scholarly journals 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

1989 ◽  
Vol 258 (1) ◽  
pp. 57-65 ◽  
Author(s):  
W Siess ◽  
E G Lapetina
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Shape Change ◽  
Ionophore A23187 ◽  
Maximal Effect ◽  
Kinase C ◽  
Dependent Protein ◽  
High Concentrations

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.

scholarly journals Platelet aggregation induced by α2-adrenoceptor and protein kinase C activation. A novel synergism

1989 ◽  
Vol 263 (2) ◽  
pp. 377-385 ◽  
Author(s):  
W Siess ◽  
E G Lapetina
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Protein Activation ◽  
Kinase C ◽  
Gi Protein ◽  
Protein Kinase C Activation

Adrenaline or UK 14304 (a specific alpha 2-adrenoceptor agonist) and phorbol ester (phorbol 12,13-dibutyrate; PdBu) or bioactive diacylglycerols (sn-1,2-dioctanoylglycerol; DiC8) synergistically induced platelet aggregation and ATP secretion. The effect on aggregation was more pronounced than the effect on secretion, and it was observed in aspirinized, platelet-rich plasma or suspensions of washed aspirinized platelets containing ADP scavengers. No prior shape change was found. In the presence of adrenaline, DiC8 induced reversible aggregation and PdBu evoked irreversible aggregation that correlated with the different kinetics of DiC8- and PdBu-induced protein kinase C activation. Adrenaline and UK 14304 did not induce or enhance phosphorylation induced by DiC8 or PdBu of myosin light chain (20 kDa), the substrate of protein kinase C (47 kDa), or a 38 kDa protein. Immunoprecipitation studies using a Gcommon alpha antiserum or a Gi alpha antiserum showed that Gi alpha is not phosphorylated after exposure of platelets to PdBu or PdBu plus adrenaline. Adrenaline, PdBu or adrenaline plus PdBu did not cause stimulation of phospholipase C as reflected in production of [32P]phosphatidic acid. Adrenaline caused a small increase of Ca2+ in the platelet cytosol of platelets loaded with Indo-1; this effect was also observed in the absence of extracellular Ca2+. However, under conditions of maximal aggregation induced by adrenaline plus PdBu, no increase of cytosolic Ca2+ was observed. Platelet aggregation induced by PdBu plus adrenaline was not inhibited by a high intracellular concentration of the calcium chelator Quin-2. These experiments indicate that alpha 2-adrenoceptor agonists, known to interact with Gi, and protein kinase C activators synergistically induced platelet aggregation through a novel mechanism. The synergism occurs distally to Gi protein activation and protein kinase C-dependent protein phosphorylation and does not involve phospholipase C activation or Ca2+ mobilization.

Activation of Endogenous Protein Kinase C by Halothane in Synaptosomes

1996 ◽  
Vol 84 (3) ◽  
pp. 652-662 ◽  
Author(s):  
Hugh C. Hemmings ◽  
Anna I. B. Adamo
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Synaptic Membranes ◽  
Dependent Protein Kinase ◽  
Kinase C ◽  
Endogenous Protein ◽  
Dependent Protein ◽  
Protein Kinase C Activation ◽  
Synapsin 1

