GTP- and inositol 1,4,5-trisphosphate-induced release of 45Ca2+ from a membrane store co-localized with pancreatic-islet-cell plasma membrane.
Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], arising from hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], is proposed as the link between membrane-receptor activation and mobilization of Ca2+ from intracellular sites in hormone-secreting cells. The location of Ins(1,4,5)P3-sensitive membranes was investigated in cultured neonatal beta-cells. Membranes were obtained after lysis of cells attached to positively charged Sephadex. After lysis the presence of the enzyme markers 5'-nucleotidase, glucose-6-phosphatase, NADH-cytochrome c reductase, UDP-galactosyltransferase and succinate dehydrogenase indicated the mixed nature of the preparation. After sonication, however, UDP-galactosyltransferase and succinate dehydrogenase activities were undetectable, but 4.8% of total cellular glucose-6-phosphatase and 3.4% of total cellular NADH-cytochrome c reductase remained with 5'-nucleotidase in the preparation, indicating endoplasmic-reticulum association. ATP-dependent 45Ca2+ accumulation was shown in this preparation (410 +/- 24 pmol/mg of protein at 150 nM free Ca2+) and was inhibited by vanadate (100 microM). Ca2+ release was effected by Ins(1,4,5)P3, with half-maximal release at 0.5 +/- 0.14 microM-Ins(1,4,5)P3, t1/2 11.2 +/- 1.1 s. GTP- and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG)-promoted release of 45Ca2+ was demonstrated in this preparation, but the kinetics of release (half-maximal Ca2+ release at 5.4 +/- 0.7 microM, with t1/2 77.3 +/- 6.9 s, and at 51.1 +/- 4.2 microM, with t1/2 19.0 +/- 2.2 s, for GTP and p[NH]ppG respectively), and the ability of neomycin sulphate to block p[NH]ppG-induced release only, are indicative of separate release mechanisms after treatment with these agents. A close association between plasma membrane and elements of the endoplasmic reticulum is indicated in this model, providing a possible mechanism for local alterations in free Ca2+ in the sub-plasma-membrane region.
Dunlop
M E
ME
University of Melbourne, Department of Medicine, Royal Melbourne Hospital, Parkville, Victoria, Australia.
Larkins
R G
RG
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002132:Calcium Radioisotopes; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D003911:Dextrans; D006160:Guanosine Triphosphate / Q000494:pharmacology*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate; D007295:Inositol Phosphates / Q000494:pharmacology*; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism*; D008863:Microspheres; D051381:Rats; D013010:Sonication; D013403:Sugar Phosphates / Q000494:pharmacology*
D002132:Calcium Radioisotopes; D003911:Dextrans; D007295:Inositol Phosphates; D013403:Sugar Phosphates; D006165:Guanylyl Imidodiphosphate; C052839:Cytodex; D015544:Inositol 1,4,5-Trisphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D002118:Calcium