Introduction

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Reperfusion of previously ischemic myocardium is associated with the generation of large amounts of reactive oxygen species (Slezak et al., 1995). H₂O₂, a small, uncharged and relatively stable molecule that diffuses easily through tissue (Hoffman et al., 1984; Welsh et al., 1985), is considered to be an important mediator of reperfusion-induced abnormalities (Beresewicz and Horackova, 1991; Duan and Moffat, 1992). H₂O₂ is formed in mitochondria as a dismutation product of the superoxide radical (O₂ ) under physiological conditions. However, under ischemic conditions, there is proteolytic modification of xanthine dehydrogenase to xanthine oxidase (McCord and Roy, 1982) which may produce a burst of O₂  and H₂O₂ when oxygen is reintroduced during reperfusion.

H₂O₂ has several adverse effects on the myocardium including induction of cardiac arrhythmias (Beresewicz and Horackova, 1991; Duan and Moffat, 1992). These effects may be the result of its ability to induce lipid peroxidation (Rubin and Farber, 1984), enzyme activation (Mekhfi et al., 1996), altered energy metabolism (Spragg et al., 1985), protein oxidation (Fliss et al., 1988) or changes in intracellular calcium concentration (Hyslop et al., 1986; Ward and Moffat, 1995) singly or in combinations of these factors. It has been postulated that the calcium overload observed following exposure to H₂O₂ may contribute to oxidant-induced cellular damage (Kaneko et al., 1994). However, the mechanism by which H₂O₂ increases [Ca⁺⁺]_i concentration is not clear. It is possible that H₂O₂ increases [Ca⁺⁺]_i by altering the activity of ion channels and/or transport proteins, either directly or through effects on other systems that modulate their activity. Several studies have reported the effects of H₂O₂ on various ion channels and exchangers. H₂O₂ has been reported to alter the function of delayed rectifier K⁺ current (Goldhaber et al., 1989), inward rectifier K⁺ current (Matsuura and Shattock, 1991), adenosine triphosphate-sensitive K⁺ current (Goldhaber and Liu, 1994), Na⁺-Ca⁺⁺ exchange (Goldhaber and Liu, 1994) and tetrodotoxin-sensitive sodium current (Beresewicz and Horackova, 1991; Ward and Giles, 1997). A subject of considerable controversy is the effect of H₂O₂ on cardiac calcium currents. In some studies, H₂O₂ caused a rapid decrease in the amplitude of I_Ca,L in guinea pig ventricular myocytes (Goldhaber et al., 1989; Goldhaber and Liu, 1994; Satoh and Matsui, 1997). However, Sato et al. (1989) have reported a brief augmentation and subsequent attenuation of Ca⁺⁺ current by t-butyl hydroperoxide, although others have reported that Ca⁺⁺ currents are unaltered by H₂O₂ (Beresewicz and Horackova, 1991; Cerbai et al., 1991). One possible explanation for this variability could be related to different recording conditions used in these studies. This is especially important when the marked differences in the effects of H₂O₂ on action potentials under different conditions are considered (Ward and Giles, 1997; Barrington, 1994). In our study, we examined the effect of micromolar concentrations (comparable to the levels of H₂O₂ under conditions of ischemia/reperfusion) on I_Ca,L under conventional whole cell and nystatin perforated patch configurations. The former rendered the interior of the cells vulnerable to dialysis although the latter maintained a more physiological intracellular milieu for a considerably longer period. Adenosine, through the activation of adenosine A1 receptors, has been shown to exert significant cardioprotective effects (Thornton et al., 1992). Previous work from our laboratory has shown that A1 receptor activation protects against the deleterious effects of H₂O₂ in terms of attenuation of cardiodepression produced by this oxidative stressor (Karmazyn and Cook, 1992). In addition, ischemic preconditioning confers protection against H₂O₂ via an adenosine-dependent mechanism (Gan et al., 1996). Accordingly, we also examined the influence of adenosine A1 receptor activation on the effects of H₂O₂ in terms of potential ability to modulate changes in I_Ca,L.

