Jacobo Sarah Melissa P, Guerra Marcy L, Hockerman Gregory H
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana, USA.
J Pharmacol Exp Ther. 2009 Nov;331(2):724-32. doi: 10.1124/jpet.109.158519. Epub 2009 Aug 26.
The incretin peptides, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), potentiate glucose-stimulated insulin secretion (GSIS) and beta-cell proliferation and differentiation. Ca(2+) influx via voltage-gated L-type Ca(2+) channels is required for GLP-1 and GIP potentiation of GSIS. We investigated the role of the L-type Ca(2+) channels Ca(v)1.2 and Ca(v)1.3 in mediating GLP-1- and GIP-stimulated events in INS-1 cells and INS-1 cell lines expressing dihydropyridine-insensitive (DHPi) mutants of either Ca(v)1.2 or Ca(v)1.3. Ca(v)1.3/DHPi channels supported full potentiation of GSIS by GLP-1 (50 nM) compared with untransfected INS-1 cells. However, GLP-1-potentiated GSIS mediated by Ca(v)1.2/DHPi channels was markedly reduced compared with untransfected INS-1 cells. In contrast, GIP (10 nM) potentiation of GSIS mediated by both Ca(v)1.2/DHPi and Ca(v)1.3/DHPi channels was similar to that observed in untransfected INS-1 cells. Disruption of intracellular Ca(2+) release with thapsigargin, ryanodine, or 2-aminoethyldiphenylborate and inhibition of protein kinase A (PKA) or protein kinase C (PKC) significantly reduced GLP-1 potentiation of GSIS by Ca(v)1.3/DHPi channels and by endogenous L-type channels in INS-1 cells, but not by Ca(v)1.2/DHPi channels. Inhibition of glucose-stimulated phospholipase C activity with 1-(6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122) did not inhibit potentiation of GSIS by GLP-1 in INS-1 cells. In contrast, wortmannin, an inhibitor of phosphatidylinositol 3-kinase, and 2'-amino-3'-methoxyflavone (PD98059), an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase, both markedly inhibited GLP-1 potentiation of GSIS by endogenous channels in INS-1 cells and Ca(v)1.3/DHPi channels, but not by Ca(v)1.2/DHPi channels. Thus, Ca(v)1.3 is preferentially coupled to GLP-1 potentiation of GSIS in INS-1 cells via a mechanism that requires intact intracellular Ca(2+) stores, PKA and PKC activity, and activation of ERK1/2.
肠促胰岛素肽、葡萄糖依赖性促胰岛素多肽（GIP）和胰高血糖素样肽-1（GLP-1）可增强葡萄糖刺激的胰岛素分泌（GSIS）以及β细胞的增殖和分化。GLP-1和GIP增强GSIS需要通过电压门控L型Ca(2+)通道的Ca(2+)内流。我们研究了L型Ca(2+)通道Ca(v)1.2和Ca(v)1.3在介导GLP-1和GIP刺激INS-1细胞及表达Ca(v)1.2或Ca(v)1.3二氢吡啶不敏感（DHPi）突变体的INS-1细胞系中的事件中的作用。与未转染的INS-1细胞相比，Ca(v)1.3/DHPi通道支持GLP-1（50 nM）对GSIS的完全增强作用。然而，与未转染的INS-1细胞相比，由Ca(v)1.2/DHPi通道介导的GLP-1增强的GSIS明显降低。相反，Ca(v)1.2/DHPi和Ca(v)1.3/DHPi通道介导的GIP（10 nM）对GSIS的增强作用与未转染的INS-1细胞中观察到的相似。用毒胡萝卜素、ryanodine或2-氨基乙基二苯基硼酸盐破坏细胞内Ca(2+)释放以及抑制蛋白激酶A（PKA）或蛋白激酶C（PKC）可显著降低INS-1细胞中Ca(v)1.3/DHPi通道和内源性L型通道介导的GLP-1对GSIS的增强作用，但不影响Ca(v)1.2/DHPi通道。用1-(6-((17β-3-甲氧基雌甾-1,3,5(10)-三烯-17-基)氨基)己基)-1H-吡咯-2,5-二酮（U73122）抑制葡萄糖刺激的磷脂酶C活性并不抑制INS-1细胞中GLP-1对GSIS的增强作用。相反，磷脂酰肌醇3-激酶抑制剂渥曼青霉素和丝裂原活化蛋白激酶/细胞外信号调节激酶（ERK）激酶抑制剂2'-氨基-3'-甲氧基黄酮（PD98059）均显著抑制INS-1细胞内源性通道和Ca(v)1.3/DHPi通道介导的GLP-1对GSIS的增强作用，但不影响Ca(v)1.2/DHPi通道。因此，在INS-1细胞中，Ca(v)1.3通过一种需要完整细胞内Ca(2+)储存、PKA和PKC活性以及ERK1/2激活的机制优先与GLP-1增强GSIS相关联。
