scholarly journals The action of the protein kinase C inhibitor, staurosporine, on human platelets. Evidence against a regulatory role for protein kinase C in the formation of inositol trisphosphate by thrombin

1988 ◽  
Vol 249 (2) ◽  
pp. 345-350 ◽  
Author(s):  
S P Watson ◽  
J McNally ◽  
L J Shipman ◽  
P P Godfrey
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphatidic Acid ◽  
Phorbol Esters ◽  
Inositol Phosphates ◽  
Protein A ◽  
Polymyxin B ◽  
Human Platelets ◽  
Phosphoinositide Metabolism ◽  
Kinase C

The ability of several putative inhibitors of protein kinase C (PKC) to block dioctanoylglycerol (DC8)-induced phosphorylation of a 47 kDa protein (a recognized substrate for PKC) in human platelets was investigated. Staurosporine (1 microM) caused complete inhibition of phosphorylation, whereas the other reagents were either inactive (polymyxin B) or gave only partial inhibition (C-1, H-7, tamoxifen). Staurosporine (1 microM) fully inhibited the phosphorylation of the 47 kDa protein in platelets challenged with thrombin, but also inhibited the phosphorylation of a 20 kDa protein which is a substrate for myosin light-chain kinase. The inhibition of both kinases by staurosporine was associated with the inhibition of thrombin-induced secretion of ATP and 5-hydroxytryptamine and a slowing of the aggregation response; staurosporine, however, had no effect on the formation of phosphatidic acid and inositol phosphates induced by thrombin. Staurosporine also reversed the inhibitory action of phorbol esters on thrombin-induced formation of phosphatidic acid. These data are consistent with a role for these two kinases in secretion and aggregation (although there must be additional control signals, since aggregation was only slowed, not inhibited), but suggest that neither kinase is involved in the regulation of phosphoinositide metabolism. This latter conclusion contradicts previous observations that the activation of PKC by phorbol esters or membrane-permeable diacylglycerols alters the apparent activity of both phospholipase C and inositol trisphosphatase. Possible explanations for this discrepancy are discussed.

scholarly journals A diacylglycerol kinase inhibitor, R59022, potentiates secretion by and aggregation of thrombin-stimulated human platelets

1987 ◽  
Vol 243 (3) ◽  
pp. 809-813 ◽  
Author(s):  
D L Nunn ◽  
S P Watson
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphatidic Acid ◽  
Kinase Inhibitor ◽  
Inositol Phosphates ◽  
Protein A ◽  
Diacylglycerol Kinase ◽  
Human Platelets ◽  
Kinase C ◽  
Kinase Phosphorylation

The diacylglycerol kinase inhibitor R59022 (10 microM) potentiates secretion and aggregation responses in human platelets challenged with sub-maximal concentrations of thrombin. Potentiation correlates closely with increased formation of diacylglycerol, increased phosphorylation of a 40 kDa protein, a known substrate for protein kinase C, and with decreased formation of phosphatidic acid, the product of diacylglycerol kinase. Phosphorylation of myosin light chains, formation of inositol phosphates and the mobilization of Ca2+ by thrombin are not affected by R59022 (10 microM). These data support a role for protein kinase C in platelet aggregation and secretion, and provide further evidence that endogenous diacylglycerols bring about the activation of this enzyme. These data also add further argument against a role for phosphatidic acid in platelet activation.

scholarly journals Ethanol inhibits thrombin-induced secretion by human platelets at a site distinct from phospholipase C or protein kinase C

1990 ◽  
Vol 269 (2) ◽  
pp. 489-497 ◽  
Author(s):  
C Benistant ◽  
R Rubin
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Phorbol Ester ◽  
Inositol Phosphates ◽  
Shape Change ◽  
Protein A ◽  
Human Platelets ◽  
Kinase C ◽  
A Site

Ethanol is known to inhibit the activation of platelets in response to several physiological agonists, but the mechanism of this action is unclear. The addition of physiologically relevant concentrations of ethanol (25-150 mM) to suspensions of washed human platelets resulted in the inhibition of thrombin-induced secretion of 5-hydroxy[14C]tryptamine. Indomethacin was included in the incubation buffer to prevent feedback amplification by arachidonic acid metabolites. Ethanol had no effect on the activation of phospholipase C by thrombin, as determined by the formation of inositol phosphates and the mobilization of intracellular Ca2+. Moreover, ethanol did not interfere with the thrombin-induced formation of diacylglycerol or phosphatidic acid. Stimulation of platelets with phorbol ester (5-50 nM) resulted in 5-hydroxy[14C]tryptamine release comparable with those with threshold doses of thrombin. However, ethanol did not inhibit phorbol-ester-induced secretion. Ethanol also did not interfere with thrombin- or phorbol-ester-induced phosphorylation of myosin light chain (20 kDa) or a 47 kDa protein, a known substrate for protein kinase C. By electron microscopy, ethanol had no effect on thrombin-induced shape change and pseudopod formation, but prevented granule centralization and fusion. The results indicate that ethanol does not inhibit platelet secretion by interfering with the activation of phosphoinositide-specific phospholipase C or protein kinase C by thrombin. Rather, the data demonstrate an inhibition of a Ca2(+)-mediated event such as granule centralization.

