INTRODUCTION

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SEVERAL STUDIES have reported an increase in the formation of H₂O₂ under different pathological conditions, including ischemia-reperfusion in the heart (15, 16, 29). Such an increase in the production of this active species of oxygen has been shown to be due to dismutation of the superoxide radicals, generation in the mitochondria, and activation of the leukocytes (15, 16, 26). In fact, H₂O₂ has been suggested to be involved in promoting cardiac dysfunction and injury by inducing intracellular Ca²⁺ overload due to modification of the sarcolemmal and sarcoplasmic reticular Ca²⁺-handling mechanisms (8, 10-12, 26). Because the -adrenoceptor, G proteins, and adenylyl cyclase system are known to regulate the intracellular concentration of Ca²⁺ and cardiac function (2, 3, 30), it is likely that this signal transduction pathway may be affected by H₂O₂. In this regard, some investigators have shown a decrease in the affinity without any change in the density of -adrenoceptors (13, 18), whereas others have reported both an increase and a decrease in the density of -adrenoceptors when cardiac membranes were treated with H₂O₂ (7). Likewise, H₂O₂ was observed to decrease and increase the adenylyl cyclase activities in cardiac membranes (18) and vascular smooth muscle cells (31), respectively. Although H₂O₂ has been reported to exert no effect on G proteins in cardiac membranes and vascular smooth muscle cells (18, 31), the results are of a preliminary nature. From the conflicting and inconclusive results regarding the effects of H₂O₂ on the -adrenoceptor, G proteins, and adenylyl cyclase system as well as the lack of information concerning the action of H₂O₂ on cardiac ₁- and ₂-adrenoceptors, this study was undertaken to provide detailed information concerning changes in the -adrenoceptor linked signal transduction mechanism due to H₂O₂. For this purpose, alterations in ₁- and ₂-adrenoceptors, adenylyl cyclase activities, and G_i as well as G_s protein functions were monitored when rat heart membranes were treated with H₂O₂.

	METHODS

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Treatment of membranes with H₂O₂. Cardiac membranes were prepared from the nonperfused rat heart according to the method described earlier (4). Unless otherwise indicated, aliquots of membrane were incubated with different concentrations of H₂O₂ for 10 min at 30°C. Membranes incubated without any additions served as controls. Catalase and mannitol, when used as scavengers, were at the concentration of 10 µg/ml and 20 mM, respectively. Membranes treated with or without H₂O₂ were thoroughly washed and resuspended in 50 mM tris(hydroxymethyl)aminomethane (Tris) · HCl (pH 7.4) before their use for different assays.

-Adrenergic receptor binding. To determine ₁- and ₂-adrenergic receptor binding (24), aliquots of control or H₂O₂-treated membrane preparations (0.1 mg/ml) were incubated for 60 min at 37°C with various concentrations (5-400 pM) of [¹²⁵I]iodocyanopindolol (¹²⁵I-CYP, 2,200 Ci/mmol) in the absence or presence of either 100 µM CGP-20712A (a highly selective ₁-antagonist) or 100 µM ICI-118551 (a highly selective ₂-antagonist). Incubations were stopped by rapid vacuum filtration through Whatman GF/C filters, and the radioactivity in the absence or presence of -adrenoceptor antagonists was measured to determine the total and nonspecific receptor binding. Specific binding to ₁-receptors was calculated as the difference between ¹²⁵I-CYP binding values in the absence (total binding) and presence (nonspecific binding) of CGP-20712A, whereas the ₂-receptor specific binding was the difference between ¹²⁵I-CYP binding values in the absence (total binding) and presence (nonspecific binding) of ICI-118551. The values for maximal binding (B_max) and dissociation constant (K_d) were calculated from the Scatchard plot analysis of the binding data according to the interactive LIGAND program of Munson and Rodbard (20).

