We recently isolated and characterized 86-amino acid CCK-releasing peptide from porcine intestinal mucosa. The sequence of this peptide is identical to that of porcine diazepam-binding inhibitor (DBI). Intraduodenal administration 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) that contains CCK to examine if DBI directly acts on these cells to stimulate CCK release. We investigated the cellular mechanisms responsible for this action. We showed that DBI_33-50, a biologically active fragment of DBI_1-86, significantly stimulated CCK secretion in STC-1 cells. This action was abolished by Ca²⁺-free medium. The mean basal intracellular Ca²⁺ concentration ([Ca²⁺]_i) was 52 nM in fura 2-loaded STC-1 cells. DBI_33-50 (1-1,000 nM) elicited Ca²⁺ oscillations; DBI_33-50 (10 nM) increased the oscillation frequency 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 transient [Ca²⁺]_i followed by a small sustained [Ca²⁺]_i plateau. Withdrawal of extracellular Ca²⁺ abolished Ca²⁺ oscillations stimulated by DBI_33-50. L-type Ca²⁺ channel blockers nifedipine and diltiazem (3-10 µM) markedly attenuated DBI-stimulated Ca²⁺ oscillations. In other cell types L-type Ca²⁺ channels are activated by cAMP-protein kinase A. DBI_33-50 failed to stimulate cAMP formation in STC-1 cells. Similarly, DBI_33-50 had no effect on myo-inositol 1,4,5-trisphosphate concentration ([IP₃]), whereas bombesin caused an eightfold increase in [IP₃] over basal. In addition, inhibitors of phospholipase C (U-73122), phospholipase A₂ (ONO-RS-082), and protein tyrosine kinase (genistein) did not alter the Ca²⁺ oscillations elicited by DBI_33-50. It appears that DBI_33-50 acts directly on STC-1 cells to elicit Ca²⁺ oscillations via the voltage-dependent L-type Ca²⁺ channels, resulting in the secretion of CCK. Mediation of this action is by intracellular mechanisms independent of the traditional signal transduction pathways, including phospholipase C, phospholipase A₂, protein tyrosine kinase, and cAMP systems.

cholecystokinin-releasing peptide; calcium signal transduction; porcine intestine; stimulus-secretion coupling

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