Synergism between phorbol ester and the Ca2+ ionophore A23187 on protein kinase C translocation, [3H]PDBu binding and adenosine A2-receptor activation in Jurkat cells

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.

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Synergistic signals in the mechanism of antigen-induced exocytosis in 2H3 cells: evidence for an unidentified signal required for histamine release.

The Journal of Cell Biology ◽

10.1083/jcb.105.3.1129 ◽

1987 ◽

Vol 105 (3) ◽

pp. 1129-1136 ◽

Cited By ~ 73

Author(s):

M A Beaven ◽

D F Guthrie ◽

J P Moore ◽

G A Smith ◽

T R Hesketh ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Histamine Release ◽

Phorbol Ester ◽

Inositol Phosphates ◽

Synergistic Action ◽

Ionophore A23187 ◽

Kinase C

The aim of this study was to determine whether the increase in cytosolic free Ca2+ concentration ([Ca2+]i) in response to antigen (aggregated ovalbumin) on IgE-primed 2H3 cells was sufficient to account for exocytosis. When the [Ca2+]i responses to antigen and the Ca2+ ionophore A23187 were compared, A23187 was much less effective at releasing histamine at equivalent [Ca2+]i increases, and little or no stimulated histamine release occurred with A23187 concentrations that matched the [Ca2+]i response to antigen concentrations that stimulated maximal histamine release. The [Ca2+]i response to antigen is not, therefore, sufficient to account for exocytosis, although extracellular Ca2+ is necessary to initiate both the [Ca2+]i response and histamine release: the antigen must generate an additional, unidentified, signal that is required for exocytosis. To determine whether this signal was the activation of protein kinase C, the effects of the phorbol ester 12-0-tetradecanoyl phorbol 13-acetate (TPA) on the responses to antigen were examined. TPA blocked the antigen-induced [Ca2+]i response and the release of inositol phosphates but had little effect on histamine release and did not stimulate exocytosis by itself. The unidentified signal from the antigen is therefore distinct from the activation of protein kinase C and is generated independently of the [Ca2+]i response or the release of inositol phosphates. Taken together with other data that imply that there is very little activation of protein kinase C by antigen when the rate of histamine release is maximal, it is concluded that the normal exocytotic response to antigen requires the synergistic action of the [Ca2+]i signal together with an unidentified signal that is not mediated by protein kinase C.

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Rapid expression of protooncogenes c-fos and c-myc in B-chronic lymphocytic leukemia cells during differentiation induced by phorbol ester and calcium ionophore

Blood ◽

10.1182/blood.v73.6.1656.1656 ◽

1989 ◽

Vol 73 (6) ◽

pp. 1656-1663 ◽

Cited By ~ 17

Author(s):

HG Drexler ◽

JW Janssen ◽

MK Brenner ◽

AV Hoffbrand ◽

CR Bartram

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Chronic Lymphocytic Leukemia ◽

Phorbol Ester ◽

Messenger Rna ◽

Calcium Ionophore ◽

Lymphocytic Leukemia ◽

Calcium Flux ◽

Ionophore A23187 ◽

Kinase C

Abstract The peripheral blood mononuclear cells from patients with B-chronic lymphocytic leukemia (B-CLL) were incubated for 0.5 h to 72 h in the presence of the phorbol ester TPA, the calcium ionophore A23187, or a combination of these reagents. Using Northern blot analysis, total cellular RNA was prepared from cells harvested at different time points and hybridized with DNA clones specific for the protooncogenes c-fos and c-myc. While untreated control cells lacked detectable amounts of messenger RNA (mRNA), increase in the level of c-fos mRNA was noted as early as 0.5 h after exposure to the inducers. Peaks of c-fos and c-myc transcript accumulation were seen at 1 h and 4 h after induction, respectively. The most effective inducer was double stimulation with TPA plus A23187. The kinetics of c-fos and c-myc mRNA accumulation in B- CLL appear to be similar to those reported for normal lymphocytes that have been either activated by physiologic external stimuli or by direct activators of protein kinase C and calcium flux (such as TPA and A23187). No direct link between oncogene expression and proliferation or differentiation parameters could be established. These results document that expression of c-fos and c-myc genes, which are among the earliest events following stimulation of the protein kinase signal transduction pathway, can be successfully induced in B-CLL cells. The data provide further evidence for the hypothesis that signal transmission downstream of protein kinase C is intact in B-CLL.