Determination of adenylyl cyclase activity. Adenylyl cyclase activity was determined by measuring ³²P-labeled adenosine 3',5'-cyclic monophosphate (cAMP) formation from [-³²P]ATP as described previously (24). Unless otherwise indicated the incubation assay medium contained 50 mM glycylglycine (pH 7.5), 0.5 mM MgATP, [³²P]ATP (1-1.5 × 10⁶ counts/min), 5 mM MgCl₂ (in excess of the ATP concentration), 100 mM NaCl, 0.5 mM cAMP, 0.1 mM ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 0.5 mM 3-isobutyl-1-methylxanthine, 10 U/ml adenosine deaminase, and an ATP-regenerating system comprising 2 mM creatine phosphate and 0.1 mg creatine kinase/ml in a final volume of 200 µl. Incubations were initiated by the addition of membranes (30-70 µg) to the reaction mixture, which had been equilibrated for 3 min at 37°C. The incubation time was 10 min at 37°C, and the reaction was terminated by the addition of 0.6 ml of 120 mM zinc acetate containing 0.5 mM unlabeled cAMP. The unlabeled cAMP served to monitor recovery of [³²P]cAMP by measuring absorbance at 259 nm. The formation of cAMP was determined by coprecipitation of other nucleotides with ZnCO₃ by the addition of 0.5 ml of 144 mM Na₂CO₃ and subsequent chromatography by a double-column system as described by Salomon et al. (27). Under the assay conditions used, the adenylyl cyclase activity was linear with respect to protein concentration and time of incubation. To study the effects of pertussis toxin and cholera toxin on adenylyl cyclase activity (determination of the functional activities of G proteins), membrane preparations were treated with or without toxins for 60 min at 30°C in the same reaction mixture as that used for ADP ribosylation except that 10 mM NAD was used in place of [-³²P]NAD (24). The membranes were washed two to three times with Tris buffer and finally suspended in the same buffer for the estimation of adenylyl cyclase activity.

Toxin-catalyzed ADP ribosylation. Cholera toxin catalyzed- and pertussis toxin catalyzed-ADP ribosylation of G_s and G_i proteins, respectively, were performed according to the method described previously (24). In brief, 50 µg of the control and H₂O₂-treated membrane proteins were incubated for 60 min at 30°C in 100 µl of 100 mM Tris · HCl (pH 7.4) containing 1 mM EDTA, 1 mM EGTA, 5 mM MgCl₂, 1 mM ATP, 0.1 mM GTP, 10 mM thymidine, 2 µM [³²P]NAD (2 Ci/mmol), and activated pertussis toxin (5 µg/ml) for the determination of G_i protein activity. The G_s protein activity was assayed in an analogous fashion; the membrane protein was incubated for 90 min at 30°C in 100 mM Tris · HCl (pH 7.4) containing 1 mM EDTA, 1 mM EGTA, 5 mM MgCl₂, 1 mM ATP, 10 mM thymidine, 0.1 mM GTP, 10 mM arginine, 1 mM NADP⁺, 2 µM [³²P]NAD (20 Ci/mmol), and activated cholera toxin (20 µg/ml). Both toxins were activated by incubation in 50 mM dithiothreitol for 30 min at 30°C. The samples were applied to a 12% sodium dodecyl sulfate-polyacrylamide gel according to the method of Laemmli (17). The gels were dried and subjected to autoradiography using Kodak X-AR5 film at 70°C for 24-72 h. An imaging densitometer (Bio-Rad Laboratories, Mississauga, Canada) was used to quantitate the G_s and G_i proteins in control and experimental preparations.

Electrophoresis and immunoblot assay. The G_s and G_i proteins were quantified by an immunoblotting method described earlier (24). Control and H₂O₂-treated membranes were suspended in 50 µl H₂O and 50 µl sample buffer [6.4 M urea, 17 mM Tris · HCl, 19.5 mM glycine (pH 8.6), 10 mM dithiothreitol, 10 mM EGTA, 1 mM EDTA, 5 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 0.4% bromphenol blue] and then denatured by boiling for 3 min. The proteins were resolved in sodium dodecyl sulfate-12% polyacrylamide gel (17) and then electroblotted to nitrocellulose sheets by employing a 25 mM Na₂HPO₄ transfer buffer. After transfer, the nitrocellulose sheets were shaken for ~2 h in blocking buffer, which contained 10 mM Tris-buffered saline (TBS), 5% fat-free powdered milk, and 0.1% Tween 20. The blots were then incubated at 4°C for 14 h with specific antisera [AS/7 specific for G_i protein and RM/1 specific for G_s protein (1:3,000)] in TBS and then washed twice for 10 min each with 0.1% Tween 20 and TBS alternately. The antigen-antibody complexes were detected by chemiluminescent detection, whereby the nitrocellulose sheets were dipped in luminol substrate solution (Amersham, Oakville, Canada). To visualize the bands, chemilumigrams were developed on Hyperfilm-ECL; normal exposure times ranged from 30 s to 1 min. The specific bands for G_i and G_s proteins in control and experimental preparations were quantified by using the Bio-Rad imaging densitometer previously indicated.

