Myo-Inositol 1,4,5-trisphosphate. A second messenger for the hormonal mobilization of intracellular Ca2+ in liver.

S K Joseph,A P Thomas,R J Williams,R F Irvine,J R Williamson

doi:10.1016/s0021-9258(17)43262-5

Abstract

The stimulation of hepatocytes by alpha 1-adrenergic agonists and vasoactive peptides results in a mobilization of intracellular Ca2+ which is accompanied by breakdown of phosphatidylinositol 4,5-bisphosphate to release myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). The possible involvement of Ins(1,4,5)P3 in intracellular Ca2+ mobilization was tested using a preparation of saponin-permeabilized hepatocytes. Added Ca2+ was sequestered by intracellular organelles in the presence of ATP until the medium free Ca2+ concentration was lowered to a new steady state level. The subsequent addition of Ins(1,4,5)P3 caused a rapid Ca2+ release, which was complete within 5 s. Half-maximal and maximal Ca2+ release were obtained at concentrations of Ins(1,4,5)P3 of 0.1 and 0.5 microM, respectively. The maximal amount of Ca2+ mobilized was 450 pmol/mg of cell dry weight. Using experimental conditions designed to permit selective Ca2+ accumulation into mitochondrial or non-mitochondrial stores, it was determined that all of the Ca2+ released by Ins(1,4,5)P3 originated from non-mitochondrial, vesicular stores. After Ca2+ release was completed, reaccumulation occurred until the medium free Ca2+ concentration was restored to its original level. Experiments using 32P-labeled Ins(1,4,5)P3 indicated that Ca2+ reaccumulation was associated with dephosphorylation of this compound. From a consideration of the properties of the Ca2+ release induced by Ins(1,4,5)P3, with respect to its kinetics, dose response, specificity, and the amount of Ca2+ released, the data strongly suggest that this compound is a second messenger involved in the hormonal mobilization of Ca2+ from intracellular stores.

Highlights

The stimulation of hepatocytes by al-adrenergic ag- in phosphorylase a activity can be observed in the complete onists and vasoactive peptides results in a mobilization absence of extracellular Caz+,indicating that the initial inof intracelluiar Ca2+which is accompanied by break- crease in cytosolic free Ca2+is due to themobilization of Ca2+
Experiments using "P- lipid metabolism potentially capable of acting as a messenger labeled Ins(1,4,5)Ps indicated that Ca2+ reaccumula- for the mobilization of calcium from intracellular comparttion was associated with dephosphorylation of this compound
Ins(1,4,5)P3 Specific Ca2+Release from Permeabilized Liver Cells-In order to examine possible effects of Ins(1,4,5)P3on the release of Ca2+from intracellular pools, a preparation of hepatocytes was used in which the plasma membrane was permeabilized by saponin treatment

Summary

Introduction

The stimulation of hepatocytes by al-adrenergic ag- in phosphorylase a activity can be observed in the complete onists and vasoactive peptides results in a mobilization absence of extracellular Caz+,indicating that the initial inof intracelluiar Ca2+which is accompanied by break- crease in cytosolic free Ca2+is due to themobilization of Ca2+. In the range of 150-200 nM, which is believed to be the basal ml in the standard K+based medium supplemented with 75 p~ Quin free Ca2+ inthe cytosol of intact hepatocytes (21, 22).' Addition of MgATP, caused a substantial sequestration of Ca2+which amounted to about 2nmol/mg of cell dry weight.

Results

Conclusion