Published online before print August 29, 2002, doi: 10.1161/01.RES.0000035528.00678.D5
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© 2002 American Heart Association, Inc.
Cellular Biology |
Decreasing Cellular Hydrogen Peroxide With Catalase Mimics the Effects of Hypoxia on the Sensitivity of the L-Type Ca2+ Channel to ß-Adrenergic Receptor Stimulation in Cardiac Myocytes
From the Departments of Physiology (L.C.H.) and Biochemistry (P.G.A.), The University of Western Australia, Crawley, and The Western Australia Institute of Medical Research (L.C.H., P.G.A.).
Correspondence to Dr Livia Hool, Department of Physiology, The University of Western Australia, Stirling Highway, Crawley, WA 6009, Australia. E-mail lhool{at}cyllene.uwa.edu.au
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Abstract |
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In cardiac myocytes, hypoxia inhibits the basal L-type Ca2+ current (ICa-L) and increases the sensitivity of ICa-L to ß-adrenergic receptor stimulation. We investigated whether hydrogen peroxide (H2O2) is involved in the hypoxic response. Guinea pig ventricular myocytes were dialyzed with catalase, which specifically catalyzes the conversion of H2O2 to H2O and oxygen, and then ICa-L was recorded during exposure to isoproterenol (Iso). Catalase decreased the K0.5 for activation of ICa-L by Iso from 2.7±0.3 nmol/L (in cells dialyzed with heat-inactivated catalase) to 0.4±0.1 nmol/L. The increase in sensitivity to Iso by catalase may be attenuated when cells are preexposed to H2O2. A significant increase in sensitivity of ICa-L to Iso was recorded when mitochondrial function was inhibited with myxothiazol or FCCP, suggesting that the source of H2O2 was from the mitochondria. Prior exposure of cells to H2O2 attenuated the inhibition of basal ICa-L during hypoxia and the increase in sensitivity of ICa-L to Iso during hypoxia. Additionally, extracellularly applied catalase mimicked the effect of hypoxia on basal ICa-L. Measurement of the rate of production of hydrogen peroxide using 5- (and 6-)chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate acetyl ester indicated that hypoxia was associated with a significant decrease in the production of hydrogen peroxide in the cells. These data suggest that hypoxia mediates changes in channel activity through a lowering in H2O2 levels and that H2O2 is a key intermediate in modifying basal channel activity and the ß-adrenergic responsiveness of the channel during hypoxia.
Key Words: catalase • ß-adrenergic receptors • hypoxia • L-type Ca2+ channels
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Introduction |
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Reactive oxygen species are generated as byproducts of cellular metabolism. These molecules have long been regarded as toxic products of living in an aerobic environment, but recent evidence points to a role for reactive oxygen species as signal transducers in mammalian cells. One of these, hydrogen peroxide (H2O2), is a small and stable molecule that has been implicated in both pathological and physiological processes. These include altering the function of proteins by regulating the redox state of the protein.1
The effects of ß-adrenergic receptor (ß-AR) stimulation on the L-type Ca2+ current (ICa-L) are well documented.2 Activation of ß-AR results in an increase in peak inward current and slowing of inactivation via phosphorylation of the channel protein by protein kinase A (PKA). Previous work from this laboratory has shown that hypoxia inhibits basal channel activity and increases the sensitivity of the L-type Ca2+ channel to ß-AR stimulation.3,4 We investigated a possible role for H2O2 in the hypoxic response and whether its source was the mitochondria. Cells exposed to extracellular catalase only mimicked the effect of hypoxia on basal channel activity. In addition, cells dialyzed with catalase recorded a significant increase in the sensitivity of ICa-L to ß-AR stimulation. This effect could be attenuated with exposure of the cells to H2O2 and mimicked with inhibition of mitochondrial function by myxothiazol or FCCP. In addition, application of a ß-protein kinase C (ßPKC) peptide inhibitor attenuated the effect of catalase. Because the effect of catalase mimics that of hypoxia, these data suggest that H2O2 may play a role in modifying the response of the L-type Ca2+ channel basal activity and the sensitivity of the channel to ß-adrenergic receptor stimulation. Effects of hypoxia on the channel are mediated by changes in H2O2 levels. These data provide further insight into the effects of hypoxia on the regulation of L-type Ca2+ channel function in cardiac myocytes.
