treatment of human or sheep erythrocytes with PMA (phorbol myristate acetate) enhanced [32P]phosphate labelling of membrane polypeptides of approx. 100, 80 and 46 kDa. The 80 kDa and 46 kDa polypeptides coincided with bands 4.1 and 4.9 respectively on Coomassie-Blue-stained gels. Similar but smaller effects were obtained by treating human cells with 1-oleoyl-2-acetyl-rac-glycerol (OAG), exogenous bacterial phospholipase C or ionophore A23187 + Ca2+, each of which treatments would be expected to raise the concentration of membrane diacylglycerol. In contrast, sheep cells, which do not increase their content of diacylglycerol when treated with phospholipase C or A23187 + Ca2+, only showed enhanced phosphorylation with OAG. Neither human nor sheep cells showed any enhanced [32P]phosphate labelling of phosphoproteins when treated with 1-mono-oleoyl-rac-glycerol. It is concluded that diacylglycerol from a variety of sources can activate erythrocyte protein kinase C, but that the most effective diacylglycerol is that derived from endogenous polyphosphoinositides. In contrast with bacterial phospholipase C and A23187, which stimulate synthesis of phosphatidate by increasing the cell-membrane content of diacylglycerol in human erythrocytes, PMA, OAG or 1-mono-oleoyl-rac-glycerol caused no change in phospholipid metabolism.

| S-EPMC1152836 | biostudies-other

Phorbol ester and endothelin-1 alter functional expression of Na+/Ca2+ exchange, K+, and Ca2+ currents in cultured neonatal rat myocytes.

Project description:Endothelin-1 (ET-1) and activation of protein kinase C (PKC) have been implicated in alterations of myocyte function in cardiac hypertrophy and heart failure. Changes in cellular Ca2+ handling and electrophysiological properties also occur in these states and may contribute to mechanical dysfunction and arrhythmias. While ET-1 or PKC stimulation induces cellular hypertrophy in cultured neonatal rat ventricular myocytes (NRVMs), a system widely used in studies of hypertrophic signaling, there is little data about electrophysiological changes. Here we studied the effects of ET-1 (100 nM) or the PKC activator phorbol 12-myristate 13-acetate (PMA, 1 ?M) on ionic currents in NRVMs. The acute effects of PMA or ET-1 (?30 min) were small or insignificant. However, PMA or ET-1 exposure for 48-72 h increased cell capacitance by 100 or 25%, respectively, indicating cellular hypertrophy. ET-1 also slightly increased Ca2+ current density (T and L type). Na+/Ca2+ exchange current was increased by chronic pretreatment with either PMA or ET-1. In contrast, transient outward and delayed rectifier K+ currents were strongly downregulated by PMA or ET-1 pretreatment. Inward rectifier K+ current tended toward a decrease at larger negative potential, but time-independent outward K+ current was unaltered by either treatment. The enhanced inward and reduced outward currents also result in action potential prolongation after PMA or ET-1 pretreatment. We conclude that chronic PMA or ET-1 exposure in cultured NRVMs causes altered functional expression of cardiac ion currents, which mimic electrophysiological changes seen in whole animal and human hypertrophy and heart failure.

| S-EPMC3044063 | biostudies-literature

Antiallergic Phorbol Ester from the Seeds of Aquilaria malaccensis.

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| S-EPMC4813253 | biostudies-literature

Phorbol ester enhances KAI1 transcription by recruiting Tip60/Pontin complexes.

