scholarly journals Effects of activation of protein kinase C on the agonist-induced stimulation and inhibition of cyclic AMP formation in intact human platelets

1987 ◽  
Vol 243 (3) ◽  
pp. 667-678 ◽  
Author(s):  
K A Williams ◽  
W Murphy ◽  
R J Haslam
Keyword(s):  
Creatine Kinase ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Platelet Activating Factor ◽  
Human Platelets ◽  
Prostaglandin D2 ◽  
Guanine Nucleotide Binding Protein ◽  
Kinase C

Jakobs, Bauer & Watanabe [(1985) Eur. J. Biochem. 151, 425-430] reported that treatment of platelets with phorbol 12-myristate 13-acetate (PMA) prevented GTP- and agonist-induced inhibition of adenylate cyclase in membranes from the platelets. This was attributed to the phosphorylation of the inhibitory guanine nucleotide-binding protein (Gi) by protein kinase C. In the present study, the effects of PMA on cyclic [3H]AMP formation and protein phosphorylation were studied in intact human platelets labelled with [3H]adenine and [32P]Pi. Incubation mixtures contained indomethacin to block prostaglandin synthesis, phosphocreatine and creatine kinase to remove ADP released from the platelets, and 3-isobutyl-1-methylxanthine to inhibit cyclic AMP phosphodiesterases. Under these conditions, PMA partially inhibited the initial formation of cyclic [3H]AMP induced by prostaglandin E1 (PGE1), but later enhanced cyclic [3H]AMP accumulation by blocking the slow decrease in activation of adenylate cyclase that follows addition of PGE1. PMA had more marked and exclusively inhibitory effects on cyclic [3H]AMP formation induced by prostaglandin D2 and also inhibited the action of forskolin. Adrenaline, high thrombin concentrations and, in the absence of phosphocreatine and creatine kinase, ADP inhibited cyclic [3H]AMP formation induced by PGE1. The actions of adrenaline and thrombin were attenuated by PMA, but that of ADP was little affected, suggesting differences in the mechanisms by which these agonists inhibit adenylate cyclase. sn-1,2-Dioctanoylglycerol (diC8) had effects similar to those of PMA. The actions of increasing concentrations of PMA or diC8 on the modulation of cyclic [3H]AMP formation by PGE1 or adrenaline correlated with intracellular protein kinase C activity, as determined by 32P incorporation into the 47 kDa substrate of the enzyme. Parallel increases in phosphorylation of 20 kDa and 39-41 kDa proteins were also observed. Platelet-activating factor, [Arg8]vasopressin and low thrombin concentrations, all of which inhibit adenylate cyclase in isolated platelet membranes, did not affect cyclic [3H]AMP formation in intact platelets. However, the activation of protein kinase C by these agonists was insufficient to account for their failure to inhibit cyclic [3H]AMP formation. Moreover, high thrombin concentrations simultaneously activated protein kinase C and inhibited cyclic [3H]AMP formation. The results show that, in the intact platelet, the predominant effects of activation of protein kinase C on adenylate cyclase activity are inhibitory, suggesting actions additional to inactivation of Gi.

scholarly journals Protein kinase C and cyclic AMP regulate reversible exposure of binding sites for fibrinogen on the glycoprotein IIB-IIIA complex of human platelets

1991 ◽  
Vol 273 (1) ◽  
pp. 115-120 ◽  
Author(s):  
G van Willigen ◽  
J W N Akkerman
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Binding Sites ◽  
Platelet Activating Factor ◽  
Human Platelets ◽  
Common Mechanism ◽  
Tight Coupling ◽  
Kinase C ◽  
Fibrinogen Binding

Platelet aggregation is mediated via binding of fibrinogen to sites on the membrane glycoprotein IIB-IIIA complex which become exposed when the cells are stimulated. We report here evidence of a dynamic and reversible exposure of binding sites for fibrinogen. In the absence of fibrinogen, exposed sites (B*) gradually lose their capacity to bind fibrinogen and close (Bo). On stimulation with platelet-activating factor (PAF, 500 nM) at 22 degrees C, closing of B* is enhanced by agents that raise cyclic AMP levels (10 ng of prostaglandin I2/ml; 5 mM-theophylline), inhibit protein kinase C (PKC; 25 microM-sphingosine; 1 microM-staurosporine), or disrupt the energy supply (30 mM-2-deoxy-D-glucose + 1 mM-CN-), or by raising the temperature to 37 degrees C. Conversely, activation of PKC 1 microM-1,2-dioctanoyl-sn-glycerol; 55 nM-phorbol 12-myristate 13-acetate) and an increase in intracellular [Ca2+] (100 nM-ionomycin + extracellular Ca2+) oppose the disappearance of B*. Phosphorylation of the 47 kDa protein illustrates the tight coupling between PKC and B* under all conditions tested, except when the cyclic AMP level is raised, and B* is converted to Bo without affecting PKC activity. Although the increase in PKC activity is much smaller with ADP or even absent upon stimulation with adrenaline, the control of B* is equally sensitive to modulation of cyclic AMP and PKC activity. We conclude that PAF, ADP and adrenaline regulate exposure of fibrinogen binding sites through a common mechanism consisting of two independent pathways, one dominated by PKC and the other by an as yet unidentified cyclic AMP-sensitive step.

