| Abstrakt: |
We examined the effects of heparin, guanosine nucleotides, protein kinase C (PKC) modulators, such as phorbol 12,13-dibutylate (PDBu) and H-7 on Ca-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). Neomycin (30 μM), an inhibitor of phospholipase C, and intracellular applications of heparin (10 μg/ml) and guanosine 5′- O-(2-thiodiphosphate) (GDP[ βS]; 1 mM) partly but consistently inhibited the generation of I, whereas a higher concentration of heparin (100 μg/ml) transiently enhanced then suppressed the generation of I. Inhibition of I generation by heparin was more powerful at the holding potential of + 20 mV than at −20 mV. Inositol 1,4,5-trisphosphate (Ins P; 30 μM) continuously generated I at holding potentials more positive than −60 mV. Noradrenaline (10 μM) and caffeine (3-20 mM) transiently augmented, then reduced the generation of I. Heparin (10 μg/ml) completely inhibited responses induced by Ins P and noradrenaline, but not those induced by caffeine. Intracellular application of guanosine 5′-triphosphate (GTP; 200 μM) or low concentrations of guanosine 5′- O-(3-thiotriphosphate) (GTP[γS]; ⩽ 3 μM) continuously augmented the generation of I. High concentrations of GTP[γS] (⩾10 μM) transiently augmented, then inhibited I. Neither GTP[γS] nor noradrenaline induced the transient augmentation or the subsequent inhibition of I when applied in the presence of GDP[ βS] (1 mM), neomycin (30 μM) or heparin (10 μg/ml). PDBu (0.1 μM) reduced the generation of I 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 μM). In the presence of H-7, GTP[γS] continuously enhanced the generation of I. The suppression of the generation of I during application of noradrenaline (10 μM) was reduced by pretreatment with H-7. Thus both InsP and protein kinase C contribute to the generation of I in smooth muscle cells of the rabbit portal vein and heparin is not a specific Ins P antagonist on the Ins P-induced Ca-release channel (PIRC). Ins P opens PIRC and protein kinase C may deplete the stored Ca by either inhibiting the reuptake of Ca or by enhancement of the releasing actions of Ins P. [ABSTRACT FROM AUTHOR] |
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