Inhibitory effect of phorbol 12,13-dibutyrate on norepinephrine-induced contraction in rabbit iris dilator muscle

The hypothesis that the increase in Ca 2+ sensitivity on norepinephrine-induced contraction of smooth muscles and also the decrease of the norepinephrine-induced sustained level of intracellular Ca 2+ concentration are produced by the activation of protein kinase C was tested. Phorbol 12,13-dibutyrate (PDB; 10 −6 M) relaxed the norepinephrine-induced sustained contraction in a concentration-dependent manner. On pretreatment with PDB a transient contraction was produced by the application of norepinephrine, but the sustained contraction was significantly reduced. The sustained elevations of intracellular Ca 2+ concentration ([Ca 2+] i) and the contraction induced by norepinephrine in fura-2-loaded preparations were decreased by the application of PDB. These inhibitory effects were antagonized by potent protein kinase inhibitors, 2-(1-(3-dimethylaminopropyl)-indol-3-yl)-3-(-indol-3-yl)-maleimide (GF 109203X) (10 −6 M) and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) (10 −6 M), but were not affected by a protein kinase A/G inhibitor, N-(2-cinnamylaminoethyl)-5-isoquinolinesulfonamide (H-88) (10 −6 M). The slope of the regression line for norepinephrine for [Ca 2+] i and tension was significantly steeper than those obtained with high K +. Also, on pretreatment with PDB the Ca 2+ sensitivity of the K +-induced contraction was decreased, but the Ca 2+ sensitivity of norepinephrine-induced contraction tended to be increased. These observations indicate that PDB induces a decrease of [Ca 2+] i on Ca 2+ mobility and an increase of Ca 2+ sensitivity on contraction of smooth muscle through the activation of protein kinase C.