scholarly journals Stimulation by glucose of cyclic AMP accumulation in mouse pancreatic islets is mediated by protein kinase C

1988 ◽  
Vol 253 (1) ◽  
pp. 229-234 ◽  
Author(s):  
P Thams ◽  
K Capito ◽  
C J Hedeskov
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Pancreatic Islets ◽  
Time Course ◽  
Ionophore A23187 ◽  
Kinase C ◽  
Cyclic Amp Accumulation ◽  
Mouse Pancreatic Islets

The mechanism of glucose-stimulated cyclic AMP accumulation in mouse pancreatic islets was studied. In the presence of 3-isobutyl-1-methylxanthine, both glucose and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, enhanced cyclic AMP formation 2.5-fold during 60 min of incubation. Both TPA-stimulated and glucose-stimulated cyclic AMP accumulations were abolished by the omission of extracellular Ca2+. The Ca2+ ionophore A23187 did not affect cyclic AMP accumulation itself, but affected the time course of TPA-induced cyclic AMP accumulation, the effect of A23187 + TPA mimicking the time course for glucose-induced cyclic AMP accumulation. A 24 h exposure to TPA, which depletes islets of protein kinase C, abolished the effects of both TPA and glucose on cyclic AMP production. Both TPA-induced and glucose-induced cyclic AMP productions were inhibited by anti-glucagon antibody, and after pretreatment with this antibody glucose stimulation was dependent on addition of glucagon. Pretreatment of islets with TPA for 10 min potentiated glucagon stimulation and impaired somatostatin inhibition of adenylate cyclase activity in a particulate fraction of islets. Carbamoylcholine, which is supposed to activate protein kinase C in islets, likewise stimulated cyclic AMP accumulation in islets. These observations suggest that glucose stimulates islet adenylate cyclase by activation of protein kinase C, and thereby potentiates the effect of endogenous glucagon on adenylate cyclase.

scholarly journals Adenylate cyclase, cyclic AMP and extracellular-signal-regulated kinase-2 in airway smooth muscle: modulation by protein kinase C and growth serum

1995 ◽  
Vol 306 (3) ◽  
pp. 723-726 ◽  
Author(s):  
N Moughal ◽  
P A Stevens ◽  
D Kong ◽  
S Pyne ◽  
N J Pyne
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Airway Smooth Muscle ◽  
Phospholipase D ◽  
Extracellular Signal Regulated Kinase ◽  
Kinase C ◽  
Extracellular Signal

Bradykinin and phorbol 12-myristate 13-acetate stimulate adenylate cyclase activity in serum-depleted cultured airway smooth muscle via a protein kinase C (PKC)-dependent pathway. The probable target is the type II adenylate cyclase, which can integrate coincident signals from both PKC and Gs. Therefore, activation of Gs (by cholera-toxin pre-treatment) amplified the bradykinin-stimulated cyclic AMP signal and concurrently attenuated the partial activation of extracellular-signal-regulated kinase-2 (ERK-2) by bradykinin. We have previously demonstrated that, in order to induce full activation of ERK-2 with bradykinin, it is necessary to obliterate PKC-stimulated cyclic AMP formation. We concluded that the cyclic AMP signal limits the magnitude of ERK-2 activation [Pyne, Moughal, Stevens, Tolan and Pyne (1994) Biochem. J. 304, 611-616]. The present study indicates that the bradykinin-stimulated ERK-2 pathway is entirely cyclic AMP-sensitive, and suggests that coincident signal detection by adenylate cyclase may be an important physiological route for the modulation of early mitogenic signalling. Furthermore, the direct inhibition of adenylate cyclase activity enables bradykinin to induce DNA synthesis, indicating that the PKC-dependent activation of adenylate cyclase limits entry of cells into the cell cycle. These studies suggest that the mitogenicity of an agonist may be governed, in part, by its ability to stimulate an inhibitory cyclic AMP signal pathway in the cell. The activation of adenylate cyclase by PKC appears to be downstream of phospholipase D. However, in cells that were maintained in growth serum (i.e. were not growth-arrested), bradykinin was unable to elicit a PKC-stimulated cyclic AMP response. The lesion in the signal-response coupling was not at the level of either the receptor or phospholipase D, which remain functionally operative and suggests modification occurs at either PKC or adenylate cyclase itself. These studies are discussed with respect to the cell signal regulation of mitogenesis in airway smooth muscle.

