Department of Physiology, College of Medicine, Konyang University, Daejeon 302-718, Korea.
Korean J Physiol Pharmacol. 2011 Feb;15(1):53-9. doi: 10.4196/kjpp.2011.15.1.53. Epub 2011 Feb 28.
The secretion of insulin from pancreatic β-cells is triggered by the influx of Ca(2+) through voltage-dependent Ca(2+) channels. The resulting elevation of intracellular calcium (Ca(2+)) triggers additional Ca(2+) release from internal stores. Less well understood are the mechanisms involved in Ca(2+) mobilization from internal stores after activation of Ca(2+) influx. The mobilization process is known as calcium-induced calcium release (CICR). In this study, our goal was to investigate the existence of and the role of caffeine-sensitive ryanodine receptors (RyRs) in a rat pancreatic β-cell line, INS-1 cells. To measure cytosolic and stored Ca(2+), respectively, cultured INS-1 cells were loaded with fura-2/AM or furaptra/AM. Ca(2+) was repetitively increased by caffeine stimulation in normal Ca(2+) buffer. However, peak Ca(2+) was only observed after the first caffeine stimulation in Ca(2+) free buffer and this increase was markedly blocked by ruthenium red, a RyR blocker. KCl-induced elevations in Ca(2+) were reduced by pretreatment with ruthenium red, as well as by depletion of internal Ca(2+) stores using cyclopiazonic acid (CPA) or caffeine. Caffeine-induced Ca(2+) mobilization ceased after the internal stores were depleted by carbamylcholine (CCh) or CPA. In permeabilized INS-1 cells, Ca(2+) release from internal stores was activated by caffeine, Ca(2+), or ryanodine. Furthermore, ruthenium red completely blocked the CICR response in permeabilized cells. RyRs were widely distributed throughout the intracellular compartment of INS-1 cells. These results suggest that caffeine-sensitive RyRs exist and modulate the CICR response from internal stores in INS-1 pancreatic β-cells.
胰岛β细胞的胰岛素分泌是由电压依赖性钙通道（voltage-dependent Ca(2+) channels）内流的 Ca(2+)触发的。由此导致的细胞内钙离子浓度升高(Ca(2+))会引发内部储存库的额外 Ca(2+)释放。但对于 Ca(2+)内流激活后内部储存库中 Ca(2+)动员的相关机制，我们的了解还不够充分。这个动员过程被称为钙诱导钙释放（calcium-induced calcium release，CICR）。在这项研究中，我们的目标是研究咖啡因敏感的兰尼碱受体（ryanodine receptors，RyRs）在大鼠胰岛β细胞系 INS-1 细胞中的存在和作用。为了分别测量胞质溶胶和储存的 Ca(2+)，我们用 fura-2/AM 或 furaptra/AM 负载培养的 INS-1 细胞。在正常 Ca(2+)缓冲液中，通过咖啡因刺激可反复增加胞质溶胶 Ca(2+)浓度。然而，在无 Ca(2+)缓冲液中，第一次咖啡因刺激后才观察到峰 Ca(2+)浓度增加，并且这种增加被 RyR 阻断剂钌红（ruthenium red）显著阻断。用钌红预处理、用细胞松弛素 D（cyclopiazonic acid，CPA）或咖啡因耗尽内部 Ca(2+)储存库，都可降低 KCl 诱导的 Ca(2+)浓度增加。在耗尽内部储存库后，细胞松弛素 D（carbachol，CCh）或 CPA 引起的咖啡因诱导的 Ca(2+)动员停止。在通透化的 INS-1 细胞中，咖啡因、Ca(2+)或兰尼碱（ryanodine）均可激活内部储存库的 Ca(2+)释放。此外，钌红完全阻断了通透化细胞中的 CICR 反应。RyRs 广泛分布于 INS-1 细胞的细胞内隔室。这些结果表明，INS-1 胰岛β细胞中存在咖啡因敏感的 RyRs，并且调节来自内部储存库的 CICR 反应。
