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.

scholarly journals Requirement for diacylglycerol and protein kinase C in HeLa cell-substratum adhesion and their feedback amplification of arachidonic acid production for optimum cell spreading.

1993 ◽  
Vol 4 (3) ◽  
pp. 271-281 ◽  
Author(s):  
J S Chun ◽  
B S Jacobson
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Hela Cell ◽  
Cell Attachment ◽  
Cell Spreading ◽  
Subsequent Formation ◽  
Sequence Of Events ◽  
Kinase C ◽  
Pkc Activation

Release of arachidonic acid (AA) and subsequent formation of a lipoxygenase (LOX) metabolite(s) is an obligatory signal to induce spreading of HeLa cells on a gelatin substratum (Chun and Jacobson, 1992). This study characterizes signaling pathways that follow the LOX metabolite(s) formation. Levels of diacylglycerol (DG) increase upon attachment and before cell spreading on a gelatin substratum. DG production and cell spreading are insignificant when phospholipase A2 (PLA2) or LOX is blocked. In contrast, when cells in suspension where PLA2 activity is not stimulated are treated with exogenous AA, DG production is turned on, and inhibition of LOX turns it off. This indicates that the formation of a LOX metabolite(s) from AA released during cell attachment induces the production of DG. Consistent with the DG production is the activation of protein kinase C (PKC) which, as with AA and DG, occurs upon attachment and before cell spreading. Inhibition of AA release and subsequent DG production blocks both PKC activation and cell spreading. Cell spreading is also blocked by the inhibition of PKC with calphostin C or sphingosine. The inhibition of cell spreading induced by blocking AA release is reversed by the direct activation of PKC with phorbol ester. However, the inhibition of cell spreading induced by PKC inhibition is not reversed by exogenously applied AA. In addition, inhibition of PKC does not block AA release and DG production. The data indicate that there is a sequence of events triggered by HeLa cell attachment to a gelatin substratum that leads to the initiation of cell spreading: AA release, a LOX metabolite(s) formation, DG production, and PKC activation. The data also provide evidence indicating that HeLa cell spreading is a cyclic feedback amplification process centered on the production of AA, which is the first messenger produced in the sequence of messengers initiating cell spreading. Both DG and PKC activity that are increased during HeLa cell attachment to a gelatin substratum appear to be involved. DG not only activates PKC, which is essential for cell spreading, but is also hydrolyzed to AA. PKC, which is initially activated as consequence of AA production, also increases more AA production by activating PLA2.

Phospholipase D: enzymology, mechanisms of regulation, and function

1997 ◽  
Vol 77 (2) ◽  
pp. 303-320 ◽  
Author(s):  
J. H. Exton
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphatidic Acid ◽  
G Protein ◽  
Phospholipase D ◽  
Tyrosine Kinases ◽  
Rho Proteins ◽  
Kinase C

Phospholipase D exists in various forms that differ in their regulation but predominantly hydrolyze phosphatidylcholine. The Ca(2+)-dependent isozymes of protein kinase C regulate phospholipase D in vitro and play a major role in its control by growth factors and G protein-linked agonists in vivo. Recent studies have demonstrated that small G proteins of the ADP-ribosylation factor (ARF) and Rho families activate the enzyme in vitro, and evidence is accumulating that they also are involved in its control in vivo. Both types of G protein play important roles in cellular function, and the possible mechanisms by which they are activated by agonists are discussed. There is also emerging evidence of the control of phospholipase D and Rho proteins by soluble tyrosine kinases and novel serine/threonine kinases. The possible role of these kinases in agonist regulation of phospholipase D is discussed. The function of phospholipase D in cells is still poorly defined. Postulated roles of phosphatidic acid produced by phospholipase D action include the activation of Ca(2+)-independent isoforms of protein kinase C, the regulation of growth and the cytoskeleton in fibroblasts, and control of the respiratory burst in neutrophils. Another important function of phosphatidic acid is to act as a substrate for a specific phospholipase A2 to generate lysophosphatidic acid, which is becoming increasingly recognized as a major intercellular messenger. Finally, it is possible that the phospholipid changes induced in various cellular membranes by phospholipase D may per se play an important role in vesicle trafficking and other membrane-associated events.

scholarly journals Protein kinase C activity is not involved in N-formylmethionyl-leucyl-phenylalanine-induced phospholipase D activation in human neutrophils, but is essential for concomitant NADPH oxidase activation: studies with a staurosporine analogue with improved selectivity for protein kinase C

1993 ◽  
Vol 292 (3) ◽  
pp. 781-785 ◽  
Author(s):  
G C Kessels ◽  
K H Krause ◽  
A J Verhoeven
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Nadph Oxidase ◽  
Phospholipase D ◽  
Respiratory Burst ◽  
Feedback Inhibition ◽  
Human Neutrophils ◽  
Kinase C ◽  
Pkc Activation

