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

• Published in: American journal of respiratory cell and molecular biology Vol. 12; no. 2; pp. 149 - 161
• Main Authors: Hirst, SJ, Webb, BL, Giembycz, MA, Barnes, PJ, Twort, CH
• Format: Journal Article
• Language: English
• Published: United States Am Thoracic Soc 01.02.1995; American Thoracic Society
• Subjects: Animals; Asthma - etiology; Asthma - pathology; Cell Division - drug effects; Cell Division - physiology; Cells, Cultured; Cinnamates - pharmacology; Culture Media; Disease Models, Animal; DNA - biosynthesis; Humans; Indoles - pharmacology; Maleimides - pharmacology; Muscle, Smooth - cytology; Muscle, Smooth - drug effects; Muscle, Smooth - metabolism; Protein Kinase C - antagonists & inhibitors; Protein Kinase C - physiology; Protein-Tyrosine Kinases - antagonists & inhibitors; Protein-Tyrosine Kinases - physiology; Rabbits; Sulfides - pharmacology; Tetradecanoylphorbol Acetate - pharmacology; Trachea - cytology; Trachea - drug effects; Trachea - metabolism
• ISSN: 1044-1549; 1535-4989
• DOI: 10.1165/ajrcmb.12.2.7865214

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 Ca(2+)-activated, phospholipid-dependent PKC activity and one peak of Ca(2+)- and phospholipid-independent PKC activity. The selective PKC inhibitors, Ro31-8220 and Ro31-7549, abolished the main peak of PKC activity and the Ca(2+)- 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 microM ATP: Ro31-8220 0.026 microM, Ro31-7549 0.073 microM; 100 microM ATP: Ro31-8220 0.065 microM, Ro31-7549 0.271 microM), 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 Ca(2+)-dependent activity. The PTK inhibitor, ST638, had no effect on the kinase activity associated with any of the Ca(2+)-dependent or -independent peaks that eluted from the column. ST638, however, maximally inhibited FCS-stimulated PTK activity (IC50 25 microM). FCS-stimulated PTK was also inhibited by Ro31-8220 (IC50 0.15 microM), 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.