BOMBESIN-INDUCED GASTRIN-RELEASE FROM CANINE G-CELLS IS STIMULATED BYCA2-KINASE-C, AND IS ENHANCED BY DISRUPTION OF RHO( BUT NOT BY PROTEIN)CYTOSKELETAL PATHWAYS/

Isolated canine G cells in primary culture have been used to study cal cium, protein kinase C (PKC), and rho/cytoskeletal-dependent intracell ular pathways involved in bombesin-stimulated gastrin release. A metho d to obtain highly purified G cells by culture (64% G cells) after now cytometry on elutriated fractions of cells from digested canine gastr ic antral mucosa has been developed. Pretreatment of G cells with thap sigargin (10(-8)-10(-6) M) and release experiments in Ca2+-containing or -depleted media showed that influx of Ca2+ into the cells and not a cute release from intracellular stores plays an important role in bomb esin-stimulated gastrin release. Inhibition of PKC by the specific inh ibitor GF 109 203X did not affect bombesin-stimulated release. Rho, a small GTP-binding protein that regulates the actin cytoskeleton, is sp ecifically antagonized by Clostridium botulinum C3 exoenzyme, C3 (10 m u g/ml) enhanced basal and bombesin-stimulated gastrin release by 315 and 266%, respectively. The importance of the cytoskeleton for regulat ion of gastrin release was emphasized by a more pronounced release of gastrin when the organization of the actin cytoskeleton was disrupted by cytochalasin D (5 x 10(-7) and 10(-6) M). Wortmannin, a potent inhi bitor of phosphoinositide-3-kinase, did not alter bombesin-stimulated gastrin release, Thus, it is concluded that bombesin-induced gastrin r elease from canine G cells is stimulated by Ca2+ but not by PKC, and i s enhanced by disruption of rho/cytoskeletal pathways.