Effect of inositol-1,4,5-trisphosphate on isolated subcellular fractions of rat pancreas

Abstract

We have previously shown that inositol-1,4,5-trisphosphate (IP3) releases Ca2+ from an intracellular calcium store in permeabilized acinar cells of rat pancreas (H. Streb et al., 1983, Nature (London) 306:67-69). This observation suggests that IP3 might provide the missing link between activation of the muscarinic receptor and Ca2+ release from intracellular stores during stimulation. In order to localize the intracellular IP3-sensitive calcium pool, IP3-induced Ca2+ release was measured in isolated subcellular fractions. A total homogenate was prepared from acinar cells which had been isolated by a collagenase digestion method. Endoplasmic reticulum was separated from mitochondria, zymogen granules and nuclei by differential centrifugation. Plasma membranes and endoplasmic reticulum were separated by centrifugation on a sucrose step gradient or by precipitation with high concentrations of MgCl2. IP3-induced Ca2+ release per mg protein in the total homogenate was the same as in leaky cells and was sufficiently stable to make short separation procedures possible. In fractions obtained by either differential centrifugation at 7000 X g, sucrose-density centrifugation, or MgCl2 precipitation there was a close correlation of Ip3-induced Ca2+ release with the endoplasmic reticulum markers ribonucleic acid (r = 0.96, 1.00, 0.91, respectively) and NADPH cytochrome c reductase (r = 0.63, 0.98, 0.90, respectively). In contrast, there was a clear negative correlation with the mitochondrial markers cytochrome c oxidase (r = -0.64) and glutamate dehydrogenase (r = -0.75) and with the plasma membrane markers (Na+ + K+)-ATPase (r = -0.81) and alkaline phosphatase (r = -0.77) in all fractions analyzed. IP3-induced Ca2+ release was distributed independently of zymogen granule or nuclei content of the fractions as assessed by electron microscopy. The data suggest that inositol-1,4,5-trisphosphate releases Ca2+ from endoplasmic reticulum in pancreatic acinar cells.

Binding of inositol trisphosphate by a liver microsomal fraction

“…The time course of Ins(1,4,5)P3 formation as well as its effect on calcium metabolism in various types of permeabilized cells supports this postulate (Berridge, 1984;Williamson et al, 1985). Moreover, it was found that Ins(1,4,5)P3 is capable of releasing calcium from various microsomal preparations (Prentki et al, 1984;Streb et al, 1984;O'Rourke et al, 1985), including a rat liver microsomal fraction (Dawson & Irvine, 1984;Muallem et al, 1985). In this report we present evidence for the specific, saturable, and reversible binding of radiolabelled Ins(1,4,5)P3 to a crude microsomal fraction of rat liver, which demonstrates a specific receptor for this putative second messenger.…”

Hormonal activation of single K+ channels via internal messenger in isolated pancreatic acinar cells

“…Channel opening is controlled exclusively by the membrane potential and [Ca2+]i whereas changes in [Ca2'], have no effect. The external Ca2+ requirement in the isolated membrane-patch area in the experiments demonstrating indirect CCK5 activation of K+ channels ( fig.1) cannot therefore be explained by the action of Ca2+ on the external site of the channel but must be due to an influence on [Ca'+]i. Hormones and transmitters seem to mobilize intracellular Ca" via the intracellular messenger inositol trisphosphate (InsP3) [ 11,121 and this substance acts on the endoplasmic reticulum membrane by opening a Ca2+ pathway [ 13,141. However, many hormone and transmitterevoked responses in exocrine gland cells are dependent on extracellular Ca2+ [ 15,161 as documented for the stimulant-evoked hyperpolarization and increase in outward K+ current in pig pancreatic acinar cells [3,5] and pancreatic secretagogues increase unidirectional Ca2+ flux into acinar cells [17].…”

A quantitative investigation into the dependence of Ca2+ mobilisation on changes in inositol 1,4,5‐trisphosphate levels in the stimulated neutrophil

“…Ca2+ mobilisation by 1P3 has been studied extensively both in permeabilised cells and by micro-injection techniques and has been found to be mediated by a specific receptor associated with the endoplasmic reticulum (Streb et al, 1984). Recent work has revealed that the 1P3 receptor and the IP3-sensitive Ca2 + channel are part of the same intrinsic membrane protein (Ferris et al, 1989).…”