title: Evidence that a low-molecular-mass GTP-binding protein is required for store-activated Ca2+ inflow in hepatocytes.
abstract: The roles of a monomeric GTP-binding regulatory protein in the activation of store-activated plasma membrane Ca2+ channels and in the release of Ca2+ from the smooth endoplasmic reticulum (SER) in rat liver parenchymal cells were investigated with the use of freshly isolated rat hepatocytes and rat liver microsomes. A low concentration (approx. 130 microM intracellular) of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) activated Ca2+ inflow in intact hepatocytes in the absence of an agonist, whereas a high concentration (approx. 530 microM intracellular) of GTP-S- or guanosine 5'-[betagamma-imido]triphosphate (p[NH]ppG) inhibited the Ca2+ inflow induced by inhibitors of the activity of the endoplasmic-reticulum Ca2+-ATPase (SERCA) and by vasopressin. GTP (530 microM) prevented the inhibition of Ca2+ inflow by GTP-S- and p[NH]ppG. Brefeldin A and the peptide human Arf-1-(2-17), which inhibit many functions of ADP ribosylation factor (Arf) proteins, inhibited the Ca2+ inflow induced by SERCA inhibitors and vasopressin, and altered the profile of Ca2+ release from the SER. These effects were observed at concentrations of Brefeldin A and Arf-1-(2-17) comparable with those that inhibit the functions of Arf proteins in other systems. Succinylated Arf-1-(2-17) had a negligible effect on Ca2+ inflow. GTP[S] and Arf-1-(2-17) completely inhibited the synergistic action of GTP and Ins(1,4,5)P3 in releasing 45Ca2+ from rat liver microsomes loaded with 45Ca2+. AlF4(-) (under conditions expected to activate trimeric G-proteins) and succinylated Arf-1-(2-17) had no effect on GTP/Ins(1,4,5))3-induced 45Ca2+ release, and a mastoparan analogue caused partial inhibition. Arf-1-(2-17) did not inhibit 45Ca2+ release induced by either thapsigargin or ionomycin. It is concluded that a low-molecular-mass G-protein, most probably a member of the Arf protein family, is required for store-activated Ca2+ inflow in rat hepatocytes. The idea that the role of this G-protein is to maintain a region of the SER in the correct intracellular location is discussed briefly.
authors.affiliation: Department of Medical Biochemistry, School of Medicine, Faculty of Health Sciences, Flinders University, G.P.O. Box 2100, Adelaide, South Australia, 5001, Australia.
authors.forename: K C
authors.initials: KC
authors.lastname: Fernando
authors.forename: R B
authors.initials: RB
authors.lastname: Gregory
authors.forename: F
authors.initials: F
authors.lastname: Katsis
authors.forename: B E
authors.initials: BE
authors.lastname: Kemp
authors.forename: G J
authors.initials: GJ
authors.lastname: Barritt
mesh_terms: D020727:ADP-Ribosylation Factors; D000818:Animals; D020126:Brefeldin A; D002118:Calcium / Q000378:metabolism*; D003517:Cyclopentanes / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D017136:Ion Transport; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D051381:Rats; D019284:Thapsigargin / Q000494:pharmacology; D014667:Vasopressins / Q000494:pharmacology
chemical_list: D003517:Cyclopentanes; D010446:Peptide Fragments; D014667:Vasopressins; D020126:Brefeldin A; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors; D002118:Calcium