Dual roles of phorbol 12, 13-dibutyrate in the regulation of guinea-pig gastric contraction

Abstract

We performed experiments to investigate the actions of protein kinase C (PKC) on mechanical contraction during agonist stimulation in guinea-pig stomach. We used carbachol and high K condition to enhance mechanical contraction by mobilizing intracellular Ca²⁺ and increasing Ca²⁺ influx through voltage-dependent Ca²⁺ channel, respectively. Phorbol 12, 13-dibutyrate (PDBu) increased spontaneous contractions sensitive to verapamil (438±82.2%, n=7) and potentiated high K-induced contraction (189±22.5%, n=5). However, carbachol (CCh)-induced contractions in PDBu-treated condition depended on extracellular Ca²⁺. In the presence of extracellualr Ca²⁺, CCh-induced contraction was potentiated, while it was suppressed in the absence of extracellular Ca²⁺ in Ca²⁺-preloaded muscle strips. To prove the hypothesis that such phenomena might be related with changes of myoplasmic Ca²⁺ concentration, we investigated the effect of PDBu on voltage-dependent Ca²⁺ current (I_Ca) and CCh-induced Ca²⁺-activated K current (I_{K (Ca)} ) transient using whole-cell voltage clamp technique. For recording voltage-dependent Ca²⁺ current (I_Ca), 10mM Ba²⁺, instead of Ca²⁺, was used to enhance the current size. Voltage-dependent Ba²⁺ current (I_{K Ba}) was increased by PDBu (212±32.2% of steady state currents, n=5), while CCh-induced increase of I_{K (Ca)} transient was inhibited by PDBu (n=5), the changes of which were similar to those of muscle contractions. To analyze the steps involved in the inhibition of CCh-induced I_{K (Ca)} transient by PDBu, we investigated the effect of PDBu on I_{K (Ca)} in the cells perfused with Ins (1, 4, 5) P3. However, Ins (1, 4, 5) P3-induced I_{K (Ca)} was not inhibited by the treatment with phorbol ester. From these results, it is concluded that inhibition of phosphatidylinositol-phospholipase C (PI-PLC) system and potentiation of I_Ca by PKC are important regulatory mechanisms in agonist-induced muscle contraction.