A slowly ADP-ribosylated pertussis-toxin-sensitive GTP-binding regulatory protein is required for vasopressin-stimulated Ca2+ inflow in hepatocytes.
The roles of heterotrimeric GTP-binding regulatory proteins (G-proteins) and inositol polyphosphates in the mechanism by which vasopressin stimulates Ca2+ inflow in hepatocytes were investigated by using single cells loaded with fura2 by microinjection. Vasopressin-stimulated Ca2+ inflow was mimicked by microinjection of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) or guanosine 5'-[beta gamma-imido]triphosphate to the cells, but not adenosine 5'-[gamma-thio]triphosphate (ATP[S]) or guanosine 5'-[beta-thio]diphosphate (GDP[S]). Extracellular Gd3+ (5 microM) inhibited both vasopressin- and GTP[S]-stimulated Ca2+ inflow. GDP[S], but not GMP, administered to hepatocytes by microinjection, completely inhibited vasopressin-stimulated Ca2+ inflow and partially inhibited vasopressin-induced release of Ca2+ from intracellular stores. The microinjection of pertussis toxin had no effect either on the release of Ca2+ from intracellular stores or on Ca2+ inflow induced by vasopressin, but completely inhibited changes in these processes induced by epidermal growth factor (EGF). Hepatocytes isolated from rats treated with pertussis toxin for 24 h exhibited no vasopressin- or GTP[S]-stimulated Ca2+ inflow, whereas the vasopressin-stimulated release of Ca2+ from intracellular stores was similar to that observed for control cells. Heparin or ATP[S] inhibited, or delayed the onset of, both vasopressin-induced release of Ca2+ from intracellular stores and vasopressin-stimulated Ca2+ inflow. Vasopressin-induced oscillations in intracellular [Ca2+] were observed in some heparin-treated cells. It is concluded that the stimulation by vasopressin of Ca2+ inflow to hepatocytes requires inositol 1,4,5-trisphosphate (InsP3) and, by implication, the pertussis-toxin-insensitive G-protein required for the activation of phospholipase C beta [Taylor, Chae, Rhee and Exton (1991) Nature (London) 350, 516-518], and another G-protein which is slowly ADP-ribosylated by pertussis toxin and acts between InsP3 and the putative plasma-membrane Ca2+ channel. EGF-stimulated Ca2+ inflow involves at least one G-protein which is rapidly ADP-ribosylated and is most likely required for InsP3 formation.
Berven
L A
LA
Department of Medical Biochemistry, School of Medicine, Flinders University, Adelaide, Australia.
Hughes
B P
BP
Barritt
G J
GJ
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005456:Fluorescent Dyes; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D037342:Pertussis Toxin; D051381:Rats; D017208:Rats, Wistar; D013873:Thionucleotides / Q000494:pharmacology; D013997:Time Factors; D014667:Vasopressins / Q000494:pharmacology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D005456:Fluorescent Dyes; D013873:Thionucleotides; D010566:Virulence Factors, Bordetella; D014667:Vasopressins; D006153:Guanosine Diphosphate; D000246:Adenosine Diphosphate Ribose; C022571:adenosine 5'-O-(3-thiotriphosphate); D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004815:Epidermal Growth Factor; C023427:guanosine 5'-O-(2-thiodiphosphate); D000255:Adenosine Triphosphate; D006493:Heparin; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D002118:Calcium