scholarly journals Multiple non-specific effects of sphingosine on adenylate cyclase and cyclic AMP accumulation in S49 lymphoma cells preclude its use as a specific inhibitor of protein kinase C

1990 ◽  
Vol 268 (2) ◽  
pp. 507-511 ◽  
Author(s):  
J A Johnson ◽  
R B Clark
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Mammalian Cells ◽  
Specific Inhibitor ◽  
Lymphoma Cells ◽  
Specific Effects ◽  
Kinase C ◽  
Cyclic Amp Accumulation

Recent studies with phorbol esters have suggested that protein kinase C (PKC) may play a role in the regulation of adenylate cyclase in mammalian cells. Since D-sphingosine has been reported to specifically inhibit PKC in many cell types, we evaluated its effects on stimulation of cyclic AMP accumulation by adrenaline in S49 lymphoma cells. We found sphingosine to have multiple non-specific effects which could not be explained by an inhibition of PKC. These effects included: (i) inhibition by sphingosine (50 microM) of adrenaline-stimulated cyclic AMP accumulation and sphingosine permeation of the cells which rendered them leaky to ATP; (iii) sphingosine (20 microMs) augmentation of adrenaline-stimulated cyclic AMP accumulation; (iii) inhibition by sphingosine of adrenaline-stimulated adenylate cyclase in isolated membranes by up to 95%; and (iv) sphingosine (20 microM) inhibition of cellular mechanisms for the elimination of cyclic AMP. These results demonstrate the importance of evaluating the non-specific effects of sphingosine before concluding that its actions are the consequences of a specific inhibition of PKC.

Protein kinase C and cyclic AMP modulate thrombin-induced platelet-activating factor synthesis in human endothelial cells

1991 ◽  
Vol 1093 (1) ◽  
pp. 55-64 ◽  
Author(s):  
Regine Heller ◽  
Federico Bussolino ◽  
Dario Ghigo ◽  
Giovanni Garbarino ◽  
Henning Schröder ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Platelet Activating Factor ◽  
Human Endothelial Cells ◽  
Kinase C

Differential megakaryocytic desensitization to platelet agonists

1992 ◽  
Vol 263 (4) ◽  
pp. C864-C872 ◽  
Author(s):  
G. W. Dorn ◽  
M. G. Davis
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Calcium Signaling ◽  
Phospholipase C ◽  
Platelet Activating Factor ◽  
Dienoic Acid ◽  
Cytosolic Calcium ◽  
Peripheral Circulation ◽  
Human Platelets ◽  
Kinase C

Platelets are released into the peripheral circulation from the bone marrow where they arise as fragments of megakaryocyte cytoplasm. To characterize the effects of platelet agonists on megakaryocytes, we examined calcium signaling and desensitization to thrombin, the thromboxane A2 (TxA2) mimetic (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619), and platelet-activating factor (PAF) in cultured CHRF-288-11 megakaryocytic cells. Initially, we compared agonist-stimulated calcium transients in fura-2-loaded CHRF-288-11 cells and human platelets. The 50% effective concentration values for the agonists to increase free cytosolic calcium were as follows: thrombin (0.11 +/- 0.02 U/ml in CHRF, 0.19 +/- 0.03 U/ml in platelets), U46619 (147 +/- 33 nM in CHRF, 157 +/- 5 nM in platelets), and PAF [15 +/- 2 nM in CHRF, 16 +/- 2 nM in platelets (n = 4 each)]. CHRF-288-11 thrombin, TxA2, and PAF receptors were demonstrated to be coupled to phospholipase C because each of the agonists stimulated phosphatidylinositol hydrolysis in myo-[3H]inositol-loaded CHRF-288-11 cells and pharmacological inhibition of phospholipase C-blunted agonist-stimulated calcium signaling. CHRF-288-11 cells exposed to the three agonists for 1 h showed different patterns and extent of homologous and heterologous desensitization. Protein kinase C activation appeared to be necessary but not sufficient for desensitization because 1) activation of protein kinase C with phorbol 12-myristate 13-acetate inhibited the calcium responses to all three agonists, 2) inhibition of protein kinase C with staurosporine attenuated subsequent desensitization to each agonist, and 3) each agonist increased protein kinase C activity in CHRF-288-11 cell homogenates.

