Forskolin and prostacyclin inhibit fluoride induced platelet activation and protein kinase C dependent responses

Platelet activation (cytosolic [Ca2+] increase, aggregation and ATP secretion) was induced with A1F-4. This agent presumably interacts with a G protein which appears to mediate the coupling of the receptors for Ca mobilizing hormones and phospholipase C. All the A1F-4 evoked responses were inhibited by treatment with forskolin or prostacyclin, agents known to increase cellular cAMP. Thus the G protein-phospholipase C system appears to be the site of cAMP inhibition. Unexpectedly forskolin and prostacyclin also inhibited secretion and aggregation induced by the activators of protein kinase C, diglyceride and phorbol ester, suggesting that cAMP can also inhibit directly the protein kinase C dependent responses.     

Role of protein kinase C in mediating alpha-1-adrenoceptor-induced negative inotropic response in rat ventricles

The aim of this study was to determine the effect of protein kinase C (PKC) activation on intracellular Ca(2+) transient and its relation to alpha(1)-adrenoceptor (alpha(1)-AR)-stimulated negative inotropic response in rat ventricles. The electromechanical responses to phenylephrine (PE) in rat ventricular muscles were concomitantly examined using the conventional microelectrode method. The responses of intracellular Ca(2+) transient and cell contractions to PE in the absence of certain pharmacological interventions were ascertained in fura-2-loaded myocytes. The influence of PE on L-type Ca(2+) current (I(Ca,L)) was also examined using a voltage clamp in a whole-cell configuration. PE did not alter the action potential parameters during the negative inotropic phase. The negative inotropic effect (NIE) was inhibited by prazosin, chloroethylclonidine (CEC) and staurosporine, but was insensitive to pertussis toxin. Desensitization of PKC after prolonged pretreatment of rat ventricles with PDBu also abolished the NIE of PE. Caffeine modulated the NIE, but thapsigargin did not. The evoked intracellular Ca(2+) transient and cell contraction were initially decreased by PE, while I(Ca,L) was not altered. Prazosin and staurosporine significantly inhibited the responses. Our data indicated that alpha(1)AR-mediated NIE in rat ventricular muscles was due to the decrease of intracellular Ca(2+) transients by the modulation of PKC on Ca(2+)-releasing channels signaling through a CEC-sensitive alpha(1)AR subtype.     

Adenosine triphosphate alters the selenite-induced contracture and negative inotropic effect on cardiac muscle contractions

Selenium is known to play an important role in the physiology of many different cell types and extracellular application of selenite causes cellular dysfunction in many different types of tissues. In a previous study, we have shown that in rat ventricles, sodium selenite (≥1 mM) caused an increase in the resting tension and a decrease in contractile force, in a time-dependent manner. In the present study, we have shown that sodium selenite caused a contracture state both in Langendorff perfused hearts and isolated papillary muscles. We also showed that the application of extracellular ATP (0.1 mM) markedly reduced this detrimental effect of sodium selenite on ventricular contraction in Langendorff perfused hearts and delayed it in isolated papillary muscle preparations. In contrast, isoproterenol (0.1 μM) did not seem to influence this action of sodium selenite in papillary muscle preparations. Possible reasons for this protective effect of ATP to selenite-induced contracture are also discussed.     

Cyclosporine A-induced prostacyclin release is maintained by extracellular calcium in rat aortic endothelial cells: role of protein kinase C.

Chronic treatment with the immunosuppressive drug Cyclosporine A (CsA) is associated with increased intracellular calcium in vascular smooth muscle cells, which may activate phospholipase A2. We used rat aortic endothelial cells to investigate the role of protein kinase C (PKC) in CsA-induced prostacyclin (PGI2) release. CsA (10(-9) M) produced a significant increase in PGI2 release. CsA-induced PGI2 release were inhibited 80-85% by 10(-9) M, and 99-100% by 10(-6) M pretreatment doses of any of three different PKC inhibitors, i.e. 1-(5-isoquinolinesulfonylmethyl)piperazine(H7), staurosporine or 1-(5-isoquinolinesulfonyl)piperazine. Pretreatment with (10(-9) M) of diltiazem (a voltage-sensitive L-type calcium channel blocker) completely inhibited both CsA-induced PGI2 release. Conversely, pretreatment with (10(-9) M) of thapsigargin (an intracellular calcium channel blocker) did not alter the action of CsA. These results strongly suggest that PKC, in association with an influx of extracellular calcium, mediates CsA-induced PGI2 release in rat aortic endothelial cells.     

