Bombesin-like peptides stimulate somatostatin release from rat fundic D cells in primary culture

In several species, bombesin-like neuropeptides stimulate somatostatin release in in vitro preparations of gastric mucosa. We sought to determine if this response is due to a direct effect on fundic D cells. Rat fundic mucosal cells were isolated by pronase E (1% D cells). D cells were separated by counterflow elutriation and subsequent density-gradient centrifugation (Nycodenz) (15% D cells) and grown in primary culture 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 incubation with rhodamine-labeled anti-rabbit and fluorescein isothiocyanate-labeled anti-mouse antibodies, reactions were visualized by fluorescence microscopy. All cells positive for somatostatin had GRP receptors, whereas all non-D cells showed no expression in this G cell-free culture system. Somatostatin release from cultured cells was stimulated by sulfated cholecystokinin octapeptide (CCK-8; EC50 3 X 10(-10) M) and epinephrine (EC50 4 X 10(-8) M), which are established stimuli for 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, eliciting maximal response at 10(-9) M (400-550% above basal). Neuromedin B was less potent and effective (maximal response at 10(-8) M, 230% above basal). [D-Phe6]bombesin-(6-13)-OMe, a specific bombesin receptor antagonist, inhibited bombesin-stimulated somatostatin release in a competitive manner (IC50 9 X 10(-8) M). Potentiating interactions were observed between bombesin and dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP) or epinephrine, but not between bombesin and CCK-8. We conclude that bombesin-like peptides directly stimulate somatostatin release by interacting with specific receptors on rat fundic D cells. Bombesin-like peptides appear to induce Ca(2+)-phospholipid-dependent signal-response transduction, as is indirectly suggested by potentiating interactions with DBcAMP or epinephrine.