BOMBESIN-LIKE PEPTIDES STIMULATE SOMATOSTATIN RELEASE FROM RAT FUNDICD-CELLS IN PRIMARY CULTURE

In several species, bombesin-like neuropeptides stimulate somatostatin release in in vitro preparations of gastric mucosa, We sought to dete rmine if this response is due to a direct effect on fundic D cells. Ra t fundic mucosal cells were isolated by pronase E (1% D cells). D cell s were separated by counterflow elutriation and subsequent density-gra dient centrifugation (Nycodenz) (15% D cells) and growth in primary cu lture for 48 h (46% D cells). Cultured cells were double stained with affinity-purified rabbit-anti-gastrin-releasing peptide (GRP) receptor antibody and mouse monoclonal antibody to human somatostatin. After i ncubation with rhodamine-labeled anti-rabbit and fluorescein isothiocy anate-labeled anti-mouse antibodies, reactions were visualized by fluo rescence microscopy. All cells positive for somatostatin had GRP recep tors, whereas all non-D cells showed no expression in this G cell-free culture system. Somatostatin release from cultured cells was stimulat ed by sulfated cholecystokinin octapeptide (CCK-8; EC50 3 x 10(-10) M) and epinephrine (EC50 4 x 10(-8) M), which are established stimuli fo r canine fundic D cells. Bombesin (EC50 6 x 10(-11) M), its mammalian analog GRP-27, and neuromedin C (GRP-10) (EC50 1 x 10(-10) M, for both ) were almost equally potent stimuli of somatostatin release, elicitin g maximal response at 10(-9) M (400-550% above basal). Neuromedin B wa s less potent and effective (maximal response at 10(-8) M, 230% above basal). [D-Phe(6)]bombesin-(6-13)-OMe, a specific bombesin receptor an tagonist, inhibited bombesin-stimulated somatostatin release in a comp etitive manner (IC50 9 x 10(-8) M). Potentiating interactions were obs erved between bombesin and dibutyryladenosine 3',5'-cyclic monophospha te (DBcAMP) or epinephrine, but not between bombesin and CCK-8. We con clude that bombesin-like peptides directly stimulate somatostatin rele ase by interacting with specific receptors on rat fundic D cells. Bomb esin-like peptides appear to induce Ca2+-phospholipid-dependent signal -response transduction, as is indirectly suggested by potentiating int eractions with DBcCAMP or epinephrine.