Interactions of bradykinin,calcium, G-protein and protein kinase in the activation of phospholipase A2 in bovine pulmonary artery endothelial cells |
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Authors: | Dennis Ricupero Linda Taylor Peter Polgar |
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Institution: | (1) Boston University School of Medicine, 02118 Boston, MA, USA |
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Abstract: | Rise in free cytosolic calcium concentrations Ca2+]i in response to bradykinin and guanosine 5 -O-thiotriphosphate (GTP S) was related to the action of phospholipase A2 (arachidonic acid release). At 900 M extracellular CaCl2, bradykinin induced a typical Ca2+ movement consisting of an initial Ca2+]i peak at approximately 400 nM followed by a sustained increase in the steady-state cytosolic Ca2+ level at approximately 290 nM. As the extracellular CaCl2 concentration was reduced to 100 M, the bradykinin induced initial spike was reduced followed by only a marginal increase in steady-state cytosolic Ca2+ levels. Treatment of endothelial cells with saponin (0.002% w/w) did not increase Ca2+]i and saponin treated cells exhibited a very similar pattern of Ca2+ mobilization in response to bradykinin. However, with saponin treatment, GTP S (100 M) increased Ca2+]i at an almost identical tracing exhibited with 50 nM bradykinin stimulation (in either the presence or absence of 0.002% saponin). No additive increase in Ca2+]i was observed in cells stimulated with both 100 M GTP S and 50 nM bradykinin or in bradykinin stimulated cells subsequently exposed to GTP S. Pertussis toxin (PTX) did not affect the bradykinin induced Ca2+ mobilization. However, as we showed previously 1], PTX inhibited bradykinin stimulated arachidonic acid release. These results indicate transduction of the bradykinin signal by G-protein for both phospholipase A2 (PLA2) activation and Ca2+ mobilization but likely by different G subunits, a PTX sensitive and an insensitive subunit. Furthermore, the bradykinin and GTP S stimulated release of arachidonic acid appears to be only partially dependent on Ca2+]i. For example, 10 M ionomycin, a calcium ionophore, did not release arachidonic acid at extracellular CaCl2 concentrations below 300 M while GTP S stimulated a greater release of arachidonic acid at 300 and 100 M CaCl2 than at 900 M CaCl2. However, at 100 M CaCl2, ionomycin increased Ca2+]i to the same level as bradykinin or GTP S stimulated cells incubated in 900 M CaCl2.In previously published experiments 1], we showed that phorbol 12-myristate 13-acetate (TPA) augments bradykinin activated arachidonic acid release in endothelial cells. In the absence of bradykinin, TPA had little effect on arachidonic acid release by endothelial cells. However, in the saponin treated cells, TPA alone (in the absence of bradykinin) caused a marked release of arachidonic acid. The bradykinin and TPA activated arachidonic acid releases were additive. The TPA activated release did not require an increase in Ca2+]i and occurred in the absence of any added extracellular CaCl2. TPA did not induce an increase in Ca2+]i in either saponin treated or untreated endothelial cells. This TPA stimulated release of arachidonic acid was totally down-regulated by an 18 h preincubation of the cells in 500 nM TPA but was not inhibited by protein kinase C inhibitor H7. |
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