Project description:Down-regulation of the KAI1 (CD82) metastasis suppressor is common in advanced human cancer, but underlying mechanism(s) regulating KAI1 expression are only now being elucidated. Recent data provide evidence that low levels of KAI1 mRNA in LNCaP cells are caused by binding of beta-catenin/Reptin complexes to a specific motif in the proximal promoter, which prevents binding of Tip60/Pontin activator complexes to the same motif, thus inhibiting transcription. Here, we explored a pathway by which phorbol 12-myristate 13-acetate (PMA) up-regulates KAI1 transcription in LNCaP prostate cancer cells. Pretreatment with specific inhibitors showed that induction of KAI1 by PMA uses classic isoforms of protein kinase C (cPKC), is independent of Ras and Raf, and requires activation of MEK1/2 and ERK1/2, but does not involve p38MAPK. Induction of KAI1 transcription by PMA was associated with enhanced overall acetylation of histones H3 and H4, but only acetylation of H3 was blocked by a PKC inhibitor. Chromatin immunoprecipitation showed that PMA induces recruitment of Tip60/Pontin activator complexes to NFkappaB-p50 motifs in the proximal promoter, and this was blocked by a PKC inhibitor. These changes were not associated with differences in overall levels of Tip60, Pontin, beta-catenin, or Reptin protein expression but with PMA-induced nuclear translocation of Tip60.

| S-EPMC2586693 | biostudies-literature

Phorbol ester degradation in Jatropha seedcake using white rot fungi.

Project description:White rot fungi are well known for their ability to degrade a wide range of xenobiotics due to their enzymatic systems. Therefore, the present investigation was aimed at screening ten different white rot fungi for degradation of phorbol esters from Jatropha seedcake (JSC). The JSC was fermented with pure cultures of white rot fungi for 20 days under solid state condition. All the white rot fungi tested exhibited degradation of phorbol ester during fermentation of JSC without adversely influencing the nutritional properties of the seedcake. Ganoderma lucidum and Trametes zonata were found to degrade phorbol ester in JSC to undetectable levels. This study demonstrates the potential of white rot fungi for degradation of phorbol esters, a major anti-nutritional factor, in JSC preventing its utilization as cattle feed.

| S-EPMC4145619 | biostudies-literature

Resveratrol inhibits phorbol ester-induced membrane translocation of presynaptic Munc13-1.

Project description:BackgroundResveratrol (1) is a naturally occurring polyphenol that has been implicated in neuroprotection. One of resveratrol's several biological targets is Ca2+-sensitive protein kinase C alpha (PKCα). Resveratrol inhibits PKCα by binding to its activator-binding C1 domain. Munc13-1 is a C1 domain-containing Ca2+-sensitive SNARE complex protein essential for vesicle priming and neurotransmitter release.MethodsTo test if resveratrol could also bind and inhibit Munc13-1, we studied the interaction of resveratrol and its derivatives, (E)-1,3-dimethoxy-5-(4-methoxystyryl)benzene, (E)-5,5'-(ethene-1,2-diyl)bis(benzene-1,2,3-triol), (E)-1,2-bis(3,4,5-trimethoxyphenyl)ethane, and (E)-5-(4-(hexadecyloxy)-3,5-dihydroxystyryl)benzene-1,2,3-triol with Munc13-1 by studying its membrane translocation from cytosol to plasma membrane in HT22 cells and primary hippocampal neurons.ResultsResveratrol, but not the derivatives inhibited phorbol ester-induced Munc13-1 translocation from cytosol to membrane in HT22 cells and primary hippocampal neurons, as evidenced by immunoblot analysis and confocal microscopy. Resveratrol did not show any effect on Munc13-1H567K, a mutant which is not sensitive to phorbol ester. Binding studies with Munc13-1 C1 indicated that resveratrol competes with phorbol ester for the binding site. Molecular docking and dynamics studies suggested that hydroxyl groups of resveratrol interact with phorbol-ester binding residues in the binding pocket.Conclusions and significanceThis study characterizes Munc13-1 as a target of resveratrol and highlights the importance of dietary polyphenol in the management of neurodegenerative diseases.

| S-EPMC5623624 | biostudies-literature

MicroRNA Regulation by Phorbol-ester and cytokines in Human Lung Fibroblasts

Project description:Human Fetal pulmonary Fibroblasts (CCL153) were treated by PMA, IL1, TGFbeta and TNFalpha for 24 hours and compared for microRNA expression with control cells.

2009-11-01 | GSE12035 | GEO

Structural determinants of phorbol ester binding activity of the C1a and C1b domains of protein kinase C theta.

