The effects of phorbol 12,13-diacetate on responses of guinea-pig isolated trachea to methylxanthines, isoprenaline and ryanodine.

1. Using guinea-pig isolated trachea, we have studied how phorbol 12,13-diacetate (PDA) modulates mechanical responses of the tissue to methylxanthines, isoprenaline and ryanodine. 2. Caffeine (10 microM-5 mM), theophylline (10 microM-5 mM) and isoprenaline (1 nM-1 microM), each inhibited the spontaneous tone of the trachea. Pretreatment with PDA (0.1-10 microM) converted relaxant responses to high concentrations of the methylxanthines into contractions. PDA produced no equivalent effect against isoprenaline. Pretreatment with verapamil (1 or 10 microM), nifedipine (0.1 microM) or incubation with Ca(2+)-free, EGTA (0.1 mM)-containing physiological salt solution (PSS) suppressed the contraction produced by caffeine or theophylline in PDA (5 microM)-treated tissues. 3. The ability of PDA (5 microM) to convert caffeine-induced relaxation into caffeine-induced contraction was retained in tissues pretreated with a combination of atropine (1 microM) and mepyramine (1 microM) and in tissues denuded of the airway epithelium. 4. Caffeine (10 microM-5 mM), theophylline (10 microM-5 mM) and isoprenaline (1 nM-1 microM), each relaxed trachea contracted with histamine (0.1 mM). The relaxation induced by caffeine, theophylline and isoprenaline was markedly reduced in the presence of PDA (5 microM) and the responses to high concentrations of caffeine and theophylline, but not those to isoprenaline, were reversed to contractions. Verapamil (10 microM) prevented the effects of PDA against caffeine- or theophylline-induced relaxation. 5. PDA (1 microM) enhanced the tracheal spasm produced by caffeine (10 mM) and theophylline (10 mM) in indomethacin (2.8 microM)-treated trachea maintained at 20 degrees C. This enhancement was reduced in the presence of verapamil (10 microM). 6. Tested in trachea bathed by K+-rich (40 mM), Ca2+-free PSS, CaCl2 (0.1-20 mM) caused concentration-dependent spasm. PDA (1-5 MicroM) did not significantly modify the shape or position of the log concentration-effect curve for CaCl2. In contrast, verapamil (1 and 10 MicroM) antagonized CaCl2.7. Tested in trachea bathed by indomethacin (2.8 MicroM)-containing PSS, ryanodine (1-100 MicroM) caused concentration-dependent spasm. PDA (5 MicroM) potentiated ryanodine. Verapamil (10 MicroM) inhibited ryanodine in inducing spasm and suppressed the ability of PDA to potentiate ryanodine.8. It is concluded that, in guinea-pig isolated trachea, PDA augments the spasmogenic activity of the methylxanthines and ryanodine. This effect of PDA does not result from PDA-induced suppression of spontaneous tone, from increased cellular entry of Ca2+ through L-type channels or from sensitization of the intracellular contractile machinery to activator Ca2+. The evidence suggests, instead, that PDA facilitates methylxanthine- or ryanodine-induced release of Ca2+ from the intracellular store.