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Materials and Methods |
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Data Acquisition for Patch-Clamp Experiments
Ventricular myocytes were isolated as previously described.3,4 For further detail regarding the cell isolation procedure, please see the online data supplement (available at http://www.circresaha.org).
Macroscopic currents were recorded using an Axopatch 200B voltage-clamp amplifier (Axon Instruments) and an IBM-compatible computer with a Digidata 1200 interface and pClamp software (Axon Instruments). An Ag/AgCl electrode (Clark Electrodes, Clark Electromedical Instruments) was used to ground the bath. Currents were measured in extracellularly modified Tyrode’s solution containing (in mmol/L) NaCl 140, CsCl 5.4, CaCl2 2.5, MgCl2 0.5, HEPES 5.5, glibenclamide 0.01, and glucose 11 (pH was adjusted to 7.4 with NaOH). The solution was made hypoxic by bubbling with 100% nitrogen as previously described.4 All hypoxia experiments were performed at 
17 mm Hg oxygen tension, as determined by an oxygen-sensitive probe.4 The whole-cell configuration of the patch-clamp technique was used to record currents.4 For patch-clamp protocol, please see the online data supplement.
Measurement of Rate of Production of H2O2
Generation of H2O2 was assessed using the fluorescence indicator 5- (and 6-)chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA, Molecular Probes, Inc), which becomes highly fluorescent on oxidation by H2O2. Immediately after isolation, cells were placed in a solution containing (in mmol/L) potassium glutamate 110, KCl 25, KH2PO4 10, MgSO4 2, taurine 20, creatine 5, EGTA 1.0, HEPES 5, and glucose 20 (pH was adjusted to 7.4 with KOH) and allowed to sit for 5 hours. The cells were then centrifuged at 500 rpm for 10 minutes, and the pellet was resuspended in 1 mL of solution containing (in mmol/L) KCl 5.3, MgSO4 · 7H2O 0.4, NaCl 139, NaH2PO4 · 2H2O 5.6, HEPES 20, glutamine 2, and Ca(NO)2 · 4H2O 0.4, along with penicillin/streptomycin (1 mL/100 mL), 10% FCS, and 5 mmol/L glucose (pH was adjusted to 7.4 with NaOH) and plated onto prepared poly-D-lysine–coated plates. The cells were then incubated overnight in a 37°C incubator containing 5% CO2. Before experimentation, the plates were washed once with 3 mL of the same solution. The plates were placed on an inverted microscope stage in an airtight dish maintained at 37°C and gassed with humidified air heated to 37°C for 15 minutes before the addition of CM-H2DCFDA to simulate normoxia. The addition of 10 µmol/L CM-H2DCFDA was considered time 0. At 50 minutes, the cells were exposed to 90% N2 and 10% O2 (PO2 of 15 mm Hg) at 37°C through a humidifier to simulate hypoxia. Data were acquired every 4 minutes and analyzed with Metamorph Version 4.8 (Universal Imaging Corp). Exposure time was 200 ms. Background fluorescence was subtracted from raw data. Fluorescence was recorded in arbitrary units, and the rate of increase in fluorescence was determined by calculating the slope.
Statistical Analysis
Results are reported as mean±SE. Statistical comparisons of responses between unpaired data were made using the Student t test or between groups of cells using one-way ANOVA and the Tukey post hoc test (Minitab).
