The regulation of phosphatidylcholine (PtdCho) hydrolysis by Ca2+ and protein kinase C (PKC) was measured in [3H]palmitate-labelled cultured guinea-pig airway smooth-muscle cells as phosphatidylbutanol ([3H]PtdBut) and phosphatidate ([3H]PtdOH) formation in the presence of butanol. The former is a direct measure of phospholipase D (PLD) activity, whereas the latter, in airway smooth muscle, is indicative of net PtdCho-specific phospholipase C (PLC)-like/diacylglycerol (DG) kinase activity. Bradykinin-stimulated responses exhibited a requirement for extracellular Ca2+ influx, since they were inhibited in the presence of EGTA. This influx was independent of voltage-operated channels, since the L-type channel blocker nifedipine (up to 10 microM) was without effect on bradykinin-stimulated responses. In support of this, membrane depolarization with KCl (30 mM) failed to elicit either response. However, bradykinin-stimulated formation of both [3H]PtdBut and [3H]PtdOH was partially inhibited by 100 microM SKF96365. Ionomycin, a Ca2+ ionophore, induced PtdCho hydrolysis to a greater extent than bradykinin, also in an extracellular-Ca(2+)-dependent manner. Thapsigargin-induced emptying of intracellular Ca2+ pools elicited the formation of both [3H]PtdBut and [3H]PtdOH and displayed a requirement for extracellular Ca2+. Bradykinin-stimulated PtdCho-specific PLC-like/DG kinase pathway and PLD responses were unaffected by thapsigargin pretreatment, thereby questioning the role of Ins(1,4,5)P3/Ins(1,3,4,5)P4-dependent Ca2+ stores in the receptor stimulation of these activities in airway smooth-muscle cells. In this regard, we have previously demonstrated that the bradykinin-stimulated PtdCho-specific PLD and PLC-like activities can occur under conditions of apparent complete blockade of bradykinin-stimulated Ins(1,4,5)P3 formation by receptor antagonist in guinea-pig airway smooth muscle. The PKC inhibitor, Ro31-8220, selectively blocked both bradykinin- and ionomycin-stimulated PLD activity in a concentration-dependent manner (IC50 approx. 1 microM), but was without effect on bradykinin-stimulated PtdCho-PLC-like/DG kinase-derived PtdOH formation. In contrast, an inhibitor of PtdCho-PLC, D609, selectively blocked the formation of [3H]PtdOH in the presence of butanol (PtdCho-PLC-like/DG kinase activity), but not [3H]PtdBut formation. In conclusion, PtdCho hydrolysis appears to occur via two distinguishable routes which both require extracellular Ca2+, whereas only the PLD route is regulated by PKC.
Involvement of protein kinase C in the relaxation of acetylcholine-shortened isolated intact smooth muscle cells from the guinea-pig stomach