	Materials and Methods

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Animals. Male albino guinea pigs (300-350 g), obtained from Charles River (St. Constant, Quebec, Canada), were maintained in the Health Sciences Animal Care facility of The University of Western Ontario, in accordance with the guidelines of the Canadian Council on Animal Care (Ottawa, Ontario, Canada).

Cell isolation. The method used for the isolation of guinea pig myocytes is similar to that described previously (Thomas et al., 1997). Briefly, heparinized guinea pigs were decapitated and the heart was mounted on a Langendorff apparatus and perfused retrogradely through the aorta (10 ml/min) with oxygenated (100% O₂) calcium-free solution of the following composition (mM): 120 NaCl, 3.8 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄, 10 HEPES, 11 glucose (pH was adjusted to 7.4 with NaOH) at 37°C. The hearts were then perfused with calcium-free solution containing collagenase type-2 (Worthington Biochemical Corporation, Freehold, NJ) and protease (Sigma type XIV, Sigma Chemical Co., St. Louis, MO). After enzymatic dissociation, the ventricles were cut and washed in a high potassium Kraft-Brühe solution of the following composition (mM): 80 KOH, 50 glutamic acid, 30 KCl, 30 KH₂PO₄, 20 taurine, 10 HEPES, 10 glucose, 3 MgSO₄, 0.5 EGTA (pH was adjusted to 7.4 with KOH). After 1 to 2 hr incubation at room temperature, myocytes were placed in a bath (volume 2 ml) on the stage of an inverted microscope (Nikon). Cells were allowed to adhere to the bottom of the bath for 15 to 20 min and were then superfused with a solution containing (in mM), 145 NaCl, 10 HEPES, 10 glucose, 4 KCl, 1.8 CaCl₂ and 1 MgCl₂.

Conventional whole cell patch. Patch pipettes were pulled from borosilicate glass (Sutter Instrument Co., Novato, CA) using a pipette puller (model PP-83, Narishige Scientific Instrument Lab, Tokyo, Japan) and were fire polished. Pipettes exhibited 2 to 4 M resistance when filled with a pipette solution containing (in mM) 1 EGTA, 130 CsCl, 20 HEPES, 1 MgCl₂, 10 tetraethylammonium-Cl, 0.4 CaCl₂ and 4 ATP Na₂. All recordings were initiated only after 15 min to allow complete dialysis of the cytoplasm.

Nystatin perforated patch. The effects of H₂O₂ on whole cell I_Ca,L was also examined by using the nystatin perforated patch configuration. The pipette solution contained (in mM) 1 EGTA, 130 CsCl, 20 HEPES, 1 MgCl₂, 10 tetraethylammonium-Cl, 0.4 CaCl₂ and nystatin. A nystatin stock solution (50 mg/ml) in dimethyl sulphoxide was prepared and the final concentration of nystatin in the pipette solution was 200 µg/ml. Fresh nystatin solution was prepared hourly. The tip of the pipette was filled with nystatin free pipette solution, before backfilling with nystatin-containing solution. After obtaining a gigaseal, the pipette potential was set to 60 mV and voltage pulses were delivered to monitor the incorporation of nystatin and the access resistance. Recording of currents was initiated when the access resistance had stabilized at 6 to 13 M at which time much of the cell capacitance and series resistance (60-80%) was compensated electronically.

Chemicals. The chemicals used in this study were H₂O₂ (BDH), bisindolylmaleimide (Calbiochem, La Jolla, CA),2-[p-(carboxyethyl) phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680), 1- Deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl--D-ribofuranuronamide, N⁶-cyclopentyladenosine, and 8-cyclopentyl-1,3-dipropylxanthine (all from Research Biochemicals International, Natick, MA).

Statistics. Data in each group, derived from at least five individual hearts, are presented as mean ± S.E. Percentage values were log transformed for statistical analysis. Data were analyzed using two-way analysis of variance followed by Student-Newman-Keuls test to assess the significance level. Differences between treatment groups were considered significant when P < .05.

	Results

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Step depolarization of myocytes from 40 to 10 mV elicited a time- and voltage-dependent inward current which had all the characteristics of I_Ca,L. These currents were completely blocked by verapamil and enhanced by Bay K-8644 and showed characteristic current voltage relationships.