Statistical analysis of the data. All results are expressed as means ± SE and were analyzed statistically by using Student's t-test. In some experiments as indicated in the text, Duncan's post hoc test for multiple comparisons following F score on an analysis of variance was also used to determine the difference between mean values. P < 0.05 was considered to reflect a significant difference between the control and experimental preparations.

	RESULTS

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Adenylyl cyclase activity. The adenylyl cyclase activity in membrane preparations treated in vitro for 10 min with 1 mM H₂O₂ was decreased significantly compared with control (Table 1); this was the case whether the activity was measured in the absence (basal) or presence of various stimulants such as forskolin, NaF, or 5'-guanylylimidodiphosphate (Gpp(NH)p). However, when the results were expressed as percentage of values in the absence of stimulants, the activation of adenylyl cyclase by forskolin, NaF, or Gpp(NH)p was higher in the H₂O₂-treated preparations (Table 1). These alterations due to H₂O₂ were markedly attenuated in the presence of catalase plus mannitol (Table 1). To show whether the catalytic site of adenylyl cyclase is affected by 1 mM H₂O₂ treatment, the enzyme activity was measured in a medium containing 1 mM Mn²⁺ instead of Mg²⁺. The adenylyl activity under this experimental condition was also depressed in the H₂O₂-treated preparations in comparison to the control values (10.5 ± 1.5 vs. 43.0 ± 2.2 pmol cAMP · mg protein¹ · min¹; n = 4 for each observation). In another set of experiments, the effect of H₂O₂ on the cardiac adenylyl cyclase activity in the presence of different concentrations of isoproterenol was examined. The data in Fig. 1A show a marked reduction in the isoproterenol-stimulated adenylyl cyclase activity on treatment with 1 mM H₂O₂. However, the activation of adenylyl cyclase in the H₂O₂-treated preparations by different concentrations of isoproterenol (10¹⁰ to 10³ M) was greater than the respective control values (Fig. 1B). These results indicate that while the adenylyl cyclase activities in the absence or presence of different stimulants were depressed when membranes were treated with a high concentration (1 mM) of H₂O₂, the responsiveness of the enzyme to the stimulants was increased.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Effect of different concentrations of isoproterenol [isoproterenol] (1011 to 103 mM) on adenylyl cyclase activity in rat heart membranes treated with H2O2 (1 mM) or without (control) for 10 min at 30°C. Treatment of membranes with H2O2 was done before adenylyl cyclase activity was measured. Assay medium in this set of experiments contained 10 µM 5'-guanylylimidodiphosphate (Gpp(NH)p) and 0.3% ascorbic acid. Each value is mean ± SE of 6 different membrane preparations. A: absolute values of adenylyl cyclase activity. B: activation of adenylyl cyclase activity by isoproterenol, expressed as percentage of values in absence of isoproterenol. * Significantly different from control (P < 0.05).

View larger version (34K): [in this window] [in a new window]   Fig. 2.   Effect of different concentrations of H2O2 [H2O2] on isoproterenol-stimulated adenylyl cyclase activities in absence or presence of isoproterenol in rat heart membranes. Incubation with H2O2 was for 10 min at 30°C before the adenylyl cyclase assay; control preparations were incubated in absence of H2O2. Assay medium contained 10 µM Gpp(NH)p and 0.3% ascorbic acid; concentration of isoproterenol was 100 µM. Each value is mean ± SE of 6 separate membrane preparations. * Significantly different from control in presence of isoproterenol (P < 0.05). # Significantly different from control in absence of isoproterenol (P < 0.05).

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Effect of H2O2 in absence and presence of oxygen metabolite scavengers such as catalase (Cat) and D-mannitol (Man) on adenylyl cyclase activities in absence or presence of isoproterenol in rat heart membranes. Incubation of membranes with H2O2 (1 mM) with or without scavengers was for 10 min at 30°C before enzyme assay. Assay medium contained 10 µM Gpp(NH)p and 0.3% ascorbic acid. Concentrations of Cat and Man were 10 µg/ml and 20 mM, respectively, whereas that of isoproterenol was 100 µM. Each value is mean ± SE of 6 separate preparations. Data were analyzed statistically by using Duncan's post hoc test. * Significantly different from respective control value (P < 0.05). # Significantly different from respective value in H2O2 group (P < 0.05).