Stimulation of human neutrophils by the receptor agonist N-formylmethionyl-leucyl-phenylalanine (fMLP) results in a respiratory burst, catalysed by an NADPH oxidase. Concomitantly, phospholipase D (PLD) is activated. To investigate the role of protein kinase C (PKC) in these neutrophil responses, we have compared the effects of staurosporine and a structural analogue of staurosporine (cgp41251), that reflects a higher selectivity towards PKC [Meyer, Regenass, Fabbro, Alteri, Rösel, Müller, Caravatti and Matter (1989) Int. J. Cancer 43, 851-856]. Both staurosporine and cgp41251 dose-dependently inhibited the production of superoxide induced by phorbol 12-myristate 13-acetate (PMA). Both compounds also caused inhibition of the fMLP-induced respiratory burst, but with a lower efficacy during the initiation phase of this response. This latter observation cannot be taken as evidence against PKC involvement in the activation of the respiratory burst, because pretreatment of neutrophils with ionomycin before PMA stimulation also results in a lower efficacy of inhibition. Activation of PLD by fMLP was enhanced in the presence of staurosporine, but not in the presence of cgp41251. Enhancement of PLD activation was also observed in the presence of H-89, an inhibitor of cyclic-AMP-dependent protein kinase (PKA). Both staurosporine and H-89 reversed the dibutyryl-cyclic-AMP-induced inhibition of PLD activation, whereas cgp41251 was without effect. These results indicate that the potentiating effect of staurosporine on PLD activation induced by fMLP does not reflect a feedback inhibition by PKC activation, but instead a feedback inhibition by PKC activation. Taken together, our results indicate that in human neutrophils: (i) PKC activity is not essential for fMLP-induced activation of PLD; (ii) PKC activity does play an essential role in the activation of the respiratory burst by fMLP, other than mediating or modulating PLD activation; (iii) there exists a negative-feedback mechanism on fMLP-induced PLD activation by concomitant activation of PKA.

scholarly journals Inhibition of mitogen-activated protein kinase kinase does not impair primary activation of human platelets

1996 ◽  
Vol 318 (1) ◽  
pp. 207-212 ◽  
Author(s):  
Angelika G. BÖRSCH-HAUBOLD ◽  
Ruth M. KRAMER ◽  
Steve P WATSON
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Substrate Inhibition ◽  
Human Platelets ◽  
Mitogen Activated Protein Kinase ◽  
Ionophore A23187 ◽  
Kinase C ◽  
Mitogen Activated Protein

Mitogen-activated protein kinases (MAPKs), a family of protein serine/threonine kinases regulating cell growth and differentiation, are activated by a dual-specificity kinase through phosphorylation at threonine and tyrosine. We used a recently described selective inhibitor of the p42/p44mapk-activating enzyme, PD 98059 [2-(2´-amino-3´-methoxyphenyl)-oxanaphthalen-4-one], to investigate the role of the p42/p44mapk pathway in human platelets. PD 98059 inhibited p42/p44mapk activation in thrombin-, collagen- and phorbol ester-stimulated platelets, as determined from in-gel renaturation kinase assays, with an IC50 of approx. 5 µM (thrombin stimulation). It also prevented activation of MAPK kinase, which was measured in whole-cell lysates with glutathione S-transferase/p42mapk fusion protein (GST–MAPK) as substrate. Inhibition of p42/p44mapk did not affect platelet responses to thrombin or collagen such as aggregation, 5-hydroxytryptamine release and protein kinase C activation. In addition, PD 98059 did not interfere with release of arachidonic acid, a response mediated by cytosolic phospholipase A2 (cPLA2), or with cPLA2 phosphorylation. This suggests that platelet cPLA2 is not regulated by p42/p44mapk after stimulation with physiological agonists. In contrast, phorbol ester-induced phosphorylation of cPLA2 and potentiation of arachidonic acid release stimulated by Ca2+ ionophore A23187 were inhibited by PD 98059, indicating that p42/p44mapk phosphorylates cPLA2 after activation of protein kinase C by the non-physiological tumour promoter.

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 Interleukin 1-β-induced protein kinase C-ζ activation is mimicked by exogenous phospholipase D

1997 ◽  
Vol 321 (2) ◽  
pp. 497-502 ◽  
Author(s):  
Cristina LIMATOLA ◽  
Benedetta BARABINO ◽  
Anna NISTA ◽  
Angela SANTONI
Keyword(s):  
Signal Transduction ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Biological Effects ◽  
Interleukin 1 ◽  
Signal Transduction Pathways ◽  
Kinase C ◽  
Pkc Ζ

Interleukin 1-α (IL1-α) is a pleiotropic cytokine that stimulates a number of signal transduction pathways in cells, leading to different cellular responses. In this study we investigated the signal transduction pathways activated by IL1-α in two different human cell lines: RD/TE671, a rhabdomyosarcoma, and EJ, a bladder-derived carcinoma. We showed that this cytokine induced the activation of protein kinase C-ζ (PKC-ζ) and the accumulation of a putative physiological PKC-ζ activator, phosphatidic acid [Limatola, Schaap, Moolenaar and van Blitterswijk (1994) Biochem. J. 304, 1001Ő1008]. Exogenously supplied phospholipase D, which generated cellular phosphatidic acid, was able to mimic the cytokine effect, supporting the hypothesis that this lipid second messenger might contribute to cytokine-induced PKC-ζ activation. In addition, we show that IL1-α stimulation of BOSC23 cells, transiently overexpressing PKC-ζ, induced an increase in PKC-ζ autophosphorylation. These results give the first direct evidence that IL1-α can activate this atypical PKC isoform and suggest that this enzyme might be involved in mediating some of the biological effects induced by IL1-α.

