The effect of inositol trisphosphate on Ca2+ fluxes in insulin-secreting tumor cells.

S K Joseph,R J Williams,B E Corkey,F M Matschinsky,J R Williamson

doi:10.1016/s0021-9258(18)90638-1

Abstract

An early event associated with the stimulation of various secretory cells is the breakdown of phosphatidylinositol 4,5-bisphosphate and the mobilization of cellular calcium. Hydrolysis of this inositol lipid by a phosphodiesterase produces inositol trisphosphate (InsP3), a small water-soluble molecule which may serve a messenger function to release Ca2+ from internal stores. In order to assess the role of inositol lipid breakdown in the stimulation of insulin secretion we have examined the effect of InsP3 on Ca2+ fluxes in three different insulin-secreting tumor cells permeabilized by the addition of saponin. A rapid, transient release of Ca2+ from a non-mitochondrial pool occurred upon addition of InsP3 to all three cell types. Half-maximal Ca2+ release from the RIN-1046-38 and RIN-m5F cells was obtained in the concentration range 0.1-0.2 microM. However, the cells obtained from a transplantable tumor of the Syrian hamster were far more sensitive to InsP3 with half-maximal release being observed at 0.025 microM. A partially purified preparation of vesicles was isolated from this tumor which retained its responsiveness to InsP3. Half-maximal Ca2+ release from the vesicles was obtained at 0.2 microM InsP3. Our data are consistent with a role for InsP3 in mediating the increase in cytosolic free Ca2+ which occurs in response to a number of stimuli that promote the secretion of insulin.

Highlights

From the Departmentof Biochemistry and Biophysics and Diabetes Research Center, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
A general characteristic of most of these cell lines is that thedyisplay a poor secretory response to glucose when compared with the native islet(s8).some response to other secretagogues such as glyceraldehyde, aketoisocaproate,certainamino acids, or glucagon (8-11) is retained. In this studywe have tested the effects of InsP3 on three different insulin-secreting tumor cells
Our results show that all threetypesrespond to InsP3 by mobilizing Ca” from intracellular, nonmitochondrial stores, with the Syrian hamsterinsulinoma cell being the mostsensitive toInsP3.In addition,itis shown that a partially purifiedmicrosomal preparation, enrichedin marker enzymes forthe endoplasmic reticulum and almost free of mitochondria, responds directly to InsP3 addition

Summary

EXPERIMENTAL PROCEDURES

Insulin release‘ from the pancreatic @-cellin response to fuels and hormones such as acetylcholine is thought to be triggered by an increase in the concentratiofncytosolic free detached from the culture flasks using EDTA and trypsin (8) and were washed three times in a Ca2+- andM e - f r e e Hanks’ basal salt solution buffered with 25 mM Na+ HEPES (pH 7.2). Cells were prepared from the Syrian hamster insulinoma,approximatelyone. Homogenates (1:15, w/v) of the insulinoma and the liver were prepared using 0.25 M sucrose, 10 mM Tris HEPES (pH 7.2). Procedures for the down of a particular phospholipid, notably phosphatidylinos- preparation of InsPs from erythrocytes and measurements of InsPs it01 4,5-bisphosphate (2-4). In all these cases, the breakdown degradation and Ca2+fluxes are detailed elsewhere (6). ’The abbreviations used are:InsP3, myo-inositol 1,4,5-trisphosphate; EGTA, ethylene glycol bis(j3-aminoethyl ether)-N,N,N‘,N’tetraacetic acid; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesuldressed.

InoTsriitsopl hospahnadte

RESULTS

DISCUSSION