Stress-induced protein phosphatase 2C is a negative regulator of a mitogen-activated protein kinase

Protein phosphatases of type 2C (PP2Cs) play important roles in eukaryotic signal transduction. In contrast to other eukaryotes, plants such as Arabidopsis have an unusually large group of 69 different PP2C genes. At present, little is known about the functions and substrates of plant PP2Cs. We have previously shown that MP2C, a wound-induced alfalfa PP2C, is a negative regulator of mitogen-activated protein kinase (MAPK) pathways in yeast and plants. In this report, we provide evidence that alfalfa salt stress-inducible MAPK (SIMK) and stress-activated MAPK (SAMK) are activated by wounding and that MP2C is a MAPK phosphatase that directly inactivates SIMK but not the wound-activated MAPK, SAMK. SIMK is inactivated through threonine dephosphorylation of the pTEpY motif, which is essential for MAPK activity. Mutant analysis indicated that inactivation of SIMK depends on the catalytic activity of MP2C. A comparison of MP2C with two other PP2Cs, ABI2 and AtP2CHA, revealed that although all three phosphatases have similar activities toward casein as a substrate, only MP2C is able to dephosphorylate and inactivate SIMK. In agreement with the notion that MP2C interacts directly with SIMK, the MAPK was identified as an interacting partner of MP2C in a yeast two-hybrid screen. MP2C can be immunoprecipitated with SIMK in a complex in vivo and shows direct binding to SIMK in vitro in protein interaction assays. Wound-induced MP2C expression correlates with the time window when SIMK is inactivated, corroborating the notion that MP2C is involved in resetting the SIMK signaling pathway.     

Increased Kit/SCF receptor induced mitogenicity but abolished cell motility after inhibition of protein kinase C.

The product of the c-kit proto-oncogene, denoted Kit/SCF-R, encodes a tyrosine kinase receptor for stem cell factor (SCF). Kit/SCF-R induces proliferation, differentiation or migration of cells within the hematopoietic, gametogenic and melanogenic lineages at different developmental stages. We report here that protein kinase C (PKC) mediates phosphorylation of Kit/SCF-R on serine residues in response to SCF or PMA in intact cells. The phosphorylation inhibits SCF-induced tyrosine autophosphorylation of Kit/SCF-R. In vitro studies showed that PKC phosphorylated the Kit/SCF-R directly on serine residues and inhibited autophosphorylation of Kit/SCF-R, as well as its kinase activity towards an exogenous substrate. The PKC-induced phosphorylation did not affect Kit/SCF-R ligand binding affinity. Inhibition of PKC led to increased SCF-induced tyrosine autophosphorylation, as well as increased SCF-induced mitogenicity. In contrast, PKC was necessary for SCF-induced motility responses, including actin reorganization and chemotaxis. Our data suggest that PKC is involved in a negative feedback loop which regulates the Kit/SCF-R and that the activity of PKC determines whether the effect of SCF will be preferentially mitogenic or motogenic.     

Endothelin-induced prostacyclin production in rat aortic endothelial cells is mediated by protein kinase C.

Endothelin (ET) is a vasoconstrictor peptide released from endothelial cells that is known to cause prostaglandin (PG) release. The mechanism remains unclear. To determine whether the protein kinase C (PKC) signaling pathway is stimulated by endothelin, we pretreated rat aortic endothelial cells with either PKC activator or inhibitors and measured the release of prostacyclin (PGI2) by radioimmunoassay. ET (10(-9) M) produced a 10-fold increase in PGI2 release. Pretreatment with 10(-9) M of three different PKC inhibitors: 1-(5-isoquinolinesulfonyl) piperazine (CL), staurosporine, and 1-(5-isoquinolinesulfonyl-methyl) piperazine (H7) blocked ET induced PGI2 release. ET induced prostacyclin release was also blocked by pretreatment with inhibitors of either phospholipase A2 (7,7,dimethyleicosadienoic acid or trifluoromethyl ketone analogue) (10(-9) M) or cyclooxygenase (indomethacin) (10(-9) M). We conclude that ET activates PKC which activates phospholipase A2 which liberates arachidonic acid which increases PGI2 production and release.     