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Effect of phorbol ester and calcium ionophore on chloride secretion in canine tracheal epithelium

AJP Cell Physiology ◽

10.1152/ajpcell.1987.253.6.c828 ◽

1987 ◽

Vol 253 (6) ◽

pp. C828-C834 ◽

Cited By ~ 40

Author(s):

M. J. Welsh

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phorbol Ester ◽

Calcium Ionophore ◽

Tracheal Epithelium ◽

Ionophore A23187 ◽

Regulatory Pathway ◽

Cl Secretion ◽

Prostaglandin Production ◽

Kinase C

The control of Cl- secretion was examined by two of the terminal events in the phosphoinositide regulatory pathway: activation of protein kinase C and an increase in cell Ca2+. The phorbol ester phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C, stimulated Cl- secretion in both native and cultured monolayers of tracheal epithelium. Approximately 1.5 nM of mucosal PMA was required for half-maximal stimulation. Stimulation was not dependent on prostaglandin production nor was it accompanied by an increase in cellular levels of cAMP. Although the maximal rate of PMA-induced Cl- secretion was less than that induced by cAMP, there was a synergistic effect between PMA and forskolin, an agent that activates adenylate cyclase. The response to PMA was at least partly transient and PMA may also attenuate Cl- secretion under some circumstances. Thus the response to PMA, and presumably protein kinase C activation, may be complex. An increase in cell Ca2+ produced by addition of the Ca2+ ionophore A23187 also stimulated Cl- secretion. However, the effect was at least partly indirect. A23187 enhanced prostaglandin E2 production and the prostaglandin synthesis inhibitor, indomethacin, blocked A23187-induced secretion in native epithelia and attenuated the effect of A23187 in cultured monolayers. These results indicate the presence of a non-cAMP-dependent regulatory pathway capable of controlling Cl- secretion in tracheal epithelium. Activation of protein kinase C may stimulate secretion directly or modulate the response to cAMP. An increase in cell Ca2+ may stimulate secretion at least partly by stimulating endogenous prostaglandin production.

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Preconditioning cultured human pediatric myocytes requires adenosine and protein kinase C

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1997.272.3.h1220 ◽

1997 ◽

Vol 272 (3) ◽

pp. H1220-H1230 ◽

Cited By ~ 10

Author(s):

J. S. Ikonomidis ◽

T. Shirai ◽

R. D. Weisel ◽

B. Derylo ◽

V. Rao ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phorbol Ester ◽

Adenosine Receptor ◽

Receptor Activation ◽

Receptor Stimulation ◽

Kinase C ◽

Low Volume ◽

Adenosine Receptor Activation ◽

Phosphorylation Rate

We showed previously that 20 min of low-volume anoxia ("ischemia") and 20 min of "reperfusion" preconditions quiescent pediatric myocyte cultures against damage resulting from 90 min of subsequent prolonged ischemia and 30 min of reperfusion. The purpose of this study was to assess the roles of adenosine and protein kinase C (PKC) in this preconditioning model. Our results suggest that 1) preconditioned myocytes secrete a protective mediator(s) into the "ischemic" supernatant that is transferable to other cells, and adenosine is released into the supernatant in quantities sufficient for adenosine-receptor activation (2) preconditioning is inhibited by adenosine-receptor antagonism, and myocyte protection similar to preconditioning can be achieved with exogenously administered adenosine or adenosine-receptor stimulation; (3) brief ischemic and adenosine-induced myocyte preconditioning is mimicked by the phorbol ester 4beta-phorbol 12-myristate 13-acetate (PKC agonist) and inhibited by PKC antagonists; and (4) brief ischemic and adenosine-induced myocyte preconditioning both induce PKC translocation to myocyte membranes and increase the PKC phosphorylation rate. These data suggest that adenosine released from ischemic human pediatric myocytes mediates preconditioning through activation of PKC.

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Comparison of the effects of NGF, activators of protein kinase C, and a calcium ionophore on the expression of Thy-1 and N-CAM in PC12 cell cultures.

The Journal of Cell Biology ◽

10.1083/jcb.107.1.333 ◽

1988 ◽

Vol 107 (1) ◽

pp. 333-340 ◽

Cited By ~ 48

Author(s):

P Doherty ◽

D A Mann ◽

F S Walsh

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Pc12 Cells ◽

Cell Cultures ◽

Phorbol Ester ◽

Pc12 Cell ◽

Calcium Ionophore ◽

Cell Surface Expression ◽

Ionophore A23187 ◽

Kinase C

The addition of nerve growth factor (NGF) to PC12 cells induces an approximate doubling in the cell surface expression of the Thy-1 glycoprotein and the neural cell adhesion molecule (N-CAM) after 24 h of culture. Although both responses are measured at the same time point, their sensitivity to NGF differed with half-maximal induction of Thy-1 apparent at NGF concentrations (approximately 0.1 ng/ml NGF) that had little effect on N-CAM expression. Phorbol ester derivatives capable of activating Ca2+/phospholipid-dependent protein kinase (protein kinase C) and the calcium ionophore A23187 were found to mimic the NGF induction of Thy-1, but not N-CAM. Similar results were observed when a synthetic diacylglycerol was added to PC12 cell cultures. Increased expression of Thy-1 consequent to phorbol ester, calcium ionophore, or NGF treatment was associated with an increase in the expression of the mRNA species that encodes Thy-1. Increased expression of Thy-1 consequent to all three treatments was also reduced by treatment with the transcription inhibitor cordycepin. Treatment of PC12 cells with high concentrations of phorbol esters was found to inhibit the NGF induction of Thy-1, but not N-CAM. Whereas the above results are consistent with activation of protein kinase C underlying the NGF induction of Thy-1, the same data are not consistent with this pathway being important in the N-CAM response.