scholarly journals Bombesin and platelet-derived growth factor stimulate formation of inositol phosphates and Ca2+ mobilization in Swiss 3T3 cells by different mechanisms

1989 ◽  
Vol 258 (1) ◽  
pp. 177-185 ◽  
Author(s):  
D M Blakeley ◽  
A N Corps ◽  
K D Brown
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Inositol Phosphates ◽  
Platelet Derived Growth Factor ◽  
3T3 Cells ◽  
Kinase C ◽  
Swiss 3T3 ◽  
Stimulation Of

Highly purified platelet-derived growth factor (PDGF) or recombinant PDGF stimulate DNA synthesis in quiescent Swiss 3T3 cells. The dose-response curves for the natural and recombinant factors were similar, with half-maximal responses at 2-3 ng/ml and maximal responses at approx. 10 ng/ml. Over this dose range, both natural and recombinant PDGF stimulated a pronounced accumulation of [3H]inositol phosphates in cells labelled for 72 h with [3H]inositol. In addition, mitogenic concentrations of PDGF stimulated the release of 45Ca2+ from cells prelabelled with the radioisotope. However, in comparison with the response to the peptide mitogens bombesin and vasopressin, a pronounced lag was evident in both the generation of inositol phosphates and the stimulation of 45Ca2+ efflux in response to PDGF. Furthermore, although the bombesin-stimulated efflux of 45Ca2+ was independent of extracellular Ca2+, the PDGF-stimulated efflux was markedly inhibited by chelation of external Ca2+ by using EGTA. Neither the stimulation of formation of inositol phosphates nor the stimulation of 45Ca2+ efflux in response to PDGF were affected by tumour-promoting phorbol esters such as 12-O-tetradecanoylphorbol 13-acetate (TPA). In contrast, TPA inhibited phosphoinositide hydrolysis and 45Ca2+ efflux stimulated by either bombesin or vasopressin. Furthermore, whereas formation of inositol phosphates in response to both vasopressin and bombesin was increased in cells in which protein kinase C had been down-modulated by prolonged exposure to phorbol esters, the response to PDGF was decreased in these cells. These results suggest that, in Swiss 3T3 cells, PDGF receptors are coupled to phosphoinositidase activation by a mechanism that does not exhibit protein kinase C-mediated negative-feedback control and which appears to be fundamentally different from the coupling mechanism utilized by the receptors for bombesin and vasopressin.

The role of protein kinase C in LHRH-induced LH and FSH release and LHRH self-priming in rat anterior pituitary glands in vitro

1988 ◽  
Vol 116 (2) ◽  
pp. 231-239 ◽  
Author(s):  
M. S. Johnson ◽  
R. Mitchell ◽  
G. Fink
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Polymyxin B ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Kinase C ◽  
Pituitary Glands

ABSTRACT We have investigated the role of protein kinase C (PKC) in LHRH-induced LH and FSH secretion and LHRH priming. Hemipituitary glands from pro-oestrous rats were incubated with agents known to affect PKC and with or without LHRH, during which time the secretion of gonadotrophins was measured. Phorbol esters and phospholipase C, activators of PKC, released LH and FSH in a concentration-dependent manner and potentiated the LHRH-induced secretion of gonadotrophins in parallel with their ability to release these hormones alone. Inhibitors of PKC had either no effect on LH release (1-(5-isoquinolinesulphonyl)-2-methylpiperazine hydrochloride) or they augmented LHRH-induced gonadotrophin release (polymyxin B and 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate). Neither the activators nor the inhibitors of PKC, when present with LHRH, caused any change in LHRH priming, even though the activators alone produced a release of gonadotrophins that showed a temporal pattern similar to that produced by LHRH priming. The profiles of effects on LH and FSH secretion were always qualitatively similar. These results show that PKC may be involved in general regulation of gonadotrophin release but that it is not important in acute responses to LHRH nor in LHRH self-priming. J. Endocr. (1988) 116, 231–239

scholarly journals Activation of human platelets by peroxovanadate is associated with tyrosine phosphorylation of phospholipase Cγ and formation of inositol phosphates

