Hara N, Ichinose M, Sawada M, Maeno T
Department of Physiology, Shimane Medical University, Izumo, Japan.
Pflugers Arch. 1993 Apr;423(1-2):140-8. doi: 10.1007/BF00374971.
In mouse peritoneal macrophages, alpha 1-adrenoceptor stimulation evokes a Ca(2+)-dependent K+ current [Io(Adr)][Hara et al. (1991) Pflügers Arch 419:371-379]. The roles of D-myo-inositol 1,4,5-trisphosphate (InsP3) and a GTP-binding protein (G protein) in Io(Adr) were investigated with tight-seal whole-cell recordings and fura-2 fluorescence measurements. Intracellular injection of InsP3 (5-50 microM) evoked transient outward currents [Io(InsP3)] with or without damped oscillations in membrane currents at -40 mV. Dialysis with 0.2 mM guanosine 5'-[3-thio]triphosphate (GTP[gamma S], a poorly hydrolysable GTP analogue) at -40 mV activated oscillatory outward currents or a slowly developing steady current on which such oscillations were superimposed after a delay of 10-90 s. Io(InsP3) and the GTP[gamma S]-induced current [Io(GTP[gamma S])] were accompanied by an increase in conductance. Reversal potentials of both responses closely depended on the extracellular K+ concentration. Fura-2 measurements revealed that Io(InsP3) and Io(GTP[gamma S]) result from a rise in intracellular free Ca2+ concentration ([Ca2+]i). Removal of extracellular Ca2+ did not abolish Io(InsP3) and Io(GTP[gamma S]). Both were blocked by bath-applied charybdotoxin. Intracellular D-myo-inositol 1,3,4,5-tetrakisphosphate (InsP4, 50 microM) did not evoke any responses, whereas D-myo-inositol 2,4,5-trisphosphate [InsP3(2,4,5), 20 microM] elicited an outward current at -40 mV. Io(InsP3) was completely blocked by prior dialysis with the InsP3 receptor antagonist heparin (5 mg/ml). Inclusion of guanosine 5'-[2-thiol] diphosphate (GDP[beta S], 2 mM) or heparin (5 mg/ml) together with GTP[gamma S] in the patch pipette solution completely blocked Io(GTP[gamma S]). These results indicate that intracellular injection of InsP3 or GTP[gamma S] mimic Io(Adr).(ABSTRACT TRUNCATED AT 250 WORDS)
在小鼠腹腔巨噬细胞中，α1 - 肾上腺素能受体刺激可引发一种依赖钙离子的钾电流[Io(Adr)][原田等人（1991年）《普弗吕格尔氏文献》419:371 - 379]。利用全细胞膜片钳记录和fura - 2荧光测量技术，研究了D - 肌醇1,4,5 - 三磷酸（InsP3）和一种鸟苷酸结合蛋白（G蛋白）在Io(Adr)中的作用。在 - 40 mV时，向细胞内注射InsP3（5 - 50微摩尔）可引发瞬时外向电流[Io(InsP3)]，膜电流有或无衰减振荡。在 - 40 mV时，用0.2 mM鸟苷5'-[3 - 硫代]三磷酸（GTP[γS]，一种难水解的GTP类似物）进行透析，可激活振荡外向电流或缓慢发展的稳定电流，在延迟10 - 90秒后会叠加这种振荡。Io(InsP3)和GTP[γS]诱导的电流[Io(GTP[γS])]伴随着电导增加。两种反应的反转电位都密切依赖于细胞外钾离子浓度。Fura - 2测量显示，Io(InsP3)和Io(GTP[γS])是由细胞内游离钙离子浓度（[Ca2+]i）升高引起的。去除细胞外钙离子并没有消除Io(InsP3)和Io(GTP[γS])。两者都被浴槽施加的蝎毒素阻断。细胞内注射D - 肌醇1,3,4,5 - 四磷酸（InsP4，50微摩尔）未引发任何反应，而D - 肌醇2,4,5 - 三磷酸[InsP3(2,4,5)，20微摩尔]在 - 40 mV时引发外向电流。先用InsP3受体拮抗剂肝素（5毫克/毫升）进行透析可完全阻断Io(InsP3)。在膜片钳微电极溶液中加入鸟苷5'-[2 - 硫代]二磷酸（GDP[βS]，2 mM）或肝素（5毫克/毫升）与GTP[γS]一起，可完全阻断Io(GTP[γS])。这些结果表明，向细胞内注射InsP3或GTP[γS]可模拟Io(Adr)。（摘要截断于250字）