Background Protein kinase C is a signal transducing enzyme that is an important regulator of multiple physiologic processes and a potential molecular target for general anesthetic actions. However, the results of previous studies of the effects of general anesthetics on protein kinase C activation in vitro have been inconsistent. Methods The effects of halothane on endogenous brain protein kinase C activation were analyzed in isolated rat cerebrocortical nerve terminals (synaptosomes) and in synaptic membranes. Protein kinase C activation was monitored by the phosphorylation of MARCKS, a specific endogenous substrate. Results Halothane stimulated basal Ca2+ dependent phosphorylation of MARCKS (Mr = 83,000) in lysed synaptic membranes (2.1-fold; P< 0.01) and in intact synaptosomes (1.4-fold; P< 0.01). The EC50 for stimulation of MARCKS phosphorylation by halothene in synaptic membranes was 1.8 vol%. A selective peptide protein kinase C inhibitor, but not a protein phosphatase inhibitor (okadaic acid) or a peptide inhibitor of Ca2+/calmodulin-dependent protein kinase II, another Ca2+/-dependent signal transducing enzyme, blocked halothane-stimulated MARCKS phosphorylation in synaptic membranes. Halothane did not affect the phosphorylation of synapsin 1, a synaptic vesicle-associated protein substrate for Ca2+/calmodulin-dependent protein kinase II and AMP-dependent protein kinase, in synaptic membranes or intact synaptosomes subjected to KC1-evoked depolarization. However, halothane stimulated synapsin 1 phosphorylation evoked by ionomycin (a Ca2+ ionophore that permeabilizes membranes to Ca2+) in intact synaptosomes. Conclusions Halothane acutely stimulated basal protein kinase C activity in synaptosomes when assayed with endogenous nerve terminal substrates, lipids, and protein kinase C. This effect appeared to be selective for protein kinases C, because two other structurally similar second messenger-regulated protein kinases were not affected. Direct determinations of anesthetic effects on endogenous protein kinase C activation, translocation, and/or down-regulation are necessary to determine the ultimate effect of anesthetics on the protein kinase C signaling pathway in intact cells.

ANP stimulates phospholipase C in cultured RIMCT cells: roles of protein kinases and G protein

1991 ◽  
Vol 260 (4) ◽  
pp. F590-F595 ◽  
Author(s):  
T. Berl ◽  
J. Mansour ◽  
I. Teitelbaum
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Kinases ◽  
G Protein ◽  
Pertussis Toxin ◽  
Cyclic Nucleotide ◽  
Dependent Protein Kinase ◽  
Kinase C ◽  
Dependent Protein ◽  
Stimulation Of

We examined the possibility that, in addition to stimulation of guanylate cyclase (GC), atrial natriuretic peptide (ANP) also activates phospholipase C (PLC) in cultured rat inner medullary collecting tubule (RIMCT) cells. ANP (10(-12)M) causes marked release of inositol trisphosphate (IP3) at a concentration that does not stimulate GC. Concentrations of ANP that stimulate GC (greater than or equal to 10(-10) M) result in attenuated IP3 release. Similarly, exogenous dibutyryl guanosine 3',5'-cyclic monophosphate (10(-6) M) markedly inhibits the response to 10(-10) M ANP. Inhibition of cyclic nucleotide-dependent protein kinase by H 8, but not inhibition of protein kinase C by H 7, restores the response to 10(-8) and 10(-6) M ANP. Therefore, activation of cyclic nucleotide-dependent protein kinase inhibits ANP-stimulated PLC activity. Activation of protein kinase C by phorbol 12-myristate-13-acetate (PMA) decreases ANP-stimulated IP3 production. Pretreatment with H 7, but not H 8, prevents inhibition by PMA. To explore a potential role for G proteins, we examined the effect of guanine nucleotide analogues on ANP-stimulated IP3 production in saponin-permeabilized cells. ANP-stimulated IP3 production is enhanced by GTP gamma S and is inhibited by GDP beta S. Similarly, preincubation with pertussis toxin prevents ANP-stimulated IP3 release. We conclude that ANP stimulates PLC in RIMCT cells via a pertussis toxin-sensitive G protein. Stimulation of PLC is inhibited on activation of either cyclic nucleotide or Ca2+-phospholipid dependent protein kinases.

scholarly journals Platelet phospholipase D is activated by protein kinase C via an integrin αIIbβ3-independent mechanism

1995 ◽  
Vol 310 (2) ◽  
pp. 623-628 ◽  
Author(s):  
E A Martinson ◽  
S Scheible ◽  
A Greinacher ◽  
P Presek
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Phospholipase D ◽  
Major Protein ◽  
Ionophore A23187 ◽  
Type I ◽  
Specific Product ◽  
Kinase C ◽  
Fibrinogen Binding

