Calcium influx mediated by the Escherichia coli heat-stable enterotoxin B (STB).

The heat-stable enterotoxin B (STB) of Escherichia coli is a 48-amino acid extracellular peptide that induces rapid fluid accumulation in animal intestinal models. Unlike other E. coli enterotoxins that elicit cAMP or cGMP responses in the gut [heat-labile toxin (LT) and heat-stable toxin A (STA), respectively], STB induces fluid loss by an undefined mechanism that is independent of cyclic nucleotide elevation. Here we studied the effects of STB on intracellular calcium concentration ([Ca2+]i), another known mediator of intestinal ion and fluid movement. Ca2+ and pH measurements were performed on different cell types including Madin-Darby canine kidney (MDCK), HT-29/C1 intestinal epithelial cells, and primary rat pituitary cells. Ca2+ and pH determinations were performed by simultaneous real-time fluorescence imaging at four emission wavelengths. This allowed dual imaging of the Ca(2+)- and pH-specific ratio dyes (indo-1 and SNARF-1, respectively). STB treatment induced a dose-dependent increase in [Ca2+]i with virtually no effect on internal pH in all of the cell types tested. STB-mediated [Ca2+]i elevation was not inhibited by drugs that block voltage-gated Ca2+ channels including nitrendipine, verapamil (L-type), omega-conotoxin (N-type), and Ni2+ (T-type). The increase in [Ca2+]i was dependent on a source of extracellular Ca2+ and was not affected by prior treatment of MDCK cells with thapsigargin or cyclopiazonic acid, agents that deplete and block internal Ca2+ stores. In contrast to these results, somatostatin and pertussis toxin pretreatment of MDCK cells completely blocked the STB-induced increase in [Ca2+]i. Taken together, these data suggest that STB opens a GTP-binding regulatory protein-linked receptor-operated Ca2+ channel in the plasma membrane. The nature of the STB-sensitive Ca2+ channel is presently under investigation.

大肠埃希菌（Escherichia coli）的热稳定肠毒素B（STB）是一种含48个氨基酸的胞外肽，可在动物肠道模型中诱导快速体液蓄积。与可在肠道内分别激活环腺苷酸（cAMP）、环鸟苷酸（cGMP）通路的其他大肠埃希菌肠毒素——不耐热肠毒素（LT）与耐热肠毒素A（STA）——不同，STB通过不依赖环核苷酸水平升高的未知机制诱导体液流失。本研究聚焦STB对细胞内钙离子浓度（[Ca2+]i）的调控作用，而[Ca2+]i是介导肠道离子与体液转运的另一关键信号分子。
研究针对多种细胞类型开展钙离子与pH值检测，涵盖马-达二氏犬肾（MDCK）细胞、HT-29/C1肠道上皮细胞以及原代大鼠垂体细胞。检测采用四发射波长同步实时荧光成像技术，可同时成像钙离子、pH特异性比率荧光染料（分别为Indo-1与SNARF-1）。
实验结果显示，STB处理可在所有受试细胞系中引发[Ca2+]i的剂量依赖性升高，而对细胞内pH值几乎无影响。可阻断电压门控钙离子通道的各类药物——包括尼群地平、维拉帕米（L型）、ω-芋螺毒素（N型）及Ni²+（T型）——均无法抑制STB介导的[Ca2+]i升高。该[Ca2+]i升高依赖于细胞外钙离子来源，预先用毒胡萝卜素（thapsigargin）或环匹阿尼酸（cyclopiazonic acid）处理MDCK细胞（二者可耗竭并阻断内钙库）对该过程无显著影响。
与之形成鲜明对比的是，预先采用生长抑素（somatostatin）或百日咳毒素（pertussis toxin）处理MDCK细胞，可完全阻断STB诱导的[Ca2+]i升高。综上，本研究数据表明STB可在细胞质膜上激活一类与GTP结合调节蛋白（GTP-binding regulatory protein）偶联的受体操纵性钙离子通道。目前，STB敏感性钙离子通道的具体分子机制仍在研究之中。