scholarly journals Differential down-regulation of protein kinase C selectively affects IgE-dependent exocytosis and inositol trisphosphate formation

1990 ◽  
Vol 270 (3) ◽  
pp. 679-684 ◽  
Author(s):  
G Gat-Yablonski ◽  
R Sagi-Eisenberg
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Term Treatment ◽  
Short Term Treatment ◽  
Kinase C ◽  
Serotonin Secretion ◽  
Long Term Treatment ◽  
Ic50 Values

Short-term treatment of rat basophilic leukaemia (RBL-2H3) cells with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) activates protein kinase C (PKC) and results in the inhibition of the IgE-dependent formation of inositol phosphates, but in the potentiation of serotonin secretion. Long-term treatment with TPA, which depletes the cells of their endogenous PKC, eliminates both Ca2(+)-ionophore- and TPA- as well as IgE-dependent secretion, but it potentiates by 1.7-fold IgE-induced inositol phosphate formation. Taken together, these observations strongly suggest that the dual actions of TPA on IgE-dependent responses are both mediated by PKC. The opposing effects of TPA are differentially down-regulated. Following TPA treatment, the rate by which the cells lose their ability to undergo exocytosis is faster than the rate at which inhibition of inositol phosphates formation is relieved and their production potentiated. In addition, both processes show different sensitivities to inhibitors of PKC action. Whereas IgE-dependent secretion is completely blocked by the PKC inhibitors K252a, H-7 and sphingosine [concns. causing 50% inhibition (IC50 values) = 25 ng/ml 80 microns and 30 microns respectively], these inhibitors do not relieve inhibition of inositol phosphate formation by TPA, nor do they potentiate this response. These results may imply that the bidirectional control exerted by PKC on IgE-dependent responses is mediated by its different isoenzymes.

scholarly journals Effect of secretagogues on chromogranin A synthesis in bovine cultured chromaffin cells. Possible regulation by protein kinase C

1989 ◽  
Vol 260 (3) ◽  
pp. 915-922 ◽  
Author(s):  
J P Simon ◽  
M F Bader ◽  
D Aunis
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Chromogranin A ◽  
Chromaffin Cells ◽  
Cell Types ◽  
Term Treatment ◽  
Synthesis Rate ◽  
Kinase C ◽  
Long Term Treatment ◽  
Bovine Chromaffin Cells

Chromogranin A is a major component of storage granules in many different secretory cell types. After [35S]methionine labelling of proteins from cultured bovine chromaffin cells, chromogranin A was immunoprecipitated with specific antibodies, and the radioactivity incorporated into chromogranin A was determined and used as an index of its synthesis rate. Depolarization of cells with nicotine or high K+ evoked a Ca2+-dependent increase in chromogranin A synthesis, whereas muscarine, which does not evoke significant Ca2+ influx from bovine chromaffin cells, had no effect on chromogranin A synthesis. Forskolin, an activator of adenylate cyclase, affected neither the basal nor the nicotine-stimulated rate of chromogranin A synthesis. In contrast, 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, significantly enhanced the incorporation of radioactivity into chromogranin A. Sphingosine, an inhibitor of protein kinase C, abolished both nicotine-stimulated and TPA-induced chromogranin A synthesis. In addition, long-term treatment of chromaffin cells with TPA decreased protein kinase C activity and inhibited the nicotine-stimulated chromogranin A synthesis. These results suggest that protein kinase C may play an important role in the control of chromogranin A synthesis.

Differential effects of phorbol esters on PGE2 and bradykinin-induced elevation of [Cai2+] in MDCK cells

1989 ◽  
Vol 256 (6) ◽  
pp. F1135-F1143 ◽  
Author(s):  
A. Aboolian ◽  
M. Vander Molen ◽  
E. P. Nord
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Mdck Cells ◽  
Exchange Chromatography ◽  
Adenylate Cyclase System ◽  
Transduction Pathway ◽  
Kinase C

Prostaglandin (PG) activation of the phosphoinositol transduction pathway in MDCK cells and modulation of this process by phorbol esters was studied by monitoring changes in cytosolic free Ca2+ concentration, [Cai2+], with the Ca2+-sensitive fluorescent probe, fura-2 and measurement of stimulation of inositol phosphates by anion-exchange chromatography. Cells challenged with PGE1 or PGE2 responded with a prompt and transient increase in [Cai2+] that was independent of extracellular Ca2+. The K0.5 for PGE2 for the process was 6.1 X 10(-7) M. PGE1 and PGE2 appeared to be recognized by a common receptor. PGF2 alpha was without effect. 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) but not verapamil, a Ca2+ channel inhibitor, blocked the PGE2-evoked Ca2+ transient. Under identical conditions PGE2 increased inositol phosphate accumulation by 54 +/- 8% (inositol-1-monophosphate), 23 +/- 6% (inositol-1,4-bisphosphate), and 49 +/- 3% (total inositol trisphosphate), above control values. Brief (30-60 s) exposure of cells to phorbol-12,13-myristate (PMA) or phorbol-12,13-dibutyrate (PDB) completely blocked the PGE2-induced Ca2+ transient. The K0.5 for the process for PMA and PDB was 0.3 +/- 0.1 and 4.5 +/- 2.2 nM, respectively. Neither 4 alpha-nor 4 beta-phorbol, which lack the ability to activate protein kinase C, were effective in this regard. In contrast to complete blockade by 10(-8) PMA of the PGE2 (10(-5) M)-elicited Ca2+ transient, this concentration of PMA inhibited the Ca2+ transient evoked by 10(-9) M bradykinin (BK) by 50%. In fact 10(-4) M PMA only partially blocked the BK-elicited Ca2+ transient. In summary, in MDCK cells, the PG receptor is coupled both to the adenylate cyclase system and inositol phospholipid transduction pathway. The PG receptor appears to be regulated by protein kinase C. In addition to protein kinase C other factors regulate the BK receptor.

