title: 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.
abstract: 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.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Division of Medicine UMDS, St. Thomas' Hospital, London, United Kingdom.
authors.forename: S J
authors.initials: SJ
authors.lastname: Hirst
authors.forename: B L
authors.initials: BL
authors.lastname: Webb
authors.forename: M A
authors.initials: MA
authors.lastname: Giembycz
authors.forename: P J
authors.initials: PJ
authors.lastname: Barnes
authors.forename: C H
authors.initials: CH
authors.lastname: Twort
mesh_terms: D000818:Animals; D001249:Asthma / Q000209:etiology / Q000473:pathology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002934:Cinnamates / Q000494:pharmacology; D003470:Culture Media; D004247:DNA / Q000096:biosynthesis; D004195:Disease Models, Animal; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D009130:Muscle, Smooth / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000502:physiology; D011817:Rabbits; D013440:Sulfides / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014132:Trachea / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism
chemical_list: D002934:Cinnamates; D003470:Culture Media; D007211:Indoles; D008301:Maleimides; D013440:Sulfides; C060747:ST 638; D004247:DNA; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; C064758:Ro 31-8220; C064755:Ro 31-7549