scholarly journals Parathyroid hormone induces protein kinase C but not adenylate cyclase in adult cardiomyocytes and regulates cyclic AMP levels via protein kinase C-dependent phosphodiesterase activity

1995 ◽  
Vol 310 (2) ◽  
pp. 439-444 ◽  
Author(s):  
K D Schlüter ◽  
M Weber ◽  
H M Piper
Keyword(s):  
Protein Kinase C ◽  
Parathyroid Hormone ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Phosphodiesterase Activity ◽  
Beta Adrenoceptor ◽  
Kinase C ◽  
Adult Cardiomyocytes ◽  
Cyclic Amp Accumulation

Adult ventricular cardiomyocytes have been identified as target cells for parathyroid hormone (PTH) but little is known about its signal transduction in these cells. In the present study the influence of PTH on cyclic AMP accumulation and the activity of protein kinase C (PKC) in cardiomyocytes was evaluated. A mid-regional synthetic fragment of PTH, PTH-(28-48), which exerts a hypertrophic effect on cardiomyocytes, increased the activity of membrane-associated PKC in a dose-dependent manner (1-100 nM). Activated membranous PKC was dependent on Ca2+ and sensitive to an inhibitor of Ca(2+)-dependent isoforms of PKC. When adenylate cyclase was stimulated by the addition of isoprenaline, a beta-adrenoceptor agonist, PTH-(28-48) antagonized cyclic AMP accumulation. This antagonistic effect of PTH-(28-48) could be mimicked by activation of PKC with a phorbol ester and inhibited by isobutylmethylxanthine, a phosphodiesterase inhibitor. An N-terminal synthetic fragment, PTH-(1-34), which includes an adenylate cyclase-activating domain, did not stimulate the accumulation of cyclic AMP in cardiomyocytes. The results demonstrate that in adult cardiomyocytes PTH (1) is able to stimulate PKC, (2) is not able to cause accumulation of cyclic AMP and (3) functionally antagonizes the effect of beta-adrenoceptor stimulation to increase cellular cyclic AMP concentrations via PKC-dependent phosphodiesterase activity.

scholarly journals Okadaic acid identifies a phosphorylation/dephosphorylation cycle controlling the inhibitory guanine-nucleotide-binding regulatory protein Gi2

1991 ◽  
Vol 274 (2) ◽  
pp. 317-321 ◽  
Author(s):  
M Bushfield ◽  
B E Lavan ◽  
M D Houslay
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Okadaic Acid ◽  
Regulatory Protein ◽  
Nucleotide Binding ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Kinase C ◽  
Guanine Nucleotide Binding

Recently, the alpha-subunit of the inhibitory guanine-nucleotide-binding protein Gi2 (alpha-Gi2) has been shown to be a substrate for phosphorylation both by protein kinase C and also by other unidentified kinase(s) which are activated as a result of elevated cyclic AMP levels in intact rat hepatocytes [Bushfield, Murphy, Lavan, Parker, Hruby, Milligan & Houslay (1990) Biochem. J. 268, 449-457]. Here we show that the incorporation of [32P]Pi into alpha-Gi2 was enhanced 3-fold by incubation of intact hepatocytes with the tumour promoter and protein phosphatase (1 and 2A) inhibitor, okadaic acid. This action was both time- and concentration-dependent and was accompanied by a loss of guanine-nucleotide-induced inhibition of adenylate cyclase. The increased labelling of alpha-Gi2 induced by okadaic acid was partially additive with that elicited by 8-bromo cyclic AMP, but not with that elicited by the protein kinase C activator phorbol 12-myristate 13-acetate. We suggest that, in the absence of hormones, the activity of alpha-Gi2 is under the control of a dynamic phosphorylation/dephosphorylation system involving protein kinase C and protein phosphatases 1 and/or 2A. This highlights the regulation of kinases and phosphatases as both providing potentially important mechanisms for causing ‘cross-talk’ between different signalling systems, in this instance controlling cellular responsiveness through regulation of alpha-Gi2 phosphorylation.

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