Effect of H₂O₂ on I_Ca,L under conventional whole cell patch configuration. Guinea pig ventricular myocytes were superfused with H₂O₂ (100 µM) and I_Ca,L was recorded under whole cell configuration. Figure 1A shows recordings from a typical experiment under conventional whole cell patch configuration, before and after the myocyte was exposed to H₂O₂. Very little changes were observed in the peak I_Ca,L when the myocyte was exposed to H₂O₂ for 25 min. Figure 2 depicts the effect of H₂O₂ on I_Ca,L under conventional whole cell configuration (n = 5). Figure 2A shows the current voltage relationship of I_Ca,L under whole cell configuration. H₂O₂ did not significantly change the magnitude of the peak or cause any significant voltage shift. The mean results obtained from five myocytes exposed to H₂O₂ for a period of 25 min (fig. 2B) revealed that H₂O₂ has no significant effect of on I_Ca,L under conventional whole cell configuration.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Effects of H2O2 (100 µM) on ICa,L in guinea pig ventricular myocytes under different recording conditions. ICa,L was elicited by 200 msec voltage step to 10 mV from a holding potential of 40 mV. A, Individual ICa,L recordings from a typical experiment under conventional ruptured patch method showing the absence of any significant effect of H2O2 after 25 min exposure. B, H2O2 induced a large increase in ICa,L when recorded (25 min exposure to H2O2) under perforated patch configuration.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Absence of effect of H2O2 on ICa,L under conventional whole cell configuration. A, Peak current voltage relationship from six ventricular myocytes. Test potentials ranged from 50 to 50 mV in 10 mV steps. Symbols represent , control and , H2O2 100 µM. H2O2 did not alter the I-V relationship. Values are expressed as mean ± S.E.M. B, Effect of H2O2 (100 µM) on peak ICa,L (n = 6). Twenty-five min exposure of H2O2 did not cause any significant change in ICa,L under whole cell ruptured patch method. Values are expressed as % ± S.E.M. with control values taken as 100%.

Effect of H₂O₂ under perforated patch configuration. In contrast to its failure to alter I_Ca,L when using whole cell recording, H₂O₂ caused a large increase in peak I_Ca,L in ventricular myocytes when perforated patch configuration was used. Figure 1B shows tracings of I_Ca,L from an experiment in which the I_Ca,L was recorded under nystatin perforated patch and the results of six experiments in which the myocytes were exposed to H₂O₂ are shown in figure 3. Figure 3A shows the current-voltage relationships indicating an H₂O₂-induced increase in I_Ca,L and a shift in the peak current to slightly more negative values, measured after 25-min exposure to H₂O₂. The H₂O₂ effect observed in six ventricular myocytes is summarized in figure 3B, showing a time-dependent increase in I_Ca,L during exposure to H₂O₂.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   H2O2 stimulated ICa,L recorded under nystatin perforated patch configuration. A, Peak current voltage relationship from 6 myocytes. Voltage clamp protocols were as in figure 2. H2O2 (100 µM) induced a large increase in ICa,L and this was associated with a slight shift in the I-V relationship to more negative values (25-min exposure time). Values are expressed as mean ± S.E.M. B, Effect of H2O2 (100 µM) on peak ICa,L in six ventricular myocytes. H2O2 caused a significant increase in ICa,L. The enhancement was significant from 5-min exposure and continued to increase with time. Values are expressed as % ± S.E.M. with control values taken as 100%.

