PANCREASTATIN ACTIVATES PERTUSSIS-TOXIN-SENSITIVE GUANYLATE-CYCLASE AND PERTUSSIS TOXIN-INSENSITIVE PHOSPHOLIPASE-C IN RAT-LIVER MEMBRANES

We have recently found the calcium dependent glycogenolytic effect of pancreastatin on rat hepatocytes and the mobilization of intracellular calcium. To further investigate the mechanism of action of pancreastatin on liver we have studied its effect on guanylate cyclase, adenylate cyclase, and phospholipase C, and we have explored the possible involvement of GTP binding proteins by measuring GTPase activity as well as the effect of pertussis toxin treatment of plasma liver membranes on the pancreastatin stimulated GTPase activity and the production of cyclic GMP and myo-inositol 1,4,5-triphosphate. Pancreastatin stimulated GTPase activity of rat liver membranes about 25% over basal. The concentration dependency curve showed that maximal stimulation was achieved at 10(-7) M pancreastatin (EC(50) = 3 nM). This stimulation was partially inhibited by treatment of the membranes with pertussis toxin. The effect of pancreastatin on guanylate cyclase and phospholipase C were examined by measuring the production of cyclic GMP and myo-inositol 1,4,5-triphosphate respectively. Pancreastatin increased the basal activity of guanylate cyclase to a maximum of 2.5-fold the unstimulated activity at 30 degrees C, in a time- and dose-dependent manner, reaching the maximal stimulation above control with 10(-7) M pancreastatin at 10 min (EC(50) = 0.6 nM). This effect was completely abolished when rat liver membranes had been ADP-ribosylated with pertussis toxin. On the other hand, adenylate cyclase activity was not affected by pancreastatin. Phospholipase C activity of rat liver membranes was rapidly stimulated (within 2-5 min) at 30 degrees C by 10(-7) M pancreastatin, reaching a maximum at 15 min. The dose response curve showed that with 10(-7) M pancreastatin, maximal stimulation was obtained (EC(50) = 3 nM). GTP (10(-5) M) stimulated the membrane-bound phospholipase C as expected. However, the incubation of rat liver membranes with GTP partially inhibited the stimulation of phospholipase C activity produced by pancreastatin, whereas GTP enhanced the activation of phospholipase C by vasopressin. This inhibition by GTP was dose dependent and 10(-5) M GTP obtained the maximal inhibition (about 40%). The inhibitory effect of GTP on the stimulatory effect of pancreastatin on phospholipase C activity was completely abolished when rat liver membranes had previously been ADP-ribosylated with pertussis toxin. The presence of 8-Br-cGMP mimics the effect of GTP, whereas CMP-PNP increased both basal and pancreastatin-stimulated phospholipase C, suggesting a role of the cyclic CMP as a feed-back regulator of the synthesis of myo-inositol 1,4,5-triphosphate. However, the pretreatment of membranes with pertussis toxin did not modify the production of myo-Inositol 1,4,5-triphosphate stimulated by pancreastatin. In conclusion, pancreastatin activates guanylate cyclase activity and phospholipase C involving different pathways,pertussis toxin-sensitive, and -insensitive, respectively. (C) 1994 Wiley-Liss, Inc.