Roles of inositol trisphosphate and protein kinase C in the spontaneous outward current modulated by calcium release in rabbit portal vein

We examined the effects of heparin, guanosine nucleotides, protein kinase C (PKC) modulators, such as phorbol 12,13-dibutylate (PDBu) and H-7 on Ca²⁺-dependent K⁺ currents in smooth muscle cells of the rabbit portal vein using the whole-cell patch-clamp technique, to explore the effects of PKC on the oscillatory outward current (I_oo). Neomycin (30 M), an inhibitor of phospholipase C, and intracellular applications of heparin (10 g/ml) and guanosine 5-O-(2-thiodiphosphate) (GDP[S]; 1 mM) partly but consistently inhibited the generation of I_oo, whereas a higher concentration of heparin (100 g/ml) transiently enhanced then suppressed the generation of I_oo. Inhibition of I_oo generation by heparin was more powerful at the holding potential of + 20 mV than at –20 mV. Inositol 1,4,5-trisphosphate (InsP₃; 30 M) continuously generated I_oo at holding potentials more positive than –60 mV. Noradrenaline (10 M) and caffeine (3–20 mM) transiently augmented, then reduced the generation of I_oo. Heparin (10 g/ml) completely inhibited responses induced by InsP₃ and noradrenaline, but not those induced by caffeine. Intracellular application of guanosine 5-triphosphate (GTP; 200 M) or low concentrations of guanosine 5-O-(3-thiotriphosphate) (GTP[S]; 3 M) continuously augmented the generation of I_oo. High concentrations of GTP[S] (10 M) transiently augmented, then inhibited I_oo. Neither GTP[S] nor noradrenaline induced the transient augmentation or the subsequent inhibition of I_oo when applied in the presence of GDP[S] (1 mM), neomycin (30 M) or heparin (10 g/ml). PDBu (0.1 M) reduced the generation of I_oo but failed to produce an outward current following application of caffeine (3–5 mM). This action of PDBu was inhibited by pretreatment with H-7 (20 M). In the presence of H-7, GTP[S] continuously enhanced the generation of I_oo. The suppression of the generation of I_oo during application of noradrenaline (10 M) was reduced by pretreatment with H-7. Thus both InsP₃ and protein kinase C contribute to the generation of I_oo in smooth muscle cells of the rabbit portal vein and heparin is not a specific InsP₃ antagonist on the InsP₃-induced Ca²⁺-release channel (PIRC). InsP₃ opens PIRC and protein kinase C may deplete the stored Ca²⁺ by either inhibiting the reuptake of Ca²⁺ or by enhancement of the releasing actions of InsP₃.