Masuda H, Kaneko M, Hong R B, Ikegaya T, Hayashi H, Kobayashi A, Yamazaki N
Third Department of Internal Medicine, Hamamatsu University School of Medicine, Japan.
Jpn Circ J. 1993 Oct;57(10):1007-15. doi: 10.1253/jcj.57.1007.
This study was undertaken to examine the effects of hydrogen peroxide on stimulatory guanine nucleotide-binding protein (Gs), and coupling in the beta-adrenergic receptor-Gs-adenylate cyclase system in rat heart, in vitro. Cardiac membranes were preincubated with various concentrations (0.1, 1, and 10 mM) of hydrogen peroxide at 30 degrees C for 5, 10, 30 and 60 min. Although the assay of beta-adrenergic receptors involving [3H]-dihydroalprenolol ([3H]-DHA) binding revealed that the maximal number of binding sites (Bmax) was not altered, the dissociation constant (Kd) for [3H]-DHA was increased in the presence of 1 mM and 10 mM hydrogen peroxide (control 0.68 +/- 0.16 nM, vs 1 mM H2O2 1.13 +/- 0.16, 10 mM H2O2 1.01 +/- 0.12). Conversely, no significant changes in Gs activities were observed in hydrogen peroxide-treated groups. Adenylate cyclase activity (stimulated by forskolin) was significantly reduced by 10 mM hydrogen peroxide after a 5 min preincubation period (control 277.1 +/- 19.2 pmol cAMP/mg protein/min, H2O2 230.3 +/- 14.9). The amounts of cyclic AMP produced by the stimulation of membranes with GTP, GTP+(l)-isoproterenol, guanylimidodiphosphate (Gpp(NH)p) or Gpp(NH)p+(l)-isoproterenol were significantly lower in 10 mM hydrogen peroxide-treated groups than those in controls (GTP: control 57.6 +/- 5.6 pmol cAMP/mg protein/min vs H2O2 46.4 +/- 6.9, GTP+(l)-isoproterenol: control 83.9 +/- 10.2 vs H2O2 67.7 +/- 10.3, Gpp(NH)p: control 77.5 +/- 8.8 vs H2O2 61.0 +/- 8.6, Gpp(NH)p+(l)-isoproterenol: control 105.0 +/- 13.1 vs H2O2 83.9 +/- 12.2, forskolin: control 223.2 +/- 13.8 vs H2O2 182.8 +/- 18.4).(ABSTRACT TRUNCATED AT 250 WORDS)
本研究旨在体外检测过氧化氢对大鼠心脏β-肾上腺素能受体-Gs-腺苷酸环化酶系统中刺激性鸟嘌呤核苷酸结合蛋白（Gs）及其偶联的影响。心脏膜在30℃下用不同浓度（0.1、1和10 mM）的过氧化氢预孵育5、10、30和60分钟。尽管涉及[3H]-二氢阿普洛尔（[3H]-DHA）结合的β-肾上腺素能受体测定显示最大结合位点数（Bmax）未改变，但在1 mM和10 mM过氧化氢存在下，[3H]-DHA的解离常数（Kd）增加（对照组0.68±0.16 nM，1 mM H2O2为1.13±0.16，10 mM H2O2为1.01±0.12）。相反，在过氧化氢处理组中未观察到Gs活性有显著变化。在5分钟预孵育期后，10 mM过氧化氢显著降低了由福斯高林刺激的腺苷酸环化酶活性（对照组277.1±19.2 pmol cAMP/mg蛋白/分钟，H2O2为230.3±14.9）。在10 mM过氧化氢处理组中，用GTP、GTP +（l）-异丙肾上腺素、鸟苷亚氨二磷酸（Gpp(NH)p）或Gpp(NH)p +（l）-异丙肾上腺素刺激膜产生的环磷酸腺苷量显著低于对照组（GTP：对照组57.6±5.6 pmol cAMP/mg蛋白/分钟，H2O2为46.4±6.9；GTP +（l）-异丙肾上腺素：对照组83.9±10.2，H2O2为67.7±10.3；Gpp(NH)p：对照组77.5±8.8，H2O2为61.0±8.6；Gpp(NH)p +（l）-异丙肾上腺素：对照组105.0±13.1，H2O2为83.9±12.2；福斯高林：对照组223.2±13.8，H2O2为182.8±18.4）。（摘要截断于250字）