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Cellular signals regulating the release of ANF

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y91-227 ◽

1991 ◽

Vol 69 (10) ◽

pp. 1514-1524 ◽

Cited By ~ 16

Author(s):

Heikki Ruskoaho ◽

Päivi Kinnunen ◽

Pentti Mäntymaa ◽

Paavo Uusimaa ◽

Tarja Taskinen ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phorbol Ester ◽

Phorbol Esters ◽

Hormone Secretion ◽

Ionophore A23187 ◽

Atrial Myocytes ◽

Kinase C ◽

Ventricular Cells ◽

Intracellular Ca

Atrial natriuretic factor (ANF), a peptide hormone that regulates salt and water balance and blood pressure, is synthesized, stored, and secreted from mammalian myocytes. Stretching of atrial myocytes stimulates ANF secretion, but the cellular processes involved in linking mechanical distension to ANF release are unknown. We reported that phorbol esters, which mimic the action of diacylglycerol by acting directly on protein kinase C and the Ca2+ ionophore A23187, which introduces free Ca2+ into the cell, both increase basal ANF secretion in the isolated perfused rat heart. Phorbol ester also increased responsiveness to Ca2+ channel agonists, such as Bay k8644, and to agents that increase cAMP, such as forskolin and membrane-permeable cAMP analogs. In neonatal cultured rat atrial myocytes, protein kinase C activation by 12-O-tetradecanoylphorbol 13-acetate stimulated ANF secretion, whereas the release was unresponsive to changes in intracellular Ca2+. Endothelin, which stimulates phospholipase C mediated hydrolysis of phosphoinositides and activates protein kinase C, increased both basal and atrial stretch-induced ANF secretion from isolated perfused rat hearts. Similarly, phorbol ester enhanced atrial stretch-stimulated ANF secretion, while the increase in intracellular Ca2+ appeared to be negatively coupled to the stretch-induced ANF release. Finally, phorbol ester stimulated ANF release from the severely hypertrophied ventricles of hypertensive animals but not from normal rat myocardium. These results suggest that the protein kinase C activity may play an important role in the regulation of basal ANF secretion both from atria and ventricular cells, and that stretch of atrial myocytes appears to be positively modulated by phorbol esters.Key words: hormone secretion, atrial stretch, protein kinase C, phosphoinositide hydrolysis, cellular calcium.

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Rapid expression of protooncogenes c-fos and c-myc in B-chronic lymphocytic leukemia cells during differentiation induced by phorbol ester and calcium ionophore

Blood ◽

10.1182/blood.v73.6.1656.bloodjournal7361656 ◽

1989 ◽

Vol 73 (6) ◽

pp. 1656-1663

Author(s):

HG Drexler ◽

JW Janssen ◽

MK Brenner ◽

AV Hoffbrand ◽

CR Bartram

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Chronic Lymphocytic Leukemia ◽

Phorbol Ester ◽

Messenger Rna ◽

Calcium Ionophore ◽

Lymphocytic Leukemia ◽

Calcium Flux ◽

Ionophore A23187 ◽

Kinase C

The peripheral blood mononuclear cells from patients with B-chronic lymphocytic leukemia (B-CLL) were incubated for 0.5 h to 72 h in the presence of the phorbol ester TPA, the calcium ionophore A23187, or a combination of these reagents. Using Northern blot analysis, total cellular RNA was prepared from cells harvested at different time points and hybridized with DNA clones specific for the protooncogenes c-fos and c-myc. While untreated control cells lacked detectable amounts of messenger RNA (mRNA), increase in the level of c-fos mRNA was noted as early as 0.5 h after exposure to the inducers. Peaks of c-fos and c-myc transcript accumulation were seen at 1 h and 4 h after induction, respectively. The most effective inducer was double stimulation with TPA plus A23187. The kinetics of c-fos and c-myc mRNA accumulation in B- CLL appear to be similar to those reported for normal lymphocytes that have been either activated by physiologic external stimuli or by direct activators of protein kinase C and calcium flux (such as TPA and A23187). No direct link between oncogene expression and proliferation or differentiation parameters could be established. These results document that expression of c-fos and c-myc genes, which are among the earliest events following stimulation of the protein kinase signal transduction pathway, can be successfully induced in B-CLL cells. The data provide further evidence for the hypothesis that signal transmission downstream of protein kinase C is intact in B-CLL.

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