Verma V, Carter C, Keable S, Bennett D, Thorn P
Department of Pharmacology, Cambridge University, U.K.
Biochem J. 1996 Oct 15;319 ( Pt 2)(Pt 2):449-54. doi: 10.1042/bj3190449.
Reverse transcription-PCR (RT-PCR) techniques were used to identify the expression of ryanodine receptor (RyR) isoforms in gut epithelial cells. Restriction digest and sequence analysis of the PCR product showed the presence of RyR 2 and RyR 3. [3H]Ry binding studies on a microsome preparation, in a high-salt buffer, showed specific binding with an EC50 of 15 microM. In order to determine a potential functional role for these RyRs, we first characterized the response of the cells to acetylcholine. At all concentrations used acetylcholine induced sinusoidal cytosolic Ca2+ concentration ([Ca2+]i) oscillations. In response to 10(-4) M acetylcholine, levels of inositol 1,4,5-trisphosphate (InsP3) showed a peak of six times the basal level, at 30 s after stimulation. Application of caffeine alone failed to elicit a rise in cytosolic Ca2+. However, caffeine (5-50 mM) did rapidly and reversibly inhibit the acetylcholine-induced [Ca2+]i oscillations. The effects of Ry were more complex. Applied alone, Ry had no effect on the [Ca2+]i signal. When applied during agonist-evoked [Ca2+]i oscillations, Ry (10 microM) slowly blocked the response. In the continuous presence of Ry (10 microM) a short application of acetylcholine elicited a [Ca2+]i response that continued as oscillations even when the agonist was removed. The oscillations, in the presence of Ry (10 microM) but absence of agonist, were blocked either by removal of extracellular Ca2+ or by an application of a higher concentration of Ry (100 microM). These effects are consistent with the known use-dependence and dose-dependence for Ry action at the RyR. We conclude that the RyR 2 and RyR 3, identified by RT-PCR, play a central role in [Ca2+]i oscillations in gut epithelial cells.
采用逆转录聚合酶链反应（RT-PCR）技术鉴定肠道上皮细胞中兰尼碱受体（RyR）亚型的表达。对PCR产物进行限制性酶切和序列分析，结果显示存在RyR 2和RyR 3。在高盐缓冲液中对微粒体制剂进行的[3H]Ry结合研究表明，特异性结合的半数有效浓度（EC50）为15微摩尔。为了确定这些RyR的潜在功能作用，我们首先对细胞对乙酰胆碱的反应进行了表征。在所有使用的浓度下，乙酰胆碱均诱导正弦状胞质钙离子浓度（[Ca2+]i）振荡。在10^(-4)M乙酰胆碱刺激下，肌醇1,4,5-三磷酸（InsP3）水平在刺激后30秒达到基础水平的6倍峰值。单独应用咖啡因未能引起胞质钙离子升高。然而，咖啡因（5 - 50毫摩尔）确实能快速且可逆地抑制乙酰胆碱诱导的[Ca2+]i振荡。Ry的作用更为复杂。单独应用时，Ry对[Ca2+]i信号无影响。当在激动剂诱发的[Ca2+]i振荡期间应用时，Ry（10微摩尔）会缓慢阻断反应。在持续存在Ry（10微摩尔）的情况下，短暂应用乙酰胆碱会引发[Ca2+]i反应，即使去除激动剂，该反应仍会以振荡形式持续。在存在Ry（10微摩尔）但不存在激动剂的情况下，振荡可通过去除细胞外钙离子或应用更高浓度的Ry（100微摩尔）来阻断。这些效应与RyR上Ry作用的已知使用依赖性和剂量依赖性一致。我们得出结论，通过RT-PCR鉴定的RyR 2和RyR 3在肠道上皮细胞的[Ca2+]i振荡中起核心作用。