scholarly journals Synthesis of intracellular histamine in platelets is associated with activation of protein kinase C, but not with mobilization of Ca2+

1991 ◽  
Vol 273 (2) ◽  
pp. 405-408 ◽  
Author(s):  
S P Saxena ◽  
C Robertson ◽  
A B Becker ◽  
J M Gerrard
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Human Platelets ◽  
Ionophore A23187 ◽  
Dependent Manner ◽  
Kinase C ◽  
Histamine Synthesis ◽  
Ic50 Values ◽  
Induced Aggregation ◽  
Pkc Activation

In previous reports, we have provided evidence indicating that newly formed histamine is an intracellular messenger in human platelets. The involvement of protein kinase C (PKC) and intracellular calcium (Ca2+i) in the synthesis of histamine was investigated. Human platelets were stimulated by phorbol 12-myristate 13-acetate (PMA), collagen and the Ca2+ ionophore A23187, with or without the PKC inhibitor staurosporine. Aggregation, histamine synthesis and phosphorylation of pleckstrin (47 kDa; P47) and myosin light chain (20 kDa; P20) proteins were monitored. Staurosporine inhibited PMA- and collagen-induced aggregation, histamine synthesis and phosphorylation of 47 kDa and 20 kDa proteins in a dose-dependent manner. For PMA, median inhibitory concentrations (IC50 values) for staurosporine inhibition of aggregation, histamine synthesis and phosphorylation were similar, suggesting that histamine synthesis induced by this agonist may be a consequence of PKC activation. Conversely, collagen-stimulated histamine synthesis was inhibited by staurosporine at concentrations significantly higher than those required to inhibit aggregation (P less than 0.005) or pleckstrin phosphorylation (P less than 0.01), indicating the possible involvement of non-PKC mechanism(s) in the synthesis of histamine induced by this agonist. A23187 failed to induce the synthesis of intracellular histamine in platelets, whereas staurosporine blocked A23187-induced aggregation and phosphorylation of the 20 kDa protein at significantly higher concentrations than those needed to inhibit PKC. When platelets were stimulated with a combination of A23187 and PMA, the increase in platelet histamine was less than that with PMA alone. The results provide evidence that the synthesis of intracellular histamine in platelets occurs as a consequence of PKC activation and may be down-regulated under conditions where there is a substantial rise in [Ca2+]i.

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.

Angiotensin-mediated phosphatidylcholine hydrolysis and protein kinase C activation in mesangial cells

1993 ◽  
Vol 265 (4) ◽  
pp. C1100-C1108 ◽  
Author(s):  
R. L. Barnett ◽  
L. Ruffini ◽  
L. Ramsammy ◽  
R. Pasmantier ◽  
M. M. Friedlaender ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Angiotensin Ii ◽  
Protein Kinase ◽  
Phospholipase D ◽  
Mesangial Cells ◽  
Ang Ii ◽  
Kinase C ◽  
Pi Hydrolysis ◽  
Pkc Activation ◽  
Protein Kinase C Activation

Angiotensin II (ANG II) in mesangial cells (MC) promotes phosphatidylinositol (PI) hydrolysis resulting in diacylglycerol (DAG)-mediated increases in protein kinase C (PKC) activity. The paucity of MC inositol lipid prompted us to consider whether phosphatidylcholine (PC) could sustain DAG formation. ANG II released choline and increased phosphatidylethanol (PEt) via PC-phospholipase D (PC-PLD). ANG II also stimulated phosphorylcholine consequent to PC-phospholipase C (PC-PLC) activation. ANG II-mediated PC hydrolysis augmented DAG for 30 min. PC breakdown was influenced by extracellular Ca2+, because Ni2+ partially inhibited ANG II-induced PEt and obliterated agonist-mediated DAG formation. The consequence of Ca2+ modulation of PC metabolism was investigated by measuring PKC activity. Ni2+ had no effect on early (PI-associated) activation by ANG II at 90 s but obviated translocation from cytosol to the membrane at 10 min. The pathway responsible for PC-associated DAG was studied in PKC downregulated cells. Whereas downregulation prevented PLD-mediated PEt elevation, ANG II-stimulated DAG formation in myristate-labeled cells was unaltered, indicating PC-PLC activation. In summary, ANG II stimulates PC-PLD and PC-PLC in MC. PC-PLD is tightly regulated by PKC, whereas PC-PLC is stringently controlled by extracellular Ca2+. ANG II mediated PC breakdown principally via PC-PLC provides a mechanism for maintaining elevated DAG levels and PKC activation.

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