Effect of adenosine A1 receptor activation. We previously reported an inhibitory effect of adenosine A1 receptor activation on the cardiac effects of H₂O₂ (Karmazyn and Cook, 1992). Consequently, we next examined the effect of CPA, an adenosine A1 agonist on the H₂O₂-induced enhancement of I_Ca,L. Figure 4A shows typical data obtained in the presence of CPA (5 µM), and demonstrates an almost complete inhibition of H₂O₂-induced activation of I_Ca,L. The data for the inhibitory effect of CPA, observed in six ventricular myocytes are summarized in figure 4B. CPA by itself was without effect on basal I_Ca,L. However, as figure 4B demonstrates, CPA significantly reduced the effects of H₂O₂ on I_Ca,L.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   H2O2-induced ICa,L stimulation is inhibited by adenosine A1 agonist CPA. A, Individual traces obtained from a representative experiment demonstrating the inhibition of H2O2 effect by CPA. a, Control ICa,L recorded in a ventricular myocyte in the presence of 5 µM of CPA. b, ICa,L from the same myocyte after 25 min exposure to H2O2. CPA was present in the solution throughout the experiment. B, Overall effect of CPA on H2O2 stimulation of ICa,L (n = 6). Values are expressed as % of control levels ± S.E.M. * P < .05 compared to H2O2 (analysis of variance with Student-Newman-Keuls test).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Adenosine A1 antagonist DPCPX blocks the effect of CPA on H2O2 stimulation of ICa,L. A, Individual traces were obtained at times marked a, b, c, d and e in B. CPA did not inhibit the ICa,L increase caused by H2O2 in the presence of DPCPX. B, Time course of the experiment shown in A. CPA was added 5 min after DPCPX and 5 min before H2O2.

Effect of A2A and A3 receptor activation. We further assessed receptor specificity and determined the possible role of other adenosine receptor subtypes on H₂O₂-induced response. As shown in figure 6, neither the A2A receptor agonist CGS 21680 or the A3 agonist IB-MECA (0.5 µM each) had any effect on H₂O₂-induced I_Ca,L activation.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Adenosine A2A agonist CGS-21680 and A3 agonist IB-MECA have no significant effect on H2O2 stimulation of ICa,L. CGS-21680 and IB-MECA (0.5 µM each) were added to the superfusion fluid 5 min before H2O2 (n = 3 each).

Effect of PKC inhibition. There is evidence that PKC may modulate some of the effects of H₂O₂ in cardiac tissues (Ward and Moffat, 1995). Accordingly, the last set of experiments were done to determine the effect of PKC inhibitor BIS. In these experiments, BIS (20 nM) was added to the superfusion solution, 5 min before H₂O₂ administration and was without effect on its own on I_Ca,L. As shown in figure 7, BIS attenuated the H₂O₂ effect on I_Ca,L significantly. Figure 7A shows the current voltage relationship obtained from four ventricular myocytes in which BIS was administered before H₂O₂. Figure 7B represents the peak I_Ca,L data from five myocytes demonstrating the inhibition by BIS of H₂O₂induced I_Ca,L enhancement. We further examined whether BIS can modulate the effects of CPA on the H₂O₂ effects. In two experiments (not shown) addition of CPA to BIS-treated cells resulted in an almost 100% inhibition in the ability of H₂O₂ to activate I_Ca,L.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   PKC inhibition with bisindolylmaleimide (BIS) attenuates the effect of H2O2. A, Mean current-voltage relationship of ICa,L obtained from five ventricular myocytes. There was no significant enhancement of ICa,L when H2O2 was administered for 25 min in the presence of BIS (20 nM). B, Effect of H2O2 on peak ICa,L in the presence and absence of BIS. The effect of H2O2 was significantly lower in the presence of BIS. Values are expressed as % ± S.E.M. of control. * P < .05 compared to H2O2 (analysis of variance with Student-Newman-Keuls test).

	Discussion

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Our results demonstrate for the first time, to our knowledge, a pronounced elevation in I_Ca,L by H₂O₂, an effect dependent on recording configurations. Thus the increase in I_Ca,L observed with H₂O₂ under nystatin perforated patch configuration was not observed using conventional whole cell ruptured patch technique. Because calcium channel function depends substantially on cytoplasmic factors, in the conventional ruptured patch method calcium currents can "run down" significantly (Belles et al., 1988; Kameyama et al., 1988). This is mainly due to the loss of low molecular weight cellular components by dialysis to the pipette solution. However, some of the alterations in drug response could be due to the constituents of pipette solution interfering with the signal transduction pathways. The perforated patch configuration minimizes the washout or dilution of cytoplasmic constituents that either modulate or are required for ion channel activity and avoids the disruption of normal intracellular Ca⁺⁺ buffering mechanisms (Korn et al., 1991). Previous reports have shown that H₂O₂ can exert divergent effects under different recording conditions. For example, Barrington (1994) demonstrated the differences in the effect of H₂O₂ on action potentials in the absence and presence of EGTA in the pipette. This study also demonstrated that H₂O₂ prolonged action potential duration when recorded using high resistance standard microelectrodes where the dialysis of internal cytoplasm does not occur. Similarly, in another study, H₂O₂ did not exert any significant effects on action potentials or cell shortening under whole cell recording conditions, but induced marked prolongation of the action potential duration and an increase in cell shortening under amphotericin perforated patch configuration (Ward and Giles, 1997). Our results underline the importance of the recording conditions on the H₂O₂ response and shows that this is also true in the case of I_Ca,L.