1993 ◽  
Vol 290 (2) ◽  
pp. 471-475 ◽  
Author(s):  
R A Blake ◽  
T R Walker ◽  
S P Watson
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Inositol Phosphates ◽  
Human Platelets ◽  
Functional Responses ◽  
Phosphorylation Of Proteins ◽  
Kinase C

Vanadate ions in the presence of H2O2 (peroxovanadate) induce a marked increase in the degree of tyrosine phosphorylation of proteins in human platelets. This increase preceded the onset of platelet shape change and aggregation, and is associated with activation of phospholipase C and increased [32P]phosphorylation of proteins of 47 kDa, a substrate for protein kinase C, and 20 kDa, a substrate for both myosin light-chain kinase and protein kinase C. The non-selective inhibitor of protein kinases, staurosporine, inhibits the increase in tyrosine phosphorylation of nearly all proteins and inhibits completely all other functional responses, suggesting that these events may be linked. In support of this, peroxovanadate stimulates tyrosine phosphorylation of phospholipase C gamma 1, suggesting that this may underlie its mechanism of platelet activation. Staurosporine also inhibited activation of phospholipase C by collagen, suggesting that tyrosine phosphorylation has an important role in the early stages of collagen-induced platelet activation.

scholarly journals Protein kinase C activators selectively inhibit insulin-stimulated system A transport activity in skeletal muscle at a post-receptor level

1990 ◽  
Vol 268 (3) ◽  
pp. 633-639 ◽  
Author(s):  
A Gumà ◽  
M Camps ◽  
M Palacín ◽  
X Testar ◽  
A Zorzano
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Insulin Receptor ◽  
Insulin Action ◽  
Phorbol Esters ◽  
Polymyxin B ◽  
Transport Activity ◽  
System A ◽  
Kinase C ◽  
Protein Kinase C Activation

We have investigated the role of phorbol esters on different biological effects induced by insulin in muscle, such as activation of system A transport activity, glucose utilization and insulin receptor function. System A transport activity was measured by monitoring the uptake of the system A-specific analogue alpha-(methyl)aminoisobutyric acid (MeAIB), by intact rat extensor digitorum longus muscle. The addition of 12-O-tetradecanoylphorbol 13-acetate (TPA, 0.5 microM) for 60 or 180 min did not modify basal MeAIB uptake by muscle, suggesting that insulin signalling required to stimulate MeAIB transport does not involve protein kinase C activation. However, TPA added 30 min before insulin (100 nM) markedly inhibited insulin-stimulated MeAIB uptake. The addition of polymyxin B (0.1 mM) or H-7 (1 mM), protein kinase C inhibitors, alone or in combination with TPA leads to impairment of insulin-stimulated MeAIB uptake. This paradoxical pattern is incompatible with a unique action of Polymyxin B or H-7 on protein kinase C activity. Therefore these agents are not suitable tools with which to investigate whether a certain insulin effect is mediated by protein kinase C. TPA did not cause a generalized inhibition of insulin action. Thus both TPA and insulin increased 3-O-methylglucose uptake by muscle, and their effects were not additive. Furthermore, TPA did not modify insulin-stimulated lactate production by muscle. In keeping with this selective modification of insulin action, treatment of muscles with TPA did not modify insulin receptor binding or kinase activities. In conclusion, phorbol esters do not mimic insulin action on system A transport activity; however, they markedly inhibit insulin-stimulated amino acid transport, with no modification of insulin receptor function in rat skeletal muscle. It is suggested that protein kinase C activation causes a selective post-receptor modification on the biochemical pathway by which insulin activates system A amino acid transport in muscle.

Alpha-adrenergic regulation of phosphoinositide metabolism and protein kinase C in isolated cardiac myocytes

1991 ◽  
Vol 260 (3) ◽  
pp. C635-C642 ◽  
Author(s):  
T. Kaku ◽  
E. Lakatta ◽  
C. Filburn
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cardiac Myocytes ◽  
Inositol Phosphates ◽  
Total Activity ◽  
Phosphoinositide Metabolism ◽  
Adrenergic Stimulation ◽  
Adrenergic Regulation ◽  
Kinase C ◽  
Isolated Cardiac Myocytes

alpha 1-Adrenergic regulation of phosphoinositide metabolism and protein kinase C translocation was studied in isolated rat cardiac myocytes. Exposure of [3H]inositol-labeled myocytes to norepinephrine in the presence of propranolol caused a dose-dependent increase in [3H]inositol phosphates. Norepinephrine also increased the level of membrane-associated protein kinase C from approximately 10% of total activity to 18%, with a dose response similar to that for generation of inositol phosphates. Depolarization of myocytes with 30 mM KCl had no effect on inositol phosphates or membrane-associated protein kinase C but potentiated the effect of submaximal norepinephrine on both parameters. The potentiation of protein kinase C translocation was amplified when extracellular Ca2+ was increased to 4 mM, resulting in membrane association of one-third of the total cellular activity. These data show that activation of protein kinase C occurs during alpha 1-adrenergic stimulation of cardiac myocytes and that elevation of intracellular Ca2+ amplifies this effect at least in part through increased phosphoinositide metabolism.