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Results |
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Hypoxia Alters the Sensitivity of ICa-L to Iso
We have reported previously that hypoxia significantly increases the sensitivity of ICa-L to isoproterenol (Iso) in cardiac myocytes. Tyrode’s solution bubbled with 100% nitrogen for at least 30 minutes (resulting in PO2
17 mm Hg oxygen) shifts the K0.5 for activation of the current from 5.3±0.7 to 1.6±0.1 nmol/L.4 In addition, hypoxia does not alter the response to 1 µmol/L Iso (a maximally stimulating concentration of the ß-AR agonist) or the current-voltage (I-V) relationship for the L-type Ca2+ channel.4
Catalase Alters the Sensitivity of ICa-L to Iso
We investigated the effect of a reduction in cellular H2O2 on the response of ICa-L to ß-AR stimulation. Cells were dialyzed with 500 U/mL pipette solution of high-specific-activity catalase, followed by exposure to 3 and 10 nmol/L Iso in room oxygen. The responses to 3 and 10 nmol/L Iso were then compared with the current elicited by 1 µmol/L Iso in the same cell; exposure to catalase resulted in currents that were 88.7±3.7% and 98.8±1.2%, respectively, of the current elicited by 1 µmol/L Iso (n=12, Figure 1A). The magnitude of this response is not significantly different from that of currents recorded in cells exposed to hypoxia in the absence of catalase.4 Additionally, the response of ICa-L to 1 µmol/L Iso and catalase was not significantly different from the response of the channel to 1 µmol/L Iso during hypoxia (9.1±1.2 pA/pF [n=6] versus 10.0±0.8 pA/pF [n=8], respectively).
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We then compared the responses recorded in the presence of active catalase with currents recorded from myocytes dialyzed with catalase that had been heat-inactivated after boiling the enzyme in a water bath for at least 1 hour (Figure 1B). In these cells, the responses to 3 and 10 nmol/L Iso were significantly (P<0.01) less than the currents recorded in cells dialyzed with the active enzyme (52.7±9.4% and 89.2±8.9%, respectively; n=6). In addition, exposing the cells to active catalase did not appear to alter the I-V relationship for ICa-L. Exposure to 3 nmol/L Iso in the presence of catalase resulted in a shift in the peak current of 8.9±1.1 mV (n=9, Figure 1C) in the negative direction relative to the peak current in control solution. This was not dissimilar from the shift in the peak current recorded in 6 cells dialyzed with inactivated catalase (-8.3±1.1 mV) in the presence of 3 nmol/L Iso or when cells were exposed to 3 nmol/L Iso and hypoxia in the absence of catalase (-11.7±1.7 mV).4 Next, the concentration dependence of ICa-L on Iso was determined. The concentration of Iso that produced a half-maximal activation (K0.5) of the Ca2+ current when cells were dialyzed with inactivated catalase was 2.7±0.3 nmol/L, and the current was maximally stimulated with 1 µmol/L Iso (Figure 2). However, when cells were dialyzed with catalase, the K0.5 for activation of the Ca2+ current was significantly decreased to 0.3±0.1 nmol/L, and the current was maximally stimulated with 10 nmol/L Iso. These data indicate that catalase increases the sensitivity of ICa-L to Iso.
