A slowly ADP-ribosylated pertussis-toxin-sensitive GTP-binding regulatory protein is required for vasopressin-stimulated Ca super(2+)-inflow in hepatocytes

The roles of heterotrimeric GTP-binding regulatory proteins (G-proteins) and inositol polyphosphates in the mechanism by which vasopressin stimulates Ca super(2+) inflow in hepatocytes were investigated by using single cells loaded with fura2 by microinjection. Vasopressin-stimulated Ca super(2+) 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 Gd super(3+) (5 mu M) inhibited both vasopressin- and GTP[S]-stimulated Ca super(2+) inflow. GDP[S], but not GMP, administered to hepatocytes by microinjection, completely inhibited vasopressin-stimulated Ca super(2+) inflow and partially inhibited vasopressin-induced release of Ca super(2+) from intracellular stores. The microinjection of pertussis toxin had no effect either on the release of Ca super(2+) from intracellular stores or on Ca super(2+) 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 Ca super(2+) inflow, whereas the vasopressin-stimulated release of Ca super(2+) 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 Ca super(2+) from intracellular stores and vasopressin-stimulated Ca super(2+) inflow. Vasopressin-induced oscillations in intracellular [Ca super(2+)] were observed in some heparin-treated cells. It is concluded that the stimulation by vasopressin of Ca super(2+) inflow to hepatocytes requires inositol 1,4,5-trisphosphate (InsP sub(3)) and, by implication, the pertussis-toxin-insensitive G-protein required for the activation of phospholipase C sub( beta ), and another G-protein which is slowly ADP-ribosylated by pertussis toxin and acts between InsP sub(3) and the putative plasma-membrane Ca super(2+) channel. EGF-stimulated Ca super(2+) inflow involves at least one G-protein which is rapidly ADP-ribosylated and is most likely required for InsP sub(3) formation.