A role for protein kinase C during rat egg activation

Upon sperm-egg interaction, an increase in intracellular calcium concentration ([Ca(2+)](i)) is observed. Several studies reported that cortical reaction (CR) can be triggered not only by a [Ca(2+)](i) rise but also by protein kinase C (PKC) activation. Because the CR is regarded as a Ca(2+)-dependent exocytotic process and because the calcium-dependent conventional PKCs (cPKC) alpha and beta II are considered as exocytosis mediators in various cell systems, we chose to study activation of the cPKC in the rat egg during in vivo fertilization and parthenogenetic activation. By using immunohistochemistry and confocal microscopy techniques, we demonstrated, for the first time, the activation of the cPKC alpha, beta I, and beta II during in vivo fertilization. All three isozymes examined presented translocation to the egg's plasma membrane as early as the sperm-binding stage. However, the kinetics of their translocation was not identical. Activation of cPKC alpha was obtained by the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) or by 1-oleoyl-2-acetylglycerol (OAG) but not by the calcium ionophore ionomycin. PKC alpha translocation was first detected 5-10 min after exposure to TPA and reached a maximum at 20 min, whereas in eggs activated by OAG, translocation of PKC alpha was observed almost immediately and reached a maximum within 5 min. These results suggest that, although [Ca(2+)](i) elevation on its own does not activate PKC alpha, it may accelerate OAG-induced PKC alpha activation. We also demonstrate a successful inhibition of the CR by a myristoylated PKC pseudosubstrate (myrPKCPsi), a specific PKC inhibitor. Our study suggests that exocytosis can be triggered independently either by a [Ca(2+)](i) rise or by PKC.     

Murine T-cell differentiation antigen CD8 is a direct substrate of protein kinase C

Murine T cell differentiation antigen CD8 alpha (Lyt-2) is phosphorylated in vivo after phorbol 12-myristate 13-acetate (PMA) treatment of cells. Concanavalin A,dibutyryl cAMP and calcium ionophore are unable to stimulate phosphate incorporation into CD8 alpha. Depletion of cellular protein kinase C (PKC) by prolonged PMA treatment abolished this phosphorylation, suggesting that PKC is required for this effect. Using the amino acid sequence derived from cloning CD8 alpha, peptides encompassing both possible intracellular phosphorylation sites were made and used to test the ability of various kinases to phosphorylate CD8 alpha sequences. Only the proximal serine peptide was a kinase substrate, and of PKC, cAMP-dependent kinase and the multifunctional calcium/calmodulin-dependent kinase, only PKC was able to phosphorylate this peptide. These studies provide the first definitive evidence that CD8 alpha is a direct substrate of PKC.     

Protein kinase Czeta is a negative regulator of protein kinase B activity

Protein kinase B (PKB), also known as Akt or RAC-PK, is a serine/threonine kinase that can be activated by growth factors via phosphatidylinositol 3-kinase. In this article we show that PKCzeta but not PKCalpha and PKCdelta can co-immunoprecipitate PKB from CHO cell lysates. Association of PKB with PKCzeta was also found in COS-1 cells transiently expressing PKB and PKCzeta, and moreover we found that this association is mediated by the AH domain of PKB. Stimulation of COS-1 cells with platelet-derived growth factor (PDGF) resulted in a decrease in the PKB-PKCzeta interaction. The use of kinase-inactive mutants of both kinases revealed that dissociation of the complex depends upon PKB activity. Analysis of the activities of the interacting kinases showed that PDGF-induced activation of PKCzeta was not affected by co-expression of PKB. However, both PDGF- and p110-CAAX-induced activation of PKB were significantly abolished in cells co-expressing PKCzeta. In contrast, co-expression of a kinase-dead PKCzeta mutant showed an increased induction of PKB activity upon PDGF treatment. Downstream signaling of PKB, such as the inhibition of glycogen synthase kinase-3, was also reduced by co-expression of PKCzeta. A clear inhibitory effect of PKCzeta was found on the constitutively active double PKB mutant (T308D/S473D). In summary, our results demonstrate that PKB interacts with PKCzeta in vivo and that PKCzeta acts as a negative regulator of PKB.     

