Diazepam-binding inhibitor(33-50) elicits Ca2+ oscillation and CCK secretion in STC-1 cells via L-type Ca2+ channels

We recently isolated and characterized 86-amino acid CCK-releasing peptide from porcine intestinal mucosa. The sequence of this peptide is identical t o that of porcine diazepam-binding inhibitor (DBI). Intraduodenal administr ation of DBI stimulates the CCK release and elicits pancreatic secretion in rats. In this study we utilized a murine tumor cell line (STC-1 cells) tha t contains CCK to examine if DBI directly acts on these cells to stimulate CCK release. We investigated the cellular mechanisms responsible for this a ction. We showed that DBI33-50, a biologically active fragment of DBI1-86, significantly stimulated CCK secretion in STC-1 cells. This action was abol ished by Ca2+-free medium. The mean basal intracellular Ca2+ concentration ([Ca2+](i)) was 52 nM in fura a-loaded STC-1 cells. DBI33-50 (1-1,000 nM) e licited Ca2+ oscillations; DBI33-50 (10 nM) increased the oscillation frequ ency to 5 cycles/10 min and elicited a net [Ca2+](i) increase (peak - basal ) to 157 nM. In contrast, bombesin and forskolin caused an initial transien t [Ca2+](i) followed by a small sustained [Ca2+](i) plateau. Withdrawal of extracellular Ca2+ abolished Ca2+ oscillations stimulated by DBI33-50 L-typ e Ca2+ channel blockers nifedipine and diltiazem (3-10 mu M) markedly atten uated DBI-stimulated Ca2+ oscillations. In other cell types L-type Ca2+ cha nnels are activated by cAMP-protein kinase A. DBI33-50 failed to stimulate cAMP formation in STC-1 cells. Similarly, DBI33-50 had no effect on myo-ino sitol 1,4,5-trisphosphate concentration ([IP3]), whereas bombesin caused an eightfold increase in [IP3] over basal. In addition, inhibitors of phospho lipase C (U-73122), phospholipase A(2) (ONO-RS-082), and protein tyrosine k inase (genistein) did not alter the Ca2+ oscillations elicited by DBI33-50 It appears that DBI33-50 acts directly on STC-1 cells to elicit Ca2+ oscill ations via the voltage-dependent L-type Ca2+ channels, resulting in the sec retion of CCK. Mediation of this action is by intracellular mechanisms inde pendent of the traditional signal transduction pathways, including phosphol ipase C, phospholipase A(2), protein tyrosine kinase, and cAMP systems.