Bradykinin stimulates phospholipase D in primary cultures of guinea-pig tracheal smooth muscle.
Conditions were established for the primary culture of guinea-pig tracheal smooth muscle cells, the identity of which was confirmed by the presence of smooth muscle alpha-actin by western blotting. Cells were preincubated with [3H]palmitate which was incorporated, almost exclusively, into phosphatidylcholine. When these cells were stimulated by either bradykinin or phorbol 12-myristate 13-acetate (PMA), in the presence of butan-1-ol, the non-metabolizable product [3H]phosphatidylbutanol ([3H]PtdBut) accumulated by virtue of the phosphatidyltransferase activity of phospholipase D. The activation of phospholipase D by bradykinin was inhibited by 86 +/- 11% (N = 3 experiments) in the presence of the protein kinase C inhibitor, staurosporine (1 microM) and by 88 +/- 11% (N = 3 experiments) in cells that had been chronically treated with PMA to down-regulate their protein kinase C. PMA-stimulated phospholipase D was similarly affected (92 +/- 2% inhibited by staurosporine, 87 +/- 6% inhibited by protein kinase C down-regulation). Removal of extracellular Ca2+ markedly reduced the bradykinin-stimulated phospholipase D response (by 73 +/- 10%, N = 3 experiments) but had only a limited effect upon PMA-stimulated phospholipase D activity (by 23 +/- 6%, N = 3 experiments). [AIF4](-)-stimulation of the cells also resulted in the activation of phospholipase D, indicating the involvement of a G-protein. However, this was not Gi since pertussis-toxin pretreatment of the cells failed to abolish either bradykinin-stimulated inositol (1,4,5)trisphosphate formation or [3H]PtdBut accumulation. Western blotting revealed the presence of Gq/G11 which couples to the inositol lipid-directed phospholipase C. Indomethacin (10 microM) was without effect upon bradykinin-stimulated phospholipase D activity, suggesting that the bradykinin effects were not mediated indirectly by cyclooxygenase products. The role of phospholipase D activation in tracheal smooth muscle may be to, indirectly, produce diacylglycerol for the activation of protein kinase C which has been implicated in sustained contraction. However, the immediate product of phospholipase D, phosphatidate, has been proposed to have a number of second messenger roles and may itself, by an undefined mechanism, be involved in the sustained contraction of airway smooth muscle.
Pyne
S
S
Department of Physiology and Pharmacology, University of Strathclyde, Royal College, Glasgow, U.K.
Pyne
N J
NJ
D000818:Animals; D001920:Bradykinin / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D019204:GTP-Binding Proteins / Q000378:metabolism; D020404:Glycerophospholipids; D006168:Guinea Pigs; D007213:Indomethacin / Q000494:pharmacology; D009130:Muscle, Smooth / Q000187:drug effects / Q000201:enzymology; D010168:Palmitates; D037342:Pertussis Toxin; D010712:Phosphatidic Acids / Q000032:analysis; D010713:Phosphatidylcholines / Q000378:metabolism; D010739:Phospholipase D / Q000096:biosynthesis*; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014132:Trachea / Q000187:drug effects / Q000201:enzymology; D014316:Tritium; D010566:Virulence Factors, Bordetella
D020404:Glycerophospholipids; D010168:Palmitates; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D010566:Virulence Factors, Bordetella; C068564:phosphatidylbutanol; D014316:Tritium; D011451:Prostaglandin-Endoperoxide Synthases; D037342:Pertussis Toxin; D011493:Protein Kinase C; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate; D001920:Bradykinin; D002118:Calcium; D007213:Indomethacin