Bradykinin stimulates phospholipase D in primary cultures of guinea-pig tracheal smooth muscle

Abstract

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 α-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 μM) 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 Ca 2+ 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). [AlF 4] −-stimulation of the cells also resulted in the activation of phospholipase D, indicating the involvement of a G-protein. However, this was not g i 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 G q/G 11 which couples to the inositol lipid-directed phospholipase C. Indomethacin (10 μM) was without effect upon bradykinin-stimulated phospholipase D activity, suggesting that the bradykinin effects were not mediated indirectly by cyclo-oxygenase 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.