Blood platelets contain phospholipase D (PLD) that is rapidly activated following platelet stimulation. It is currently unclear, however, where PLD fits into the signalling cascade that leads to aggregation and secretion. Therefore we investigated the mechanism of activation of PLD in human platelets, using the formation of the PLD-specific product phosphatidylethanol as a measure of PLD activity. PLD was activated by a number of platelet agonists that also cause the activation of protein kinase C, including thrombin, collagen, the Ca2+ ionophore A23187 and the thromboxane A2-mimetic U46619. Phorbol 12-myristate 13-acetate (PMA), a direct activator of protein kinase C, also increased PLD activity. A selective inhibitor of protein kinase C, Ro-31-8220, totally blocked the stimulation of PLD by thrombin or PMA under conditions in which it also inhibited phosphorylation of pleckstrin, the major protein kinase C substrate in platelets. Ro-31-8220 additionally inhibited A23187-stimulated PLD activity, indicating that Ca2+ activation of PLD also occurs via a protein kinase C-dependent pathway. In the presence of the fibrinogen antagonist peptide RGDS, which inhibits fibrinogen binding to integrin alpha IIb beta 3 and allows little or no aggregation to occur, thrombin- and PMA-stimulated PLD activity was still observed, indicating that PLD activation is not simply a consequence of platelet aggregation. Furthermore, these agonists were able to stimulate PLD in platelets from a Glanzmann's thrombasthenia type I patient lacking the integrin alpha IIb beta 3 complex, which indicates that activation of PLD is also independent of the recruitment of integrin alpha IIb beta 3. Taken together, our results show that PLD is activated by a pathway involving protein kinase C, and suggest that PLD might be involved in signal transduction events occurring upstream of integrin alpha IIb beta 3 activation and fibrinogen binding, which are prerequisites for full platelet aggregation.

scholarly journals Dystrophin is phosphorylated by endogenous protein kinases

1993 ◽  
Vol 293 (1) ◽  
pp. 243-247 ◽  
Author(s):  
M Luise ◽  
C Presotto ◽  
L Senter ◽  
R Betto ◽  
S Ceoldo ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinases ◽  
Cyclic Gmp ◽  
Casein Kinase ◽  
Cytoskeletal Proteins ◽  
Kinase C ◽  
Endogenous Protein ◽  
Dependent Protein

Dystrophin, the protein coded by the gene missing in Duchenne muscular dystrophy, is assumed to be a component of the membrane cytoskeleton of skeletal muscle. Like other cytoskeletal proteins in different cell types, dystrophin bound to sarcolemma membranes was found to be phosphorylated by endogenous protein kinases. The phosphorylation of dystrophin was activated by cyclic AMP, cyclic GMP, calcium and calmodulin, and was inhibited by cyclic AMP-dependent protein kinase peptide inhibitor, mastoparan and heparin. These results suggest that membrane-bound dystrophin is a substrate of endogenous cyclic AMP- and cyclic GMP-dependent protein kinases, calcium/calmodulin-dependent kinase and casein kinase II. The possibility that dystrophin could be phosphorylated by protein kinase C is suggested by the inhibition of phosphorylation by staurosporin. On the other hand dystrophin seems not to be a substrate for protein tyrosine kinases, as shown by the lack of reaction of phosphorylated dystrophin with a monoclonal antiphosphotyrosine antibody. Sequence analysis indicates that dystrophin contains seven potential phosphorylation sites for cyclic AMP- and cyclic GMP-dependent protein kinases (all localized in the central rod domain of the molecule) as well as several sites for protein kinase C and casein kinase II. Interestingly, potential sites of phosphorylation by protein kinase C and casein kinase II are located in the proximity of the actin-binding site. These results suggest, by analogy with what has been demonstrated in the case of other cytoskeletal proteins, that the phosphorylation of dystrophin by endogenous protein kinases may modulate both self assembly and interaction of dystrophin with other cytoskeletal proteins in vivo.

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