Activation of glucose transport during simulated ischemia in H9c2 cardiac myoblasts is mediated by protein kinase C isoforms

Glucose transport into cells may be regulated by a variety of conditions, including ischemia. We investigated whether some enzymes frequently involved in the metabolic adaptation to ischemia are also required for glucose transport activation. Ischemia was simulated by incubating during 3 h H9c2 cardiomyoblasts in a serum- and glucose-free medium in hypoxia. Under these conditions 2-deoxy-d-[2,6-³H]-glucose uptake was increased (57% above control levels, p < 0.0001) consistently with GLUT1 and GLUT4 translocation to sarcolemma. Tyrosine kinases inhibition via tyrphostin had no effect on glucose transport up-regulation induced by simulated ischemia. On the other hand, chelerythrine, a broad range inhibitor of protein kinase C isoforms, and rottlerin, an inhibitor of protein kinase C delta, completely prevented the stimulation of the transport rate. A lower activation of hexose uptake (19%, p < 0.001) followed also treatment with Gö6976, an inhibitor of conventional protein kinases C. Finally, PD98059-mediated inhibition of the phosphorylation of ERK 1/2, a downstream mitogen-activated protein kinase (MAPK), only partially reduced the activation of glucose transport induced by simulated ischemia (31%, p < 0.01), while SB203580, an inhibitor of p38 MAPK, did not exert any effect. These results indicate that stimulation of protein kinase C delta is strongly related to the up-regulation of glucose transport induced by simulated ischemia in cultured cardiomyoblasts and that conventional protein kinases C and ERK 1/2 are partially involved in the signalling pathways mediating this process.     

The negative inotropic action of catecholamines: role of beta3-adrenoceptors

There is now evidence for the involvement of four beta-adrenoceptor populations in the regulation of cardiac function by catecholamines. Beta1- and beta2-adrenoceptor stimulation classically produces an increase in contractility. A fourth beta-adrenoceptor, as yet uncloned and designated provisionally as a beta4-adrenoceptor, also mediates a positive inotropic effect. Beta3-adrenoceptors, which had been cloned at the end of the eighties, has been extensively studied as a potential target for antiobesity and antidiabetic drugs. Its characterization in the heart has opened new fields of investigations for the understanding of the cardiac adrenergic regulation. This review describes the cardiac electrical and mechanical effects induced by Beta3-adrenoceptor stimulation in different species (including human), as well as the signaling pathway. It also analyzes the role of these receptors in the abnormal responsiveness of catecholamines in heart failure.     

Intracellular calcium changes induced by the endozepine triakontatetraneuropeptide in human polymorphonuclear leukocytes: role of protein kinase C and effect of calcium channel blockers

Background  

The endozepine triakontatetraneuropeptide (TTN) induces intracellular calcium ([Ca⁺⁺]_i) changes followed by activation in human polymorphonuclear leukocytes (PMNs). The present study was undertaken to investigate the role of protein kinase (PK) C in the modulation of the response to TTN by human PMNs, and to examine the pharmacology of TTN-induced Ca⁺⁺ entry through the plasma membrane of these cells.     

Secretion of IL-1: role of protein kinase C.

In an attempt to define the mechanism by which endotoxin induces its biologic activity, LPS was incorporated into phospholipid vesicles (liposomes) and compared with free LPS for ability to stimulate human monocytes. Activation of human monocytes by free LPS caused the translocation of protein kinase C (PKC) from the cytosol to the plasma membranes, the production of both IL-1, alpha and beta, and IL-1 secretion. Activation by LPS presented in multilamellar vesicles (MLV)-LPS caused IL-1 production but not IL-1 secretion. Moreover, MLV-LPS did not induce PKC translocation. MLV themselves did not inhibit monocyte stimulation by LPS, since LPS presented at the surface of lyophilized liposomes behaved like free LPS in cell activation. In contrast, MLV-LPS primed monocytes for subsequent LPS stimulation. When monocytes were activated by LPS in the presence of PKC inhibitors, no plasma membrane-associated PKC or IL-1 secretion was detected, whereas IL-1 production was observed. PKC inhibitors did not affect IL-1 alpha and IL-1 beta production, showing that PKC is not involved in the production of either IL-1. It can be concluded that IL-1 production and secretion are induced independently, and that IL-1 secretion involves PKC.     

