Ohtani Masahiro, Ohura Kiyoshi, Oka Takami
Department of Pharmacology, Osaka Dental University, Hirakata, Japan.
Cell Physiol Biochem. 2011;28(2):355-66. doi: 10.1159/000331752. Epub 2011 Aug 16.
In order to clarify the functional role of ionotropic purinergic (P2X) receptors in pancreatic β-cells, we examined the effect of several P2 receptor agonists and antagonists on insulin secretion by mouse pancreatic islets, mouse Beta-TC6 cell proliferation and survival of dispersed islet cells in culture. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed the expression of mRNAs of P2X(4) receptor in mouse islets and P2X(1), P2X(2), P2X(3), P2X(4), P2X(5) and P2X(7) receptors in Beta-TC6 cells. The presence of P2X(4) receptor proteins in islets and Beta-TC6 cells was confirmed by immunofluorescent staining and Western blot analysis. We have previously found that the functional P2Y(1) receptor but not P2Y(2) and P2Y(4) receptors was present in islets. In this study we found that a nonspecific P2 receptor agonist, ATP (1 μM) stimulated insulin secretion by islets in the presence of high glucose (20 mM) in culture. The effect of ATP was partially inhibited by a P2 receptor antagonist PPADS as well as a P2Y(1) receptor antagonist MRS2179. In addition, a P2X(4) receptor potentiator ivermectin per se augmented glucose-induced insulin secretion and slightly potentiated the effect of ATP. These results suggested the involvement of P2Y(1)and P2X receptors. We also found that ATP inhibited proliferation of Beta-TC6 cells in a concentration-dependent manner during 72 h culture. The inhibitory effect of ATP was completely reversed by PPADS and partially by treating cells with small interfering RNA targeted for P2X(4) receptor mRNA. Furthermore, we found that the phosphorylation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) was suppressed by treatment with ATP in Beta-TC6 cells. In addition, we found that ATP reduced the cell viability and DNA synthesis of islet cells in culture. The effect of ATP on the cell viability was blocked by PPADS or MRS2179. These results suggested that P2X receptors as well as the P2Y(1) receptor played a role in the modulation of insulin secretion, proliferation and cell viability in mouse pancreatic β-cells.
为阐明离子型嘌呤能（P2X）受体在胰腺β细胞中的功能作用，我们研究了几种P2受体激动剂和拮抗剂对小鼠胰岛胰岛素分泌、小鼠β-TC6细胞增殖以及培养的分散胰岛细胞存活的影响。逆转录-聚合酶链反应（RT-PCR）分析显示，P2X(4)受体的mRNA在小鼠胰岛中表达，而P2X(1)、P2X(2)、P2X(3)、P2X(4)、P2X(5)和P2X(7)受体的mRNA在β-TC6细胞中表达。免疫荧光染色和蛋白质印迹分析证实了胰岛和β-TC6细胞中存在P2X(4)受体蛋白。我们之前发现胰岛中存在功能性P2Y(1)受体，但不存在P2Y(2)和P2Y(4)受体。在本研究中，我们发现非特异性P2受体激动剂ATP（1 μM）在培养物中高葡萄糖（20 mM）存在的情况下刺激胰岛分泌胰岛素。ATP的作用被P2受体拮抗剂PPADS以及P2Y(1)受体拮抗剂MRS2179部分抑制。此外，P2X(4)受体增强剂伊维菌素本身可增强葡萄糖诱导的胰岛素分泌，并略微增强ATP的作用。这些结果提示P2Y(1)和P2X受体参与其中。我们还发现，在72小时培养期间，ATP以浓度依赖性方式抑制β-TC6细胞的增殖。ATP的抑制作用被PPADS完全逆转，并用针对P2X(4)受体mRNA的小干扰RNA处理细胞可部分逆转。此外，我们发现用ATP处理β-TC6细胞可抑制细胞外信号调节激酶1和2（ERK1/2）的磷酸化。另外，我们发现ATP降低了培养的胰岛细胞的活力和DNA合成。ATP对细胞活力的作用被PPADS或MRS2179阻断。这些结果表明，P2X受体以及P2Y(1)受体在调节小鼠胰腺β细胞的胰岛素分泌、增殖和细胞活力方面发挥了作用。
