Evans M G, Marty A
Proc Natl Acad Sci U S A. 1986 Jun;83(11):4099-103. doi: 10.1073/pnas.83.11.4099.
Ca2+-dependent K+ and Cl- currents were recorded in isolated and dialyzed rat lacrimal gland cells by use of the tight-seal whole-cell recording technique. Under control conditions, application of acetylcholine (0.5-1.0 microM) resulted in the full activation of both types of current. When 50-200 microM guanosine 5'-[gamma-thio]triphosphate (GTP[S], a nonhydrolyzable GTP analogue) was added to the intracellular solution, activation of both currents was seen with 1 nM acetylcholine, a dose 1/100th that needed under control conditions. Dialysis with solutions containing 200 microM GTP or cAMP had no, or only slight, potentiation effects. The effects of GTP[S] were obtained only when ATP was included in the intracellular solution. The potentiated responses to acetylcholine were blocked by increasing 10-fold the intracellular Ca2+-buffering capacity and were not dependent on external Ca2+. Thus, the potentiated responses appeared to result from a release of Ca2+ from internal stores. GTP[S] also greatly potentiated the Ca2+-dependent adrenergic (norepinephrine) response of this preparation. In addition, GTP[S] elicited in some cells transient responses without application of acetylcholine or norepinephrine. Finally, rapid and sustained responses were seen as soon as the cells were dialyzed with inositol trisphosphate (20 microM). These findings are discussed in terms of a possible role of a GTP-binding protein as a link between activation of muscarinic or adrenergic receptors and initiation of Ca2+ release by inositol trisphosphate.
采用全细胞封接记录技术，在分离并经透析处理的大鼠泪腺细胞中记录了Ca2+依赖性K+电流和Cl-电流。在对照条件下，施加乙酰胆碱（0.5 - 1.0微摩尔）可使两种类型的电流完全激活。当向细胞内溶液中加入50 - 200微摩尔鸟苷5'-[γ-硫代]三磷酸（GTP[S]，一种不可水解的GTP类似物）时，1纳摩尔乙酰胆碱即可激活两种电流，该剂量仅为对照条件下所需剂量的1/100。用含有200微摩尔GTP或cAMP的溶液进行透析，未产生或仅产生轻微的增强作用。仅当细胞内溶液中含有ATP时，GTP[S]才会产生作用。对乙酰胆碱增强的反应可通过将细胞内Ca2+缓冲能力提高10倍来阻断，且不依赖于细胞外Ca2+。因此，增强的反应似乎是由细胞内储存库释放Ca2+所致。GTP[S]也极大地增强了该制剂中Ca2+依赖性肾上腺素能（去甲肾上腺素）反应。此外，在一些细胞中，GTP[S]在未施加乙酰胆碱或去甲肾上腺素的情况下引发了瞬时反应。最后，一旦用三磷酸肌醇（20微摩尔）对细胞进行透析，即可观察到快速且持续的反应。本文根据GTP结合蛋白在毒蕈碱或肾上腺素能受体激活与三磷酸肌醇引发Ca2+释放之间可能起的连接作用，对这些发现进行了讨论。
