Introduction

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It is now well established that oxygen-free radicals (oxyradicals) are produced in the heart under different pathological conditions including ischemia-reperfusion (Arroyo et al., 1987; Gauduel and Duvellroy, 1984; Jolly et al., 1984; Shlafer et al., 1982; Zweier, 1988). Several investigators (Blaustein et al., 1986; Burton et al., 1984; Jackson et al., 1986) have reported that exogenous oxyradicals produce functional and structural abnormalities in the heart. Treatment of cardiac sarcoplasmic reticulum and sarcolemmal membranes has been shown to depress Ca⁺⁺-pump activities and these defects have been suggested to induce intracellular Ca⁺⁺-overload and subsequent heart dysfunction (Kukreja and Hess, 1992; Rowe et al., 1983; Okabe et al., 1983; Kaneko et al., 1989a). Depression in the sarcolemmal Na⁺-K⁺ ATPase and Na⁺-Ca⁺⁺ exchange activity on treatment of heart membranes with oxyradical generating systems has also been suggested to contribute towards the occurrence of intracellular Ca⁺⁺-overload (Shao et al., 1995; Hata et al., 1991). In fact, perfusion of the isolated hearts with X plus XO, a well-known oxyradical generating system, has been shown to depress both sarcolemmal Na⁺-Ca⁺⁺ exchange and Ca⁺⁺-pump activities during the development of contractile dysfunction (Matsubara and Dhalla, 1996a, 1996b). Although a decrease in the density of Ca⁺⁺-channels in the sarcolemmal membrane (Kaneko et al., 1989b) and a decrease in the density of Ca⁺⁺-release channels in the sarcoplasmic reticulum (Holmberg et al., 1991) due to oxyradicals can be seen to result in the reduction of Ca⁺⁺ available for cardiac contraction, the contribution of depressed Ca⁺⁺-stimulated ATPase activity upon exposing myofibrils to oxyradicals (Suzuki et al., 1991) in eliciting cardiac contractile abnormalities cannot be ruled out. Accordingly, it appears that heart dysfunction due to oxyradicals may be due to their effects on both Ca⁺⁺-handling by cardiomyocytes and the interaction of Ca⁺⁺ with contractile apparatus.

Because beta-adrenoceptor mechanisms including beta₁- and beta₂-adrenoceptors, guanine nucleotide binding proteins (G_s- and G_i-proteins) and adenylyl cyclase are known to affect the entry of Ca⁺⁺ in cardiomyocytes and thus play an important role in the regulation of heart function (Dhalla et al., 1982; Tsien, 1977), some investigators have examined the effects of different oxyradical generating systems on various components of this signal transduction pathway. For example, treatment of cardiac membranes with some oxyradical generating systems increased the density but decreased the affinity of beta-adrenoceptors (Kaneko et al., 1991) whereas treatment with H₂O₂, an active species of oxygen, decreased the affinity without any changes in the density of beta-adrenoceptors (Kaneko et al., 1991; Masuda et al., 1993). However, treatment of heart membranes with H₂O₂ was reported to increase the density of beta-adrenoceptors (Haenen et al., 1988) whereas a loss in the number of beta-adrenoceptors was seen upon treating cortical membranes with iron and ascorbic acid, a hydroxyl radical generating system (Heikkila, 1983). An increase or no change in the density of beta-adrenoceptors in ventricular membranes has also been observed on treatment of membranes with some oxidants (Haenen et al., 1989, 1990). It may be noted that a decrease in the adenylyl cyclase activity was found on treating heart membranes with H₂O₂ and other oxidants by some investigators (Masuda et al., 1993; Haenen et al., 1989; Haenen et al., 1990) whereas others (Tan et al., 1995) have reported an increase in the enzyme activity due to H₂O₂ in the vascular smooth muscle cells. A transient increase followed by a decrease in the adenylyl cyclase activity was observed on treating cardiac membranes with iron-ascorbic acid system (Schimke et al., 1992). Although preliminary experiments revealed no changes in G-protein functions in heart membranes (Masuda et al., 1993) and vascular smooth muscle cells (Tan et al., 1995), an extensive study in this regard is needed for making any meaningful conclusion. Furthermore, it is pointed out that no information regarding the effect of oxyradicals and oxidants on beta₁- or beta₂-adrenoceptor is available in the literature. Thus in view of the relatively little and scattered information as well as conflicting results regarding the effects of oxyradicals and oxidants on the beta-adrenoceptor signal transduction mechanism, our study was undertaken to examine in detail if any component of the beta-adrenoceptor pathway in the heart is affected by oxyradicals. For this purpose, X plus XO was used as an oxyradical generating system for treatment of rat cardiac membranes for different time intervals under in vitro conditions. The status of beta₁- and beta₂-adrenoceptors, adenylyl cyclase activities in the absence or presence of different stimulants, as well as the G_s- and G_i-protein functions in control and experimental membranes were examined to determine the site affected by X plus XO treatment.