-Adrenergic receptors. To show whether -adrenergic receptors were altered on treatment with H₂O₂, the specific binding of ¹²⁵I-CYP to both ₁- and ₂-adrenoceptors was studied in cardiac membranes. Figure 4 shows the specific binding data for ₁-adrenoceptors as well as Scatchard plot analysis of ¹²⁵I-CYP binding to ₁-receptors in control and 1 mM H₂O₂-treated membranes. Both the density (B_max) and affinity (1/K_d) of the ₁-receptors were significantly reduced in the H₂O₂-treated membranes compared with the control values (Fig. 4 and Table 3). Although Scatchard plot analysis of the data on ¹²⁵I-CYP binding to ₂-adrenoceptors alsorevealed a depression in the density of this receptor subtype, an increase in the affinity of ₂-adrenoceptors was evident in the H₂O₂-treated membranes (Table 3). The presence of catalase alone or in combination with mannitol in the incubation mixture was able to greatly prevent the H₂O₂-induced alteration in the ₁- and ₂-adrenoceptors (Table 3). It should be noted that treatment of membranes with low concentrations of H₂O₂ (0.2 mM) for 10 min had no effect on the density or affinity of ₁- and ₂-adrenoceptors because these values (n = 2) were not different from the control values shown in Table 3.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Scatchard plot analysis of [125I]iodocyanopindolol (125I-CYP) binding in control () and H2O2 ()-treated rat heart membranes. Data represent typical experiment performed in triplicate. Inset: equilibrium specific binding of 125I-CYP with membranes using ICI-118551 (100 µM) from 5 or 6 separate preparations. * Significantly different from control (P < 0.05).

G protein activities and contents. Adenylyl cyclase activity due to changes in the G protein function was determined in the presence of cholera toxin, an activator of G_s protein, or pertussis toxin, an inhibitor of G_i protein, and the results are shown in Fig. 5. The data indicate that whereas the cholera toxin-induced increase in adenylyl cyclase was depressed in the 1 mM H₂O₂-treated membranes, the pertussis toxin-induced increase in the enzyme activity was unaffected. The adenylyl cyclase activities in the absence or presence of cholera toxin were initially increased and then decreased when membranes were treated with a low concentration (0.2 mM) of H₂O₂ for different time intervals, whereas the enzyme activities in the presence of pertussis toxin were unaltered (Table 4). It should be mentioned that cholera toxin-catalyzed ADP ribosylation and anti-G_s protein binding were seen at both 45- and 52-kDa bands (Fig. 6). Although the cholera toxin-catalyzed ADP ribosylation in the H₂O₂-treated preparations was depressed at both 45- and 52-kDa bands, G_s protein content (as determined by anti-G_s protein binding) showed an attenuation at the 45-kDa band without any change in the 52-kDa band. No changes in the pertussis toxin-catalyzed ADP ribosylation at 40-kDa and G_i protein content (as measured by anti-G_i protein binding at 40 kDa) were evident due to H₂O₂ treatment (Fig. 6).

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Effect of cholera toxin (A, 20 µg/ml) and pertussis toxin (B, 5 µg/ml) on adenylyl cyclase activity in control and H2O2-treated rat heart membranes. Membranes were treated with H2O2 for 10 min at 30°C before enzyme assay. Each value is mean ± SE of 4 separate preparations in each group. Data were analyzed statistically by using Duncan's post hoc test. * Significantly different from respective control value in absence of toxin (P < 0.05). # Significantly different from its respective value in presence of toxin (P < 0.05).

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Toxin-catalyzed ADP ribosylation and Gs protein immunoblots in control and H2O2 (1 mM)-treated rat heart membranes. A: immunoblots for cholera toxin-catalyzed ADP ribosylation and Gs protein from control and H2O2-treated membranes. In addition, bar graphs are shown for densitometric analysis of cholera toxin-catalyzed ADP ribosylation and Gs protein immunoblots at 45 and 52-kDa bands in control (filled bars) membranes and membranes treated with H2O2 (open bars). Concentration of cholera toxin was 20 µg/ml. B: immunoblots for pertussis toxin-catalyzed ADP ribosylation and Gi protein from control and H2O2-treated membranes. In addition, bar graphs are shown for densitometric analysis of pertussis toxin-catalyzed ADP ribosylation and Gi protein immunoblots at 40-kDa band in control membranes (filled bars) and membranes treated with H2O2 (open bars). Membranes were treated with or without 1 mM H2O2 for 10 min and thoroughly washed before exposure to toxins indicated. Each value is mean ± SE of 4 separate preparations. * Significantly different from control (P < 0.05).

