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

Abstract

We examined the effects of heparin, guanosine nucleotides, protein kinase C (PKC) modulators, such as phorbol 12,13-dibutyrate (PDBu) and H-7 on Ca(2+)-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 (Ioo). Neomycin (30 microM), an inhibitor of phospholipase C, and intracellular applications of heparin (10 micrograms/ml) and guanosine 5'-O-(2-thiodiphosphate) (GDP[beta S]; 1 mM) partly but consistently inhibited the generation of Ioo, whereas a higher concentration of heparin (100 micrograms/ml) transiently enhanced then suppressed the generation of Ioo. Inhibition of Ioo generation by heparin was more powerful at the holding potential of +20 mV than at -20 mV. Inositol 1,4,5-trisphosphate (InsP3; 30 microM) continuously generated Ioo at holding potentials more positive than -60 mV. Noradrenaline (10 microM) and caffeine (3-20 mM) transiently augmented, then reduced the generation of Ioo. Heparin (10 micrograms/ml) completely inhibited responses induced by InsP3 and noradrenaline, but not those induced by caffeine. Intracellular application of guanosine 5'-triphosphate (GTP; 200 microM) or low concentrations of guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma S]; < or = 3 microM) continuously augmented the generation of Ioo. High concentrations of GTP[gamma S] (> or = 10 microM) transiently augmented, then inhibited Ioo. Neither GTP[gamma S] nor noradrenaline induced the transient augmentation or the subsequent inhibition of Ioo when applied in the presence of GDP[beta S] (1 mM), neomycin (30 microM) or heparin (10 micrograms/ml). PDBu (0.1 microM) reduced the generation of Ioo 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 microM). In the presence of H-7, GTP[gamma S] continuously enhanced the generation of Ioo. The suppression of the generation of Ioo during application of noradrenaline (10 microM) was reduced by pretreatment with H-7. Thus both InsP3 and protein kinase C contribute to the generation of Ioo in smooth muscle cells of the rabbit portal vein and heparin is not a specific InsP3 antagonist on the InsP3-induced Ca(2+)-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.