	Methods

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In vitro treatment with X plus XO. To examine the effects of oxyradicals under in vitro conditions, rats were decapitated, hearts removed and the ventricular tissue used for membrane preparation (Dixon et al., 1990). Aliquots of membrane suspension were incubated for different time periods at 30°C with an oxyradical generating system consisting of X plus XO at the concentration of 2 mM and 0.03 U/ml, respectively. SOD, CAT and MAN, when used as scavengers, were at the concentrations of 80 µg/ml, 10 µg/ml and 20 mM, respectively. The selection of concentrations for X, XO, SOD, CAT and MAN in this study was based on our previous work with these agents (Hata et al., 1991; Kaneko et al., 1989a, 1989b; Shao et al., 1995; Suzuki et al., 1991). Membranes incubated without any addition for the appropriate time period served as controls. Membranes treated with X plus XO (in the presence or absence of SOD, CAT and MAN) were thoroughly washed and resuspended in 50 mM Tris-HCl (pH 7.4) before their use for various assays. In some experiments, cardiac membranes were treated with different concentrations of H₂O₂ (25-200 µM), washed and used for various assays.

Beta-adrenergic receptor binding. To determine beta₁- and beta₂-adrenoceptor binding, aliquots (0.1 mg/ml) of control or oxyradical treated membrane preparations were incubated for 60 min at 37°C with various concentrations (5-400 µM) of [¹²⁵I]-CYP (2200 Ci/mmol) in the presence or absence of either 100 µM CGP-20712A (a selective beta₁ antagonist) or 100 µM ICI-118,551 (a selective beta₂ antagonist). Incubations were stopped by rapid vacuum filtration through Whatman GF/C filters. Specific binding to beta₁-adrenoceptors was calculated as the difference between [¹²⁵I]-CYP binding values in the absence (total binding) and presence of ICI-118,551 (nonspecific binding) whereas beta₂-adrenoceptor specific binding was the difference between [¹²⁵I]-CYP binding values in the absence (total binding) and presence of CGP-20712A (nonspecific binding). The values for B_max and K_d were calculated from the Scatchard plot analysis of the data according to the interactive LIGAND program of Munson and Rodbard (1980).