	DISCUSSION

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Treatment of cardiac membranes with high concentrations of H₂O₂ (0.5-5 mM) produced a concentration-dependent depression of the isoproterenol-stimulated adenylyl cyclase activity. The depressant effect of 1 mM H₂O₂ on adenylyl cyclase activity was also evident in the presence of various concentrations of isoproterenol. Such a depression in the isoproterenol-stimulated adenylyl cyclase activity may be due to changes in -adrenoceptors when cardiac membranes were treated with H₂O₂ because a decrease in both the density and affinity (1/K_d) of ₁-adrenoceptors in cardiac membranes was observed on treatment with 1 mM H₂O₂. Although the density of ₂-adrenoceptors in cardiac membranes treated with H₂O₂ was also decreased, the affinity of this subtype of receptors was increased. Such effects of H₂O₂ on the density and affinity of ₂-adrenoceptors would be of no major significance because of the opposite changes that would be elicited in terms of stimulating the adenylyl cyclase activity with isoproterenol. Because adenylyl cyclase activity in the absence of isoproterenol was also depressed on treatment of membranes with H₂O₂, it can be argued that the observed decrease in isoproterenol-stimulated enzyme activity may be related to systems other than changes in -adrenoceptors. This explanation is supported by the fact that unaltered or even greater activation of the enzyme by isoproterenol was seen in all experimental conditions used for the treatment of membranes with H₂O₂. It is possible that changes in ₁-adrenoceptors due to H₂O₂ may not be of sufficient magnitude for attenuating the response of adenylyl cyclase to agonist when receptors are occupied with isoproterenol. Thus the depressed isoproterenol-stimulated adenylyl cyclase activity does not seem to be dependent on changes in -adrenoceptors. Nonetheless, the observed changes in the ₁- and ₂-adrenoceptors should not be due to any nonspecific action of H₂O₂ because Ca²⁺-channel antagonist binding in cardiac membranes was not affected by H₂O₂ treatment under similar conditions (14).

The evidence presented in this study indicates that alterations in both the catalytic activity of adenylyl cyclase and the function of G_s proteins may contribute to the observed depression in the isoproterenol-stimulated adenylyl cyclase activity in heart membranes on treatment with high concentrations of H₂O₂. In this regard, adenylyl cyclase activities in the absence (basal) and presence of forskolin, which is known to stimulate the catalytic subunit of the enzyme directly (1), were found to decrease when cardiac membranes were treated with 1 mM H₂O₂. Furthermore, the depressed adenylyl cyclase activity in H₂O₂-pretreated membranes, when measured in the presence of Mn²⁺ without Mg²⁺, also revealed the effect of H₂O₂ on the catalytic site of adenylyl cyclase. On the other hand, depression in adenylyl cyclase activities in the presence of both NaF and Gpp(NH)p, which are known to activate the enzyme through their interaction with G proteins (5), indicates changes in G proteins on treatment of cardiac membranes with H₂O₂. Because the activation of adenylyl cyclase by forskolin, NaF, or Gpp(NH)p in the H₂O₂-treated membranes was greater than that in the control preparations, it is possible that these responses are determined by changes in the ratio of G_s and G_i proteins on treatment with H₂O₂. A depression in the G_s protein function by high concentrations of H₂O₂ is suggested from our observations that adenylyl cyclase activity in the presence of cholera toxin, an activator of G_s proteins (24), was decreased by treatment of cardiac membranes with 1 mM H₂O₂. In addition, the cholera toxin-catalyzed ADP ribosylation at both 45- and 52-kDa bands of G_s proteins was also depressed by H₂O₂ treatment. The inability of some investigators (18, 31) to detect changes in cholera toxin-catalyzed ADP ribosylation when vascular smooth cells and cardiac membranes were treated with H₂O₂ may be due to differences in tissue specificity and experimental design used in these studies. Nonetheless, our results are supported by the fact that the immunoreactivity of the 45-kDa band to antibodies for G_s proteins was markedly depressed when cardiac membranes were treated with H₂O₂. This action of H₂O₂ on the anti-G_s protein binding at the 45-kDa band may be selective on this subunit because the decrease in the anti-G_s protein binding at 52-kDa band was not significant. In addition, the depressant effect of H₂O₂ on cholera toxin-catalyzed ADP ribosylation at the 52-kDa band was less than that seen at the 45-kDa band for G_s proteins in the H₂O₂-treated membranes. The specificity of the action of H₂O₂ on G_s protein functions is also evident from the results of this study, indicating no significant changes in adenylyl cyclase activity in the presence of pertussis toxin, an inhibitor of G_i protein (24), as well as in the pertussis toxin-catalyzed ADP ribosylation and binding of antibodies for G_i protein at the 40-kDa band when membranes were treated with H₂O_2. Thus it appears that a change occurs in the ratio of G_s and G_i proteins on treatment of membranes with H₂O₂ and that the site of action of H₂O₂ for reducing G_s protein functions may primarily be at the 45-kDa band of G_s protein.