Project description:The PKC isozymes represent the most prominent family of signaling proteins mediating response to the ubiquitous second messenger diacylglycerol. Among them, PKCθ is critically involved in T-cell activation. Whereas all the other conventional and novel PKC isoforms have twin C1 domains with potent binding activity for phorbol esters, in PKCθ only the C1b domain possesses potent binding activity, with little or no activity reported for the C1a domain. In order to better understand the structural basis accounting for the very weak ligand binding of the PKCθ C1a domain, we assessed the effect on ligand binding of twelve amino acid residues which differed between the C1a and C1b domains of PKCθ. Mutation of Pro9 of the C1a domain of PKCθ to the corresponding Lys9 found in C1b restored in vitro binding activity for [3H]phorbol 12,13-dibutyrate to 3.6 nM, whereas none of the other residues had substantial effect. Interestingly, the converse mutation in the C1b domain of Lys9 to Pro9 only diminished binding affinity to 11.7 nM, compared to 254 nM in the unmutated C1a. In confocal experiments, deletion of the C1b domain from full length PKCθ diminished, whereas deletion of the C1a domain enhanced 5-fold (at 100 nM PMA) the translocation to the plasma membrane. We conclude that the Pro168 residue in the C1a domain of full length PKCθ plays a critical role in the ligand and membrane binding, while exchanging the residue (Lys240) at the same position in C1b domain of full length PKCθ only modestly reduced the membrane interaction.

| S-EPMC6957261 | biostudies-literature

Receptor- and phorbol-ester-mediated redistribution of protein kinase C in human platelets. Evidence that aggregation promotes degradation of protein kinase C.

Project description:Translocation of Ca2+/phospholipid-dependent protein kinase (PKC) activity from cytosolic to membrane fractions was assessed in washed human platelet suspensions. Phorbol myristate acetate (PMA) induced a rapid loss of PKC activity from the cytosolic compartment in stirred platelets, which was not accompanied by measurable increases in membrane-associated activity, but was paralleled by a decrease in total cellular enzyme activity (cytosol plus membrane). When platelet aggregation was prevented by not stirring, (i) cytosolic activity was decreased by PMA, (ii) significant and maintained (1-15 min with PMA) increases in membrane-bound PKC were detected, and (iii) the decline in total enzyme activity was markedly slower. In stirred platelets, total and specific inhibition of PMA-induced aggregation by a fibrinogen-derived peptide (RGDS, i.e. Arg-Gly-Asp-Ser) promoted maximal increases in membrane-associated PKC in the presence of PMA and completely prevented the loss in cellular activity. Thrombin and collagen both induced a decrease in cytosolic PKC and a loss of total activity, but a significant rise in membrane activity was seen only with collagen; ADP had no detectable effect on enzyme distribution. These results demonstrate an agonist-induced redistribution of PKC and indicate that platelet aggregation may play an important role in the proteolysis, and hence persistence, of membrane-associated PKC. This observation has implications for the potency and duration of PKC-mediated responses induced by agonists and exogenous PKC activators.

| S-EPMC1133526 | biostudies-other

Novel phorbol ester-binding motif mediates hormonal activation of Na+/H+ exchanger.

Project description:Protein kinase C (PKC) is considered crucial for hormonal Na(+)/H(+) exchanger (NHE1) activation because phorbol esters (PEs) strongly activate NHE1. However, here we report that rather than PKC, direct binding of PEs/diacylglycerol to the NHE1 lipid-interacting domain (LID) and the subsequent tighter association of LID with the plasma membrane mainly underlies NHE1 activation. We show that (i) PEs directly interact with the LID of NHE1 in vitro, (ii) like PKC, green fluorescent protein (GFP)-labeled LID translocates to the plasma membrane in response to PEs and receptor agonists, (iii) LID mutations markedly inhibit these interactions and PE/receptor agonist-induced NHE1 activation, and (iv) PKC inhibitors ineffectively block NHE1 activation, except staurosporin, which itself inhibits NHE1 via LID. Thus, we propose a PKC-independent mechanism of NHE1 regulation via a PE-binding motif previously unrecognized.

| S-EPMC2924106 | biostudies-literature

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