Determination of adenylyl cyclase activity. Adenylyl cyclase activity was determined by measuring [³²P]-cAMP formation from [-³²P]-ATP as described previously (Sethi et al., 1994; Persad et al., 1997a). Unless otherwise indicated the incubation assay medium contained 50 mM glycylglycine (pH 7.5), 0.5 mM MgATP, [³²P]-ATP (1-1.5 × 10⁶ cpm), 5 mM MgCl₂ (in excess of the ATP concentration), 100 mM NaCl, 0.5 mM cAMP, 0.1 mM EGTA, 0.5 mM 3-isobutyl-1-methylxanthine, 10 U/ml adenosine deaminase and an ATP-regenerating system comprising of 2 mM creatinine phosphate, 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. Unlabeled cAMP served to monitor the recovery of [³²P]-cAMP by measuring absorbency at 259 nm. The determination of cAMP was carried out by coprecipitation of other nucleotides with ZnCO₃ upon the addition of 0.5 ml 144 mM Na₂CO₃ and subsequent chromatography by a double column system as described by others (Salmon et al., 1979). Under the assay conditions used, the adenylyl cyclase activity was linear with respect to protein concentration and time of incubation. For studying the effects of pertussis toxin and cholera toxin on the adenylyl cyclase activity for the determination of functional activities of G_i- and G_s-proteins, respectively, the 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 instead of [-³²P NAD]. 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, was performed according to the method described by previously (Sethi et al., 1994; Persad et al., 1997a). In brief, 50 µg of the control or X plus XO-treated membranes 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). G-protein substrates of cholera toxin were assayed in an analogous fashion; the membranes were 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). The reactions were stopped by addition of cold 20% TCA and pellets were resuspended in a buffer described by Laemmli (1970) and the samples were applied to a 12% SDS polyacrylamide gel according to the method of Laemmli (1970). The gels were dried and subjected to autoradiography using Kodak X-AR5 film at 70°C for 24 to 72 hr. An imaging densitometer (Bio-Rad Laboratories, Mississauga, Canada) was used to quantitate the G_s- and G_i-proteins in control and experimental preparations. Cholera toxin and pertussis toxin were activated by incubating in 50 mM dithiothreitol for 30 min at 30°C before use.

Immunoblot assays for G-proteins. The G_s- and G_i-proteins were quantified by an immunoblotting method described by Mumby et al. (1986). Control or X plus XO-treated membranes were suspended in 50 µl H₂O and 50 µl of the sample buffer described by Laemmli (1970) and then denatured by boiling for 3 min. The proteins were resolved on 12% SDS polyacrylamide gel (Laemmli, 1970), and then electroblotted to nitrocellulose sheets. After transfer, nitrocellulose sheets were shaken for approximately 2 hr in blocking buffer, which contained 10 mM TBS, 5% fat-free powdered milk and 0.1% Tween-20. The blots were then incubated at 4°C for 14 hr with specific antisera (AS/7 specific for G_i and RM/1 specific for G_s) (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 where the nitrocellulose sheets were dipped in luminol substrate solution. To visualize the bands, chemilumigrams were developed on Hyperfilm-ECL; normal exposure times ranged from 30 sec 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 (Bio-Rad Research Laboratories, Mississauga, Canada) as indicated above.

Statistical analysis of the data. The results were expressed as mean ± S.E. and the difference between the control and experimental preparations was analyzed statistically by using the Student's t test. When appropriate, Duncan's multiple-range test was used to determine the difference between mean values. P < .05 was taken to reflect a significant difference.

	Results

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Alterations in adenylyl cyclase activity. The adenylyl cyclase activities in the absence (basal) and presence of 10 µM Gpp(NH)p in membranes treated with X plus XO for different periods exhibited a biphasic pattern of changes whereby 10 min incubation increased and 30 min incubation decreased the enzyme activity compared with their respective control values (fig. 1). This was also the case when the enzyme activity was measured in the presence of 30 µM Gpp(NH)p or other stimulants of adenylyl cyclase such as 100 µM forskolin and 5 mM NaF (table 1). The presence of SOD plus CAT was able to prevent these biphasic alterations in basal as well as Gpp(NH)p-, forskolin- and NaF-stimulated adenylyl cyclase activities due to 10 and 30 min incubation with X plus XO by 85 to 90%. The inclusion of MAN did not increase the magnitude of the protection to the basal adenylyl cyclase activity afforded by SOD plus CAT. SOD in the absence of CAT did not show any protection against the X plus XO-induced changes in the enzyme activity. Analysis of the data revealed that the magnitude of increase or decrease in the adenylyl cyclase activity on treatment with X plus XO for 10 or 30 min did not differ appreciably with respect to differences in the concentrations of membrane proteins within the range of 30 to 70 µg for each assay.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Effect of different times of incubation with xanthine (2 mM) plus xanthine oxidase (0.03 U/ml) on the basal () and Gpp(NH)p (10 µM) stimulated () adenylyl cyclase activities in cardiac membranes. Control preparations were incubated in the absence of xanthine plus xanthine oxidase for different times at 30°C before the adenylyl cyclase assay. Although 10 to 15% depression in the enzyme activity was seen upon incubating the control membranes for 40 min, the changes were not significant (P > 0.05). Each value is a mean ± S.E. of 6 separate membrane preparations. *Significantly different from control (P < 0.05).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Effect of different concentrations of isoproterenol on the adenylyl cyclase activity in cardiac membranes treated with xanthine (X, 2 mM) plus xanthine oxidase (XO, 0.03 U/ml) for either a 10- or a 30-min period at 30°C. The assay medium in this set of experiments contained 10 µM Gpp(NH)p and 0.3% ascorbic acid. Each value is the mean ± S.E. of 6 different membrane preparations. *Significantly different from control (P < .05).

