REGULATION BY HYPOXIA OF ENDOTHELIN-1-STIMULATED PHOSPHOLIPASE-D ACTIVITY IN SHEEP PULMONARY-ARTERY CULTURED SMOOTH-MUSCLE CELLS

1 The aim of the study was to characterize the effects of hypoxia on agonist-stimulated phospholipase D (PLD) and phospholipase C activity of sheep pulmonary artery cultured smooth muscle cells. 2 Endothelin-1 (ET-1), 5-hydroxytryptamine (5-HT) and the protein kinase C (PKC) activator tetradecanoylphorbol acetate (TPA), stimulated a time- and concentration-dependent increase in [H-3]-phosphatidylbutanol accumulation. This was abolished by pretreatment of the cells with the PKC inhibitor, Ro-318220, suggesting that agonist-stimulated phospholipase D activity is dependent upon the activation of PKC. 3 Hypoxia (Po-2 20 mmHg for 30 min) stimulated basal [H-3]-phosphatidylbutanol accumulation by approximately 2 fold and this activity was abolished by preincubation of the cells with 10 mu M Ro-318220. 4 In cells preincubated in low O-2 containing medium for 30 min, the subsequent agonist-stimulated accumulation of [H-3]-phosphatidylbutanol was reduced. However, the decrease in stimulation was greater for ET-1 and 5-HT than for TPA. 5 ET-1 and TPA stimulated a time-dependent increase in protein kinase C- mediated psuedosubstrate phosphorylation. Following preincubation for 30 min in low O-2 containing media, basal pseudosubstrate phosphorylation increased whilst the fold stimulation by TPA and ET-1 decreased. 6 In cells preincubated in low O-2 containing medium, ET-1-stimulated [H-3]-inositol phosphate accumulation was reduced by approximately 30- 40%. This reduction was reversed by preincubation of the cells with Ro-318220. 7 These results suggest a role for PKC in the effects of hypoxia on PLD in pulmonary artery smooth muscle cells.