Abstract
This study was undertaken to examine the effects of hydrogen peroxide on stimulatory guanine nucleotide-binding protein (Gs), and coupling in the β-aderenergic 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°C for 5, 10, 30 and 60 min. Although the assay of β-adrenergic receptors involving [3H]-dihydroaloprenolol ([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 I mM and 10 mM hydrogen peroxide (control 0.68±0.16 nM, vs 1 mM H202 1.13±0.16, 10 mM H202 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, H202 230.3±14.9). The amounts of cyclic AMP produced by the stimulation of membranes with GTP, GTP + (1)-isoproterenol, guanylimidodiphosphate (Gpp(NH)p) or Gpp(NH)p+(1)-isoproterenol were significantly lower in 10 mM hydrogen peroxide-treated groups than those in controls (GTP: control 57.6±5.6pmol cAMP/mg protein/min vs H2O2 46.4±6.9, GTP+(1)-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+(1)-isoproterenol: control 105.0±13.1 vs H202 83.9±12.2, forskolin: control 223.2±13.8 vs H202 182.8±18.4). In the presence of hydrogen peroxide, the extents of the depression in cAMP production induced by GTP. GTP+isoproterenol, Gpp(NH)p, and Gpp(NH)p+ (1)-isoproterenol were similar to those induced by forskolin stimulation. These results indicate that hydrogen peroxide does not affect Gs activity or coupling in the p-receptor - Gs - adenylate cyclase system, but does depress CAMP production by inhibiting adenylate cyclase.