H₂O₂ is formed in micromolar concentrations in the mitochondria under physiological conditions (MacFurlane and Miller, 1994; Turrens et al., 1991). Human plasma levels of H₂O₂ are also in the lower micromolar range (Caverocchi et al., 1986). Accordingly, the concentration of H₂O₂ used in this study was 100 µM, as this represents a concentration comparable to that expected to occur under pathological conditions such as ischemia/reperfusion. Moreover in our preliminary studies it was observed that higher concentrations affected the integrity of the myocytes leading to nonspecific toxic effects. The rapid and steep fall in I_Ca,L observed in some studies (Goldhaber et al., 1989; Goldhaber and Liu, 1994) may reflect, at least in part, such nonspecific effects.

Several lines of evidence suggest that an elevation of [Ca⁺⁺]_i may underlie the H₂O₂-induced cardiac abnormalities and injury. For example, H₂O₂ has been shown to induce a slow increase in [Ca⁺⁺]_i in guinea pig ventricular myocytes (Hayashi et al., 1989; Kaneko et al., 1994) which could either be due to increased Ca⁺⁺ influx or an insufficient Ca⁺⁺ extrusion from the cytosol. A facilitation of the release from, and/or an inhibition of the uptake into, sarcoplasmic reticulum calcium stores also could affect [Ca⁺⁺]_i. Because of the absence of any data indicating a significant increase in Ca⁺⁺ currents by H₂O₂, it was considered highly unlikely that the influx of Ca⁺⁺ through sarcolemmal Ca⁺⁺ channels contributed to the H₂O₂-induced elevation of [Ca⁺⁺]_i (Kaneko et al., 1994). Our study clearly indicates that a facilitated entry through the sarcolemmal L-type Ca⁺⁺ channels may also contribute to the elevation of the cytoplasmic Ca⁺⁺ by H₂O₂. This increased influx of Ca⁺⁺, could, in turn, induce further release of calcium from the sarcoplasmic reticulum. Thus these results suggest that Ca⁺⁺ influx through voltage-dependent Ca⁺⁺ channels may contribute to Ca⁺⁺ overload induced by H₂O₂.

It is interesting to note that an inhibitory effect of H₂O₂ on action potential duration has also been reported (Goldhaber et al., 1989). However, Barrington (1994) has shown that the recording mode and the composition of the pipette solution can affect H₂O₂ response on the action potential. Recently, Ward and Giles (1997) have demonstrated that under amphotericin patch configuration, H₂O₂ (50-200 µM) induced a marked prolongation of action potential duration and an increase in cell shortening but the same concentrations failed to show any significant effects on action potential duration under whole cell ruptured patch configuration. Even though any generalized comparison of the data available on the effect of H₂O₂ may not be totally accurate, it is important to note that almost all of the studies using high resistance electrodes or perforated patch techniques have reported an increase in the action potential duration by H₂O₂ (Beresewicz and Horackova, 1991; Duan and Moffat, 1992; Barrington, 1994; Ward and Giles, 1997; Satoh and Matsui, 1997), whereas using whole cell ruptured patch have shown a decrease or no change in action potential duration with H₂O₂ (Goldhaber et al., 1989; Barrington, 1994; Ward and Giles, 1997). Increased action potential duration could reflect one or several of the many mechanisms that regulate action potential duration, such as outward K⁺ current, Na⁺-Ca⁺⁺ exchange, sarcolemmal Ca⁺⁺ channels and [Ca⁺⁺]_i. Our results clearly indicate that an augmented Ca⁺⁺ influx through L-type Ca⁺⁺ channels by H₂O₂ could contribute to the increase in action potential duration observed with H₂O₂.

