Pratt Evan P S, Salyer Amy E, Guerra Marcy L, Hockerman Gregory H
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN, USA; Purdue University Life Sciences Graduate Program, Purdue University, West Lafayette, IN, USA.
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN, USA.
Mol Cell Endocrinol. 2016 Jan 5;419:60-71. doi: 10.1016/j.mce.2015.09.034. Epub 2015 Oct 3.
We previously reported that INS-1 cells expressing the intracellular II-III loop of the L-type Ca(2+) channel Cav1.2 (Cav1.2/II-III cells) are deficient in Ca(2+)-induced Ca(2+) release (CICR). Here we show that glucose-stimulated ERK 1/2 phosphorylation (GSEP) is slowed and reduced in Cav1.2/II-III cells compared to INS-1 cells. This parallels a decrease in glucose-stimulated cAMP accumulation (GS-cAMP) in Cav1.2/II-III cells. Influx of Ca(2+) via L-type Ca(2+) channels and CICR play roles in both GSEP and GS-cAMP in INS-1 cells since both are inhibited by nicardipine or ryanodine. Further, the Epac1-selective inhibitor CE3F4 abolishes glucose-stimulated ERK activation in INS-1 cells, as measured using the FRET-based sensor EKAR. The non-selective Epac antagonist ESI-09 but not the Epac2-selective antagonist ESI-05 nor the PKA antagonist Rp-cAMPs inhibits GSEP in both INS-1 and Cav1.2/II-III cells. We conclude that L-type Ca(2+) channel-dependent cAMP accumulation, that's amplified by CICR, activates Epac1 and drives GSEP in INS-1 cells.
我们之前报道过，表达L型钙通道Cav1.2细胞内II-III环的INS-1细胞（Cav1.2/II-III细胞）存在钙诱导钙释放（CICR）缺陷。在此我们表明，与INS-1细胞相比，Cav1.2/II-III细胞中葡萄糖刺激的ERK 1/2磷酸化（GSEP）减慢且减少。这与Cav1.2/II-III细胞中葡萄糖刺激的cAMP积累（GS-cAMP）减少相平行。在INS-1细胞中，通过L型钙通道的Ca(2+)内流和CICR在GSEP和GS-cAMP中均起作用，因为二者均被尼卡地平或ryanodine抑制。此外，使用基于荧光共振能量转移（FRET）的传感器EKAR测量发现，Epac1选择性抑制剂CE3F4消除了INS-1细胞中葡萄糖刺激的ERK激活。非选择性Epac拮抗剂ESI-09而非Epac2选择性拮抗剂ESI-05或PKA拮抗剂Rp-cAMPs抑制了INS-1和Cav1.2/II-III细胞中的GSEP。我们得出结论，L型钙通道依赖性cAMP积累通过CICR放大，激活Epac1并驱动INS-1细胞中的GSEP。