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Effect of xanthine (X, 2 mM) plus xanthine oxidase (XO, 0.03 U/ml) in the absence and presence of the scavengers such as superoxide dismutase (SOD) and catalase (CAT) in combination with or without mannitol (MAN) on the isoproterenol-stimulated adenylyl cyclase activity. Incubations of the rat heart membranes with xanthine (X) plus xanthine oxidase (XO) with or without scavengers were carried out for 10- and 30-min periods at 30°C. The assay medium contained 10 µM Gpp(NH)p and 0.3% ascorbic acid; the concentration of isoproterenol was 100 µM. The concentrations of SOD, CAT and MAN were 80 µg/ml, 10 µg/ml and 20 mM, respectively. Each value is the mean ± S.E. of four to six preparations. *Significantly different from control (P < .05); #Significantly different from X plus XO for its respective time of incubation (P < .05).

Alterations in beta-adrenergic receptors. To demonstrate the beta-adrenergic receptors were altered in cardiac membranes upon treatment with X plus XO for 10- or 30-min periods, the specific binding of ¹²⁵I-CYP to both beta₁-adrenoceptors and beta₂-adrenoceptors was measured. Figure 4 shows the specific binding data for beta₁-adrenoceptors as well as Scatchard plot analysis of ¹²⁵I-CYP binding to beta₁-adrenergic receptors in control and X plus XO- (10 and 30 min) treated hearts. Although the affinity (1/K_d) of beta₁-adrenoceptors was increased and the density was decreased after 10 min incubation with X plus XO, the affinity and density of these receptors after 30 min incubation were reduced significantly (fig. 4; table 4). Although the Scatchard plot analysis of data for ¹²⁵I-CYP binding with beta₂-adrenoceptors revealed an increase in the affinity and a depression in the density on 30 min treatment with X plus XO, the magnitude of the alterations was significantly smaller in comparison with that seen with beta₁-adrenoceptors. A 10-min treatment with X plus XO did not change either the affinity or the density of beta₂-adrenoceptors (table 4). The presence of SOD plus CAT in the incubation medium, prevented the X plus XO-induced alterations in the density of beta₁-adrenoceptors at 10- and 30-min incubations, as well as changes in both affinity and density of the beta₂-adrenoceptors at 30-min incubation, but was unable to affect the increase in the affinity of the beta₁-adrenoceptors at 10-min incubations (table 4).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Scatchard plot analysis of [125I]-CYP binding in control rat cardiac membranes () and membranes treated with xanthine (X) plus xanthine oxidase (XO) for 10 min () and 30 min () periods. Data represents a typical experiment performed in triplicate. Inset, Equilibrium specific binding of [125I]-CYP with membranes by using IC1-118,551 (100 µM) from 5-6 preparations. *Significantly different from control (P < .05). B/F, Bound/free [125I]-CYP (iodocyanopindolol).