scholarly journals Endothelial inositol phosphate generation and prostacyclin production in response to G-protein activation by AlF4−

1989 ◽  
Vol 264 (3) ◽  
pp. 703-711 ◽  
Author(s):  
M K Magnússon ◽  
H Halldórsson ◽  
M Kjeld ◽  
G Thorgeirsson
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
G Protein ◽  
Pertussis Toxin ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Ionophore A23187 ◽  
Kinase C

In order to elucidate the role of guanine-nucleotide-binding proteins (G-proteins) in endothelial prostacyclin (PGI2) production, human umbilical vein endothelial cells, prelabelled with either [3H]inositol or [3H]arachidonic acid, were stimulated with the non-specific G-protein activator aluminium fluoride (AlF4-). AlF4- caused a dose- and time-dependent generation of inositol phosphates, release of arachidonic acid and production of PGI2. The curves for the three events were similar. When the cells were stimulated in low extracellular calcium (60 nM), they released [3H]arachidonic acid and produced PGI2, but depleting the intracellular Ca2+ stores by pretreatment with the Ca2+ ionophore A23187 totally inhibited both events, although the cells still responded when extracellular Ca2+ was added. The Ca2+ ionophore did not inhibit the generation of inositol phosphates in cells maintained at low extracellular Ca2+. Pertussis toxin pretreatment (14 h) altered neither inositol phosphate nor PGI2 production in response to AlF4-. To investigate the functional role of the diacylglycerol/protein kinase C arm of the phosphoinositide system, the cells were pretreated with the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) or the protein kinase C inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H7). TPA inhibited the AlF4(-)-induced inositol phosphate generation but stimulated both the release of arachidonic acid and the production of PGI2. H7 had opposite effects both on inositol phosphate generation and on PGI2 production. These results suggest that AlF4(-)-induced PGI2 production is mediated by a pertussis-toxin-insensitive G-protein which activates the phosphoinositide second messenger system. This production of PGI2 can be modulated by protein kinase C activation, both at the level of inositol phosphate generation and at the level of arachidonic acid release.

scholarly journals Different roles of protein kinase C-β and -δ in arachidonic acid cascade, superoxide formation and phosphoinositide hydrolysis

1993 ◽  
Vol 292 (1) ◽  
pp. 203-207 ◽  
Author(s):  
J Duyster ◽  
H Schwende ◽  
E Fitzke ◽  
H Hidaka ◽  
P Dieter
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Inositol Phosphates ◽  
Phosphoinositide Hydrolysis ◽  
Arachidonic Acid Cascade ◽  
Pkc Delta ◽  
Kinase C ◽  
Superoxide Formation ◽  
Long Term Treatment

In contrast with protein kinase C (PKC)-beta, PKC-delta is exclusively detectable in the membrane fraction of liver macrophages. After long-term treatment with phorbol 12-myristate 13-acetate (PMA) PKC-beta is depleted faster (within 3 h) than PKC-delta (> 7h). Simultaneously, pretreatment with PMA for 3 h inhibits the PMA- and zymosan-induced generation of superoxide and the PMA-induced formation of prostaglandin (PG) E2, whereas a preincubation of more than 7 h is required to affect the zymosan-induced release of PGE2 and inositol phosphates. These results support an involvement of PKC-beta in the PMA-induced activation of the arachidonic acid cascade and in superoxide formation and imply an involvement of PKC-delta in zymosan-induced phosphoinositide hydrolysis and PGE2 formation. Two phorbol ester derivates, sapintoxin A (SAPA) and 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA), which have been previously reported to activate preferentially PLC-beta but not PKC-delta in vitro [Ryves, Evans, Olivier, Parker and Evans (1992) FEBS Lett. 288, 5-9], induce the formation of PGE2 and superoxide, down-regulate PKC-delta and potentiate inositol phosphate formation in parallel SAPA, but not DOPPA, down-regulates PKC-beta and inhibits the PMA-induced formation of eicosanoids and superoxide.

scholarly journals Scrape-loaded p21ras down-regulates agonist-stimulated inositol phosphate production by a mechanism involving protein kinase C

1989 ◽  
Vol 260 (1) ◽  
pp. 157-161 ◽  
Author(s):  
B D Price ◽  
J D H Morris ◽  
C J Marshall ◽  
A Hall
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Inositol Phosphate ◽  
Platelet Derived Growth Factor ◽  
3T3 Cells ◽  
Ras Protein ◽  
Kinase C ◽  
Inositol Phosphate Production ◽  
Ligand Stimulation ◽  
Scrape Loading

The effect of scrape-loaded [Val-12]p21ras on agonist-stimulated phosphatidylinositol 4,5-bisphosphate (PIP2) turnover in Swiss-3T3 cells was studied. Previously [Morris, Price, Lloyd, Marshall & Hall (1989) Oncogene 4, 27-31] we demonstrated that [Val-12]p21ras activates protein kinase C within 10 min of scrape loading. Here, we show that [Val-12]p21ras inhibits bombesin and platelet-derived growth factor-stimulated PIP2 breakdown 1.5-4 h after scrape loading. This effect persisted for at least 18 h and could be mimicked in control cells by activation of protein kinase C with 12-O-tetradecanoyl 13-acetate (TPA) 15 min prior to ligand stimulation. When protein kinase C was down-regulated by chronic TPA treatment, [Val-12]p21ras was no longer able to inhibit agonist-stimulated inositol phosphate production. These results indicate that changes in inositol phosphate levels caused by ras protein are probably due to activation of protein kinase C and not to an interaction of ras with phospholipase C.

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.

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