Severe chronic asthma is associated with structural changes in the airway wall including airway smooth muscle (ASM) hyperplasia. We have used cultured ASM cells isolated from rabbit trachealis as a model with which to investigate possible mechanisms of accelerated ASM growth to mitogenic stimuli. To elucidate the role that protein kinase C (PKC)- and protein tyrosine kinase (PTK)-dependent pathways play in the control of ASM mitogenesis, we have investigated the effect of reportedly selective inhibitors of PKC (3-[1-[3-(amidinothio)propyl]-3-indolyl]-4-(1-methyl-3-indolyl)-1H-pyrrole-2,5-dionemethanesulfonate [Ro31-8220] and 3-[1-(aminopropyl)indolyl]-4-(1-methyl-3-indolyl)-1H-pyrrole-2,5-dione acetate [Ro31-7549]) and PTK (alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide [ST638]) on partially purified PKC, fetal calf serum (FCS)-stimulated protein phosphotyrosine content and on FCS-induced proliferation. Anion-exchange chromatography of lysed ASM cells resolved two peaks of Ca2+-activated, phospholipid-dependent PKC activity and one peak of Ca2+- and phospholipid-independent PKC activity. The selective PKC inhibitors, Ro31-8220 and Ro31-7549, abolished the main peak of PKC activity and the Ca2+- and phospholipid-independent peak that co-eluted with the main peak. The inhibition was dependent on the concentration of ATP in the reaction cocktail (IC50: 10 mu M ATP: Ro31-8220 0.026 mu M, Ro31-7549 0.073 mu M; 100 mu M ATP: Ro31-8220 0.065 mu M, Ro31-7549 0.271 mu M), consistent with these compounds inhibiting PKC at the ATP-binding site. Ro31-8220 was more potent (2- to 3-fold) than Ro31-7549. Concentrations of each inhibitor that produced maximal inhibition of the pooled kinase activity also abolished the second peak of Ca2'-dependent activity. The PTK inhibitor, ST638, had no effect on the kinase activity associated with any of the Ca2+-dependent or -independent peaks that eluted from the column. ST638, however, maximally inhibited FCS-stimulated PTK activity (IC50 25 mu M) FCS-stimulated PTK was also inhibited by Ro31-8220 (IC50 0.15 mu M), but only by 60%, revealing an Ro31-8220-insensitive component to the response. The ability of each protein kinase inhibitor to inhibit proliferation was also studied using four independent indices of ASM cell growth and division: 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) dye conversion, Coomassie blue protein determination, hemacytometer cell counts, and DNA synthesis. Ro31-8220 and Ro31-7549 produced concentration-dependent inhibition of FCS-stimulated proliferation of growth-arrested ASM cells. Ro31-8220 was again the more potent of the two PKC inhibitors (IC50 Ro31-8220: MTT 0.84 mu M, protein 1.87 mu M, cell counts 0.35 mu M, DNA synthesis 0.53 mu M; Ro31-7549: MTT 2.5 mu M, protein 2.8 mu M, cell counts 1.81 mu M), but 50- to 100-fold greater concentrations of each inhibitor were required for inhibition in intact cells compared with the isolated enzyme - again consistent with their proposed mode of action at the ATP-binding site of PKC. Prolonged exposure of ASM cells to the phorbol diester, 4 beta-phorbol 12-myristate 13-acetate (4 beta-PMA), also inhibited FCS-stimulated proliferation (IC50: MTT 7.2 nM, protein 4.9 nM, cell counts 11 nM). 4 alpha-PMA was inactive. Similar inhibition of FCS-stimulated proliferation of growth-arrested ASM cells was observed with the tyrosine kinase inhibitor, ST638 (IC50: MTT 26.4 mu M, protein 27.8 mu M, cell counts 25.2 mu M, DNA synthesis 28.7 mu M). We conclude that both PKC- and PTK-dependent pathways are important in the positive regulation of proliferation of rabbit cultured ASM cells in response to FCS.
