INHIBITION OF FETAL CALF SERUM-STIMULATED PROLIFERATION OF RABBIT CULTURED TRACHEAL SMOOTH-MUSCLE CELLS BY SELECTIVE INHIBITORS OF PROTEIN-KINASE-C AND PROTEIN-TYROSINE KINASE

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.