Alterations in G-protein activities and anti G-protein binding. The G-protein-mediated activities of adenylyl cyclase were determined in the absence or presence of CT, an activator of G_s-proteins, and PT, an inhibitor of G_i-proteins, and the results are shown in figure 5. Although the CT-induced increase in adenylyl cyclase activity was enhanced in membranes incubated with X plus XO for 10 min, it was depressed on incubation of membranes for 30 min. However, the adenylyl cyclase activity in the presence of PT was not altered on treating cardiac membranes for 10 or 30 min with X plus XO (fig. 5). It should be noted that the CT-stimulated ADP-ribosylation of the G_s-proteins as well as anti-G_s protein binding were seen at 45- and 52-kDa bands (fig. 6). Although the CT-stimulated ADP-ribosylation activity at both 45- and 52-kDa bands as well as the anti G_s-protein binding at 52 kDa were increased in membranes incubated for 10 min with X plus XO, the anti-G_s protein binding at the 45-kDa band was depressed significantly. However, the CT-stimulated ADP-ribosylation activities as well as anti G_s-protein binding at both 45- and 52-kDa bands were depressed in the membranes treated for 30 min with X plus XO (fig. 6). Although PT-stimulated ADP-ribosylation and anti G_i-protein binding were seen at 40 kDa, no modification in the PT-stimulated ADP-ribosylation of the G_i-proteins or the anti G_i-protein binding was seen upon incubating the membranes with X plus XO for 10 and 30 min (fig. 7).

View larger version (44K): [in this window] [in a new window]   Fig. 5.   Effects of cholera toxin and pertussis toxin on the adenylyl cyclase activity in control (C) and xanthine (2 mM) plus xanthine oxidase- (0.03 U/ml) treated rat cardiac membranes. Membranes were treated with xanthine plus xanthine oxidase for either 10 min (X10) or 30 min (X30) periods at 30°C. Each value is a mean ± S.E. of four preparations in each group. *Significantly different from controls (P < .05). #Significantly different from its respective value in the presence of toxin (P < .05).

View larger version (41K): [in this window] [in a new window]   Fig. 6.   Cholera toxin catalyzed ADP-ribosylation and Gs-protein immunoblots in control (C) and xanthine (2 mM) plus xanthine oxidase- (0.03 U/ml) treated rat cardiac membranes. Lower panel shows bar graphs for the densitometric analysis of cholera toxin catalyzed ADP-ribosylation and Gs-protein immunoblots at 45- and 52-kDa bands in control membranes and membranes treated with xanthine plus xanthine oxidase for 10-min (X10) and 30-min (X30) periods. Upper panel shows immunoblots for the cholera toxin catalyzed ADP-ribosylation and Gs-protein from control and xanthine plus xanthine oxidase-treated membranes. Each value is the mean ± S.E. of four preparations. *Significantly different from control (P < .05). The concentration of cholera toxin was 20 µg/ml.

View larger version (28K): [in this window] [in a new window]   Fig. 7.   Pertussis toxin catalyzed ADP-ribosylation and Gi protein immunoblots at 40-kDa band in control (C) and xanthine (2 mM) plus xanthine oxidase- (0.03 U/ml) treated rat cardiac membranes. Lower panel shows bar graphs for the densitometric analysis of the pertussis toxin catalyzed ADP-ribosylation and Gi-protein immunoblots in control membranes and membranes treated with xanthine plus xanthine oxidase for 10-min (X10) and 30-min (X30) periods. Upper panel shows immunoblots for the pertussis toxin catalyzed ADP-ribosylation and Gi-protein from control and xanthine plus xanthine oxidase-treated membranes. Each value is the mean ± S.E. of four preparations. The concentration of pertussis toxin was 5 µg/ml.