The selective effect of H₂O₂ on I_Ca,L under nystatin configuration also suggests that an intracellular moiety may be involved in this action of H₂O₂. It is possible that this messenger becomes inactivated or diluted by some components of the pipette solution under whole cell patch configuration. Activation of PKC by H₂O₂ directly (Larsson and Cerutti, 1989) and indirectly through activation of phospholipase D (Natarajan et al., 1993) has been demonstrated in noncardiac tissues, although to our knowledge this has not as yet been demonstrated in the heart. Nonetheless, it has also been reported that PKC activation mediates the H₂O₂-induced elevation in cytosolic calcium in ventricular myocytes (Ward and Moffat, 1995). Moreover, PKC-activating phorbol esters have been shown to stimulate the calcium current in neonatal rat cardiac myocytes (Dosomeci et al., 1988). Taken together, these studies support our contention that the stimulatory effect of H₂O₂ on I_Ca,L is, at least partly, mediated via PKC. The exact mechanisms for PKC-mediated H₂O₂induced activation of I_Ca,L requires further studies although it likely involves a phosphorylation-dependent process. There is also evidence that some of the salutary effects of A1 receptor activation, particularly its involvement in ischemic preconditioning, may be mediated by PKC. Moreover, A1 receptor activation stimulates PKC in rat ventricular myocytes (Henry et al., 1996). The ability of BIS, a PKC inhibitor, to mimic the effect of CPA as well as its inability to prevent the inhibitory effects of CPA were therefore of some surprise although the results may suggest that distinct PKC isoforms may be involved in regulating the modulatory role of H₂O₂ on I_Ca,L. Further studies are necessary to delineate the potential role of this family of isozymes either with respect to H₂O₂-induced effects on the calcium current or the inhibitory effects of A1 receptor activation. The inability of BIS to completely prevent I_Ca,L activation is suggestive of additional cellular mechanisms for H₂O₂-induced effects. These intracellular events may also explain the basis for the timedependent effects of H₂O₂ observed in our study as well reported by other investigators (Hayashi et al., 1989; Kimura et al., 1992).

Adenosine A1 receptor agonists have been shown in various studies to protect the ischemic and reperfused myocardium (Thornton et al., 1992). Although the precise mechanism for this protection is not known, we previously reported that CPA inhibits the cardiotoxic effects of H₂O₂ (Karmazyn and Cook, 1992) at least suggesting that this could be a contributory factor. To determine the possible mechanisms for these effects, we examined the effect of CPA, a selective adenosine A1 receptor agonist, on the enhancement of I_Ca,L induced by H₂O₂. Our results show that CPA significantly inhibits the H₂O₂-induced stimulation of I_Ca,L. The A1 receptor specificity of this effect was further confirmed by the ability of DPCPX to reverse the effects of CPA. However, it should also be stated that although A1 receptors are the predominant adenosine receptor subtype found in the ventricular myocardium, A2 and A3 receptors are also found in the ventricular myocyte [see Cook and Karmazyn (1996) for review]. A2 receptor agonists have been reported to have minimal protective influence on the heart although A2 receptor-mediated cardioprotective actions of adenosine are observed under in vivo conditions, where the antineutrophil and antiplatelet actions of A2 receptor activation can induce protection (Schlack et al., 1993). However, because it has been reported that A2 receptors are modulated under ischemic conditions (Zucchi et al., 1992), we also examined the influence of the A2A receptor-selective agonist CGS-21680 on the effect of H₂O₂. Our results suggest a lack of A2A receptor involvement on this effect. Furthermore, it is unlikely that A3 receptors are involved based on the inability of IB-MECA to affect H₂O₂ actions.

	Summary and Conclusion

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In summary, our study shows that H₂O₂ can activate I_Ca,L in guinea pig ventricular myocytes when using perforated patch configuration. This effect of H₂O₂ appears to be at least partially dependent on PKC activity. Our study also demonstrates a potent ability of A1 receptor activation to inhibit the effects of H₂O₂ which is not shared by either A2A or A3 receptor agonists. These findings suggest a novel and potentially important role of A1 receptors in the regulation of the cardiac effects of H₂O₂, particularly under pathological conditions.