Yoshida H, Tsunoda Y, Owyang C
Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109, USA.
Am J Physiol. 1999 Mar;276(3):G694-702. doi: 10.1152/ajpgi.1999.276.3.G694.
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 DBI33-50, a biologically active fragment of DBI1-86, 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. DBI33-50 (1-1,000 nM) elicited Ca2+ oscillations; DBI33-50 (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 DBI33-50. L-type Ca2+ channel blockers nifedipine and diltiazem (3-10 microM) markedly attenuated DBI-stimulated Ca2+ oscillations. In other cell types L-type Ca2+ channels 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-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 DBI33-50. It appears that DBI33-50 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.
我们最近从猪小肠黏膜中分离并鉴定了一种86个氨基酸的胆囊收缩素释放肽。该肽的序列与猪地西泮结合抑制剂（DBI）的序列相同。十二指肠内注射DBI可刺激大鼠胆囊收缩素的释放并引发胰腺分泌。在本研究中，我们利用一种含有胆囊收缩素的小鼠肿瘤细胞系（STC-1细胞）来研究DBI是否直接作用于这些细胞以刺激胆囊收缩素的释放。我们研究了介导此作用的细胞机制。我们发现，DBI1-86的生物活性片段DBI33-50能显著刺激STC-1细胞中胆囊收缩素的分泌。无钙培养基可消除此作用。在负载fura 2的STC-1细胞中，平均基础细胞内钙离子浓度（[Ca2+]i）为52 nM。DBI33-50（1 - 1000 nM）可引发钙离子振荡；DBI33-50（10 nM）可将振荡频率提高到5次/10分钟，并使[Ca2+]i净增加（峰值 - 基础值）至157 nM。相比之下，蛙皮素和福斯可林会引起初始短暂的[Ca2+]i升高，随后是较小的持续性[Ca2+]i平台期。去除细胞外钙离子可消除DBI33-50刺激的钙离子振荡。L型钙离子通道阻滞剂硝苯地平和地尔硫䓬（3 - 10 microM）可显著减弱DBI刺激的钙离子振荡。在其他细胞类型中，L型钙离子通道由cAMP-蛋白激酶A激活。DBI33-50未能刺激STC-1细胞中cAMP的形成。同样，DBI33-50对肌醇1,4,5-三磷酸浓度（[IP3]）无影响，而蛙皮素可使[IP3]比基础值增加八倍。此外，磷脂酶C抑制剂（U-73122）、磷脂酶A2抑制剂（ONO-RS-082）和蛋白酪氨酸激酶抑制剂（染料木黄酮）均未改变DBI33-50引发的钙离子振荡。似乎DBI33-50直接作用于STC-1细胞，通过电压依赖性L型钙离子通道引发钙离子振荡，从而导致胆囊收缩素的分泌。此作用的介导是通过独立于传统信号转导途径的细胞内机制，包括磷脂酶C、磷脂酶A2、蛋白酪氨酸激酶和cAMP系统。