	Discussion

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In this study treatment of cardiac membranes with X plus XO, a free radical generating system, revealed a time-dependent biphasic change in the basal activity of adenylyl cyclase, the effector enzyme involved in the beta-adrenoceptor signal transduction pathway. A dose-response of adenylyl cyclase to isoproterenol indicated an augmentation at 10 min and an attenuation on 30-min exposure of membranes to X plus XO. Because X or XO alone had no effect on the isoproterenol-stimulated adenylyl cyclase activity, the biphasic alterations observed in this study are likely to be due to metabolites generated by the interaction of X plus XO. Although Schimke et al. (1992) have observed time-dependent biphasic changes of the isoproterenol-stimulated adenylyl cyclase activity by using an iron-ascorbic acid system (maximum activity was seen in less than 30 sec but thereafter the activity declined), the results in our study, in which X plus XO was used, showed maximum activity at 10 min and minimal activity by 30 min. These differences in the time-course of biphasic changes may be due to the differences in the oxyradical generating systems used under the experimental conditions. Nonetheless, the observed increase in the affinity of beta₁-adrenoceptors at 10 min of incubation with X plus XO can be interpreted to contribute in enhancing the isoproterenol-stimulated adenylyl cyclase activity whereas the decrease in the density of these receptors at this time would diminish their contribution in this regard. In contrast, the depressed affinity as well as density of beta₁-adrenoceptors at 30 min incubation with X plus XO is consistent with the attenuated isoproterenol response of the membrane adenylyl cyclase at this time. It should be noted that both increase and decrease in beta-adrenoceptor density due to oxidative stress has been reported previously (Kaneko et al., 1991; Heikkila, 1983; Haenen et al., 1990); however, the inability of any of these investigator to demonstrate a biphasic change may be due to differences in the experimental design. Furthermore, unlike the previous studies that reported alterations in total beta-adrenoceptor status, our study has identified alterations in beta₁-adrenoceptors that may be involved in the observed biphasic alterations due to X plus XO. However, the beta₂-adrenergic receptors do not exhibit biphasic alterations in that they only undergo a small decrease in density and a marginal increase in affinity on 30-min incubation with X plus XO; these changes in two parameters may likely cancel out the effect of each other leading to a minimal contribution of beta₂-adrenoceptors toward the modifications produced by X plus XO.

Similar to the isoproterenol-stimulated adenylyl cyclase activity, the forskolin-, NaF- and Gpp(NH)p-stimulated activities in X plus XO-treated cardiac preparations also exhibited a biphasic pattern at 10- and 30-min of incubation periods. Alterations in the basal and forskolin-stimulated adenylyl cyclase activity by X plus XO can be interpreted to suggest changes at the level of catalytic site of the enzyme by oxyradicals whereas changes in NaF- and Gpp(NH)p-stimulated adenylyl cyclase activities may reflect biphasic alterations at the level of G-proteins. These results with X plus XO system are consistent with the biphasic alterations in the adenylyl cyclase catalytic activity (basal- and forskolin-stimulated) as well as in the G-protein mediated activity [NaF- and Gpp(NH)p-stimulated activities] reported previously by using the iron-ascorbic acid system (Schimke et al., 1992). Furthermore, the view that the G-protein mediated pathway is altered in a biphasic manner is supported by changes in the CT-stimulated adenylyl cyclase activity and CT-stimulated ADP-ribosylation of the G_s-protein in cardiac membranes treated with X plus XO, whereby both parameters were increased by 10 min incubation and depressed by 30 min incubation. Increased anti-G_s-protein binding to the 52-kDa band and decreased binding to the 45-kDa band of the G_s-protein at 10 min as well as decreased binding to both bands at 30 min may indicate an altered immunosensitivity of the protein, reinforcing the suggestion that the observed alterations are at the level of the G_s-protein itself. Because the time periods used in this study are not compatible with de novo synthesis of G_s-protein, the increased antibody labeling to G_s-proteins may not necessarily indicate increased content but rather may reflect an altered immunosensitivity of the G_s-proteins due to oxyradical treatment. It should be noted that it is only the 52-kDa band that is differentially modified by 10 and 30 min of incubation with X plus XO indicating that this particular subunit of the G_s-protein may be pertinently involved in the increased and decreased response of the pathway after 10- and 30-min treatments, respectively. The changes observed at the level of G_s-proteins seem specific in nature because G_i-protein associated activities including the PT-stimulated adenylyl cyclase activity, PT-catalyzed ADP ribosylation and anti-G_i-protein binding were unaltered upon treating cardiac membranes with X plus XO for 10 or 30 min.