Although high concentrations of H₂O₂ depressed the adenylyl cyclase activities in the absence or presence of isoproterenol, treatment of membranes with low concentrations of H₂O₂ (100 µM) for a period of 10 min was observed to augment the enzyme activities. An increase in the isoproterenol-stimulated adenylyl cyclase activity in H₂O₂ (100 µM)-treated vascular smooth muscle cells has also been reported in the literature (31). In fact, an increase followed by a decrease in adenylyl cyclase activity was seen when cardiac membranes were pretreated with a low concentration of H₂O₂ (200 µM) for different time intervals (5-30 min). A biphasic pattern of changes in adenylyl cyclase activities in the absence or presence of cholera toxin was also seen when cardiac membranes were treated with low concentrations of H₂O₂ (200 µM) for different intervals. Such a biphasic effect of H₂O₂ on the isoproterenol-stimulated adenylyl cyclase activities appears to depend on the concentration of H₂O₂ as well as time of treatment and may explain the conflicting results regarding the action of H₂O₂ on the -adrenoceptor-linked mechanisms (7, 13, 18, 31). Because the density and affinity of -adrenoceptors were not altered when cardiac membranes were treated with a 200 µM concentration of H₂O₂, it is unlikely that the increase in isoproterenol-stimulated adenylyl cyclase activity due to low concentrations of H₂O₂ is due to changes in -adrenoceptors per se. Because the unaltered or even greater activation of adenylyl cyclase by isoproterenol was seen during biphasic changes in the adenylyl cyclase activities in the presence or absence of isoproterenol, it appears that the observed effects of H₂O₂ on -adrenoceptor mechanisms are of a specific nature and are consistent with the view that these changes are elicited by the action of H₂O₂ on both catalytic subunit of adenylyl cyclase and G_s proteins in the membrane.

Although the exact mechanisms by which H₂O₂ may alter the -adrenoceptor, adenylyl cyclase, and G protein system are unclear, the nature of the modifications of ₁-adrenoceptors, G_s protein functions, and adenylyl cyclase activity may be suggestive of changes in different protein components of the -adrenoceptor complex. This view is supported by the fact that aldehydes (malondealdehyde and 4-hydroxyneonatal) formed during lipid peroxidation have been shown to influence the function of the -adrenoceptor-adenylyl cyclase system in the sarcolemma by reacting with NH₂ and/or SH groups of these components (6, 22). The adenylyl cyclase enzyme as well as -adrenergic receptors are known to possess SH groups in their active sites (21, 28), the modification of which may alter the characteristics of these proteins. It is also possible that the reduced ₁-adrenoceptors, G_s protein functions, and adenylyl cyclase activity due to H₂O₂ may be caused by the effect of H₂O₂-induced lipid peroxidation (15, 16) on the physical state of the membrane. This view is based on the fact that the transmembrane -adrenoceptor signal transduction has been observed to be depressed by a reduction in membrane fluidity (25), and lipid peroxidation has been reported to decrease the membrane fluidity (22). Irrespective of the exact mechanisms, the results regarding the inhibitory effect of high concentrations of H₂O₂ on the -adrenoceptor-G_s protein-adenylyl cyclase pathway may be of some pathophysiological significance because the concentrations of H₂O₂ used in this study are comparable to those reported to occur in the myocardium during ischemia-reperfusion injury (29, 32). Although the responses to catecholamines and the -adrenoceptor mechanisms are attenuated in the ischemia-reperfused hearts (9, 19, 24), the pattern of abnormalities in the -adrenoceptors, G proteins, and adenylyl cyclase system may not be exactly similar to that seen with H₂O₂ treatment. Such differences may be attributed to free radicals as well as active species of oxygen and oxidants other than H₂O₂, which may also be formed in the ischemic-reperfused myocardium (15, 16, 24). Nonetheless, scavenging of H₂O₂ by superoxide dismutase plus catalase was found to prevent the ischemia-reperfusion as well as xanthine plus xanthine oxidase-induced changes in the positive inotropic effect of isoproterenol and isoproterenol-stimulated adenylyl cyclase activity in the perfused rat heart preparations (23, 24).