Adenosine activation of a nuclear pool of protein kinase C in rat splenocytes

The stimulatory effects of adenosine analogues on a nuclear pool of protein kinase C (PKC) were examined using isolated rat splenocyte nuclei. Nuclear receptors met pharmacological criteria of A1 adenosine receptors including a potency profile in which cyclopentyladenosine (CPA), a selective A1 agonist, was more potent than 2-phenylaminoadenosine (2PAA), a selective A2 agonist. The selective A1 receptor agonist N6-1-(phenyl-2R-propyl) adenosine (R-PIA) activated PKC whereas the S diastereomer did not. The adenosine-induced PKC response could be attenuated using a monoclonal antibody to PKC, an A1 receptor antagonist, three known PKC inhibitors and pertussis toxin (PTX). The results suggest that adenosine may exert immunomodulatory effects through the activation of nuclear PKC.     

Oenocytoid cell lysis to release prophenoloxidase is induced by eicosanoid via protein kinase C

Eicosanoids mediate insect cellular immune responses, which depend largely on phenoloxidase (PO) activity. In plasma, PO is activated by the proteolytic cleavage of proPO, which is stored in oenocytoids, a specific hemocyte type, of the beet armyworm, Spodoptera exigua. Eicosanoids induce an acute cell lysis of oenocytoids, which releases proPO into the plasma. We investigated an intracellular signal pathway following a functional interaction of eicosanoid(s) to a putative membrane receptor. U-73122 (a specific inhibitor of phospholipase C) inhibited oenocytoid lysis of S. exigua significantly after bacterial infection. We concluded that oenocytoid lysis required a certain level of calcium ion because EGTA (a calcium chelator) treatment inhibited cell lysis. Two protein kinase C (PKC) inhibitors (staurosporine and calphostin C) significantly inhibited the oenocytoid lysis. Oenocytoid lysis was likely induced by Na⁺ entry and subsequent osmotic shock because juvenile hormone analog, pyriproxyfen, which activates Na⁺-K⁺ ATPase and induces subsequent cell shrinkage, antagonized the effect of eicosaniod on cell lysis. Furthermore, ouabain (a specific Na⁺ pump inhibitor) significantly inhibited oenocytoid lysis. These results suggest that eicosanoid mediates oenocytoid lysis by activating the intracellular PKC pathway.     

Hydrogen peroxide-induced c-fos expression is mediated by arachidonic acid release: role of protein kinase C.

We found previously that stimulation of c-fos and c-myc mRNA expression are early events in hydrogen peroxide-induced growth in rat aortic smooth muscle (RASM) cells. In the present study, we investigated the role of phospholipase A2 (PLA2) and protein kinase C (PKC) in mediating hydrogen peroxide-induced c-fos mRNA expression in RASM cells. Mepacrine and p-bromophenacylbromide, potent inhibitors of PLA2 activity, blocked hydrogen peroxide-induced c-fos mRNA expression. Arachidonic acid, a product of PLA2 activity, stimulated the expression of c-fos mRNA with a time course similar to that of hydrogen peroxide. PKC down-regulation attenuated both hydrogen peroxide and arachidonic acid-induced c-fos mRNA expression by 50%. Nordihydroguaiaretic acid (a lipoxygenase-cytochrome P450 monooxygenase inhibitor) significantly inhibited both hydrogen peroxide and arachidonic acid-induced c-fos mRNA expression, whereas indomethacin (a cyclooxygenase inhibitor) had no effect. Together, these findings indicate that 1) hydrogen peroxide-induced c-fos mRNA expression is mediated by PLA2-dependent arachidonic acid release, 2) both PKC-dependent and independent mechanisms are involved in hydrogen peroxide-induced expression of c-fos mRNA and 3) arachidonic acid metabolism via the lipoxygenase-cytochrome P450 monooxygenase pathway appears to be required for hydrogen peroxide-induced expression of c-fos mRNA.     

Cytoprotective effect of prostacyclin and protein synthesis

It is shown that de novo synthesis of protein is essential for the healing process of experimental gastric ulcer and that the cytoprotective effect of prostacyclin is connected with protein synthesis. In preliminary experiments the influence of prostacyclin on hepatic protein synthesis has also been shown.