scholarly journals The diacylglycerol analogue, 1,2-sn-dioctanoylglycerol, induces an increase in cytosolic free Ca2+ and cytosolic acidification of T lymphocytes through a protein kinase C-independent process

1989 ◽  
Vol 258 (3) ◽  
pp. 689-698 ◽  
Author(s):  
R Ebanks ◽  
C Roifman ◽  
A Mellors ◽  
G B Mills
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
T Lymphocytes ◽  
Cell Activation ◽  
Phorbol Esters ◽  
Inositol Phosphates ◽  
Free Calcium ◽  
Intact Cells ◽  
Kinase C ◽  
Cytosolic Acidification

In this paper, we demonstrate that low concentrations (0.5-2.5 microM) of 1,2-sn-dioctanoylglycerol (DiC8), a potent diacylglycerol used in many previous studies to probe the role of protein kinase C (PKC) in cell activation, cause cytosolic alkalinization of human, mouse and pig T lymphocytes through PKC-mediated activation of the Na+/H+ antiport. However, at higher concentrations (greater than or equal to 12.5 microM), the effect on cytosolic pH (pHi) is reversed, resulting in a marked cytosolic acidification, followed by a gradual return of pHi to baseline values. DiC8 also induces marked changes in cytosolic free calcium concentrations ([Ca2+]i), initially by releasing calcium from intracellular stores, followed by a net transmembrane influx of calcium. The DiC8-induced cytosolic acidification, the resultant return to baseline pH and the increase in [Ca2+]i are independent of activation of PKC. Unlike many other agents which increase [Ca2+]i, DiC8 does not induce phosphatidylinositol hydrolysis with the resultant production of inositol phosphates. Other compounds known to activate PKC, including the closely related diacylglycerol analogues, 1,2-sn-dihexanoylglycerol and 1,2-sn-didecanoylglycerol, phorbol esters and mezerein, did not induce changes in [Ca2+]i or cytosolic acidification in T lymphocytes. Thus the action of DiC8 on intact lymphocytes is different from that of phorbol esters and other diacylglycerols, and is specific to the length of the acyl chains. Because changes in [Ca2+]i are often associated with cell proliferation and cell differentiation, some effects of DiC8 on intact cells may be a consequence of changes in [Ca2+]i.

Phospholipase activation and secretion: evidence that PLA2, PLC, and PLD are not essential to exocytosis

1996 ◽  
Vol 270 (4) ◽  
pp. C1153-C1163 ◽  
Author(s):  
J. R. Coorssen
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Human Platelets ◽  
Pla2 Activity ◽  
Kinase C ◽  
Gamma S ◽  
Pkc Activation

Numerous studies have identified phospholipase metabolites as membrane fusogens, and phospholipase D (PLD) (J.R. Coorssen and R.J. Haslam. FEBS Lett. 316: 170-174, 1993), C (PLC), and A2 (PLA2) activities correlate with secretion. Do these enzymes have essential or modulatory roles? This study confirms that secretion does not require Ca2+ or PLC (Coorssen et al. Cell Regul. 1: 1027-1041, 1990). Arachidonic acid (AA), phosphatidic acid (PA) and analogues, exogenous metabolites of PLA2 and PLD, were tested in electropermeabilized human platelets. AA potentiated guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-induced secretion, and eicosanoids were not essential. Endogenous [3H]AA formation correlated with GTP gamma S-induced secretion, and phorbol 12-myristate 13-acetate (PMA) promoted these effects. Inhibitors were used to probe phospholipase influences on secretion. Only PLD inhibitors blocked secretion. However, PMA blocked inhibition of protein kinase C (PKC) and secretion by quercetin, suggesting that PA formed by PLD supports PKC activation and GTP gamma S-induced secretion. Thus PA analogues had no effect alone but enhanced GTP gamma S-induced PKC activity and secretion. Slower PLD activation compared with secretion also indicates a nonessential role. This is the first report of a Ca(2+)-independent PLA2 activity in human platelets, use of quercetin as a PLD inhibitor, and dissociation of PLA2, PLC, and PLD activities from secretion. No major phospholipase activities are essential to the final steps in exocytosis, but modulatory roles are indicated.

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