The fact that SOD in the presence of CAT, but not alone, was effective in protecting the X plus XO-induced biphasic alterations in adenylyl cyclase activities implies that the formation of H₂O₂ and not superoxide radicals may be involved in promoting the observed changes on treating the membranes with X plus XO. Although low concentrations of H₂O₂ produced initially from dismutation of the primary superoxide radical, may participate in producing the initial increase in the activity of the signaling pathway, the latter attenuated activity of the pathway may be due to continued production and accumulation of H₂O₂ resulting in its increased concentration around the environment of membranes. Our results indicate that this may actually be the case because we noted that lower concentrations of H₂O₂ (50-100 µM) enhanced the basal and stimulated adenylyl cyclase activities although higher concentrations significantly depressed the enzymes activities. To this end, Tan et al. (1995) have reported an enhancement of the adenylyl cyclase activity in vascular cells due to H₂O₂ at low concentrations. However, studies done by others have reported a decrease in the adenylyl cyclase activity due to H₂O₂ at higher concentrations (Masuda et al., 1993; Haenen et al., 1990). Fliss et al. (1988) have reported that although high concentrations of H₂O₂ produce negative inotropic effects on the cardiac muscle, low concentrations have been shown to be beneficial. Because the exposure of cardiac membranes to a low concentration of H₂O₂ for short and prolonged periods produced a stimulation followed by a depression in the adenylyl cyclase activity, it is possible that H₂O₂ itself may be producing the observed biphasic alterations as a concentration and time dependent phenomenon.

It should be noted that SOD plus CAT was capable of preventing biphasic changes in G_s-protein related stimulation of adenylate cyclase and ADP-ribosylation activities due to pretreatment with X plus XO. Although the observed decrease in the density of beta₁-adrenoceptors upon incubating membranes with X plus XO for 10 and 30 min as well as the depression in the affinity of beta₁-adrenoceptors at 30 min incubation were prevented by the SOD plus CAT, this intervention failed to protect X plus XO-induced increase in the affinity of beta₁-adrenoceptors at 10 min. This finding is difficult to explain; however, it is possible that the initial increase in the affinity of beta₁-adrenoceptors may be relatively more sensitive to the action of X plus XO. Alternatively, this observation may be linked to the biphasic effects of X plus XO on the adenylyl cyclase activities. It is also likely that the biphasic effects of X plus XO on different components of the beta-adrenergic pathway may be caused by changes in the lipid peroxidation (Haenen et al., 1989). Because the proteins of the beta-adrenoceptor cascade are located in the membrane, it is conceivable that alterations in membrane fluidity due to lipid peroxidation may change the profile of the cascade. It is also possible that H₂O₂ produced during the interaction of X plus XO may directly alter the protein components of the pathway. This view is supported by different studies in which oxidative stress-induced sulfhydryl group modification of Ca⁺⁺-transport proteins has been reported to disrupt cellular mechanisms for calcium homeostasis (Scherer and Deamer, 1986; Trimm et al., 1986; Hebbel et al., 1986). In fact both the adenylyl cyclase enzyme as well as beta-adrenergic receptors are known to possess sulfhydryl groups in their active site (Skurat et al., 1985; Strauss, 1984; Padersen and Ross, 1985), the modification of which may alter the characteristic of the protein. Schimke et al. (1992) have suggested that both SH group modification and lipid peroxidation may be involved in the alteration of beta-adrenoceptor-adenylyl cyclase pathway. It has also been indicated that although protein modification may be the more important factor during the initial enhanced phase of the pathway, lipid peroxidation may become more relevant in producing the loss of activity in the pathway in the later phase (Schimke et al., 1992). In view of the generation of superoxide anions, H₂O₂ and hydroxyl radicals on exposing biological membranes to X plus XO (Kukreja and Hess, 1992), the initial increase in the adenylyl cyclase activities may be an adaptive response of membranes to superoxide anions and may serve as a defense mechanism. However, prolonged exposure of cardiac membranes to X plus XO may result in the formation of cytotoxic hydroxyl radicals which may very well overcome the defense and reduce the adenylyl cyclase activities.