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To support our findings with catalase, additional experiments were undertaken to examine the effect of glutathione peroxidase (an antioxidant enzyme that uses glutathione to specifically reduce H2O2) on the response of ICa-L to Iso. In 5 of 7 cells tested, glutathione peroxidase (4 U/mL pipette solution) caused a significant increase in the response of ICa-L to Iso. Cells exposed to 3 and 10 nmol/L Iso resulted in currents that were 72.4±11.8% and 91.8±5.6%, respectively, of currents recorded in the presence of 1 µmol/L Iso. These responses were similar to the currents recorded when cells were dialyzed with catalase (Figure 1A) and when cells were exposed to hypoxia in the absence of catalase.4
We have reported previously that exposing cardiac myocytes to hypoxia results in a 22% decrease in the basal ICa-L.4 We and others4–6 have proposed that redox modification of the channel protein may be responsible for the basal inhibition. Catalase does not cross the plasma membrane, and in previous studies, we placed the enzyme in the pipette solution. Active catalase applied intracellularly did appear to decrease basal ICa-L density. The mean basal current density was 27.1% less when cells were dialyzed with active catalase than when cells were dialyzed with heat-inactivated catalase. However, to determine whether H2O2 regulates channel function directly, we exposed cells to extracellular solution containing 500 U/mL of pure catalase and recorded ICa-L in the absence and presence of Iso. In the absence of Iso, exposure of 6 cells to catalase caused a 11.7±2.6% decrease in basal ICa-L. Currents recorded in myocytes exposed to 3 and 10 nmol/L Iso in the presence of extracellular catalase were 33.6±7.8% and 73.8±8.4%, respectively, of currents re- corded in the presence of 1 µmol/L Iso. The magnitudes of these Iso-stimulated currents are not dissimilar from those of currents recorded in the absence of hypoxia4 or while the cells were dialyzed with heat-inactivated catalase (Figure 2), consistent with an effect of catalase on extracellular H2O2 levels and channel function. These data are also consistent with previous results from this laboratory in which the thiol-reducing agent dithiothreitol mimicked the effect of hypoxia on basal ICa-L and in which the membrane-impermeant oxidizing compound 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) attenuated the effect of hypoxia on the basal current.4
To determine whether the effect of catalase was specific to the ß-AR, the effect of forskolin, which directly activates adenylate cyclase, was examined. Cells were first exposed to 600 nmol/L forskolin while being dialyzed with heat-inactivated catalase (500 U/mL pipette solution), and the response was compared with that of the current elicited by a maximally stimulating concentration of forskolin (10 µmol/L) in the same cell. Under these conditions, 600 nmol/L forskolin activated a current that was 41.6±6.7% of the current recorded during exposure to 10 µmol/L forskolin (n=5). When cells were dialyzed with active catalase (500 U/mL pipette solution), the response to 600 nmol/L forskolin was significantly increased to 84.6±6.0% of the current recorded during exposure to 10 µmol/L forskolin (n=5, P<0.01). These results indicate that catalase increases the sensitivity of the channel to ß-AR stimulation by acting at a level downstream from the ß-AR. The effect of hypoxia to increase the sensitivity of the channel to ß-AR stimulation also occurs downstream from the ß-AR.4
If a reduction in cellular H2O2 levels mediates the altered sensitivity of ICa-L to Iso, then the increased responses measured in the presence of Iso and catalase should be attenuated when cells are exposed to H2O2. To prevent any possible oxidizing effects of H2O2 on Iso, H2O2 was omitted from all solutions containing Iso, and solutions containing Iso were replaced every 60 to 90 minutes. This ensures that the concentration of Iso in the external solutions remains constant.7 H2O2 freely crosses the plasma membrane.8,9 After gaining the whole-cell configuration with catalase in the pipette solution, cells were superfused with Tyrode’s solution containing H2O2 (8.8 to 88 µmol/L) for at least 5 minutes before being exposed to Iso in the absence of H2O2 (Figure 3A). In 6 cells, prior exposure to H2O2 significantly attenuated the response of ICa-L to Iso and catalase (P<0.05). Figure 3B summarizes the effects of catalase and H2O2. In addition, prior exposure to H2O2 did not alter the response of the channel to 1 µmol/L Iso. In cells dialyzed with catalase and not exposed to H2O2, the current density during exposure to 1 µmol/L Iso was 9.1±1.1 pA/pF (n=6). This was not significantly different from the current density recorded during exposure to 1 µmol/L Iso in cells dialyzed with catalase and preexposed to H2O2 (12.0±1.7 pA/pF, n=5).
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Inhibition of Mitochondrial Function Alters the Sensitivity of ICa-L to Iso
The mitochondria have been implicated as a source for the generation of reactive oxygen species during hypoxia.10–16 We investigated whether inhibition of the mitochondria altered the response of ICa-L to Iso. Cells were dialyzed with myxothiazol, an inhibitor of electron flow at complex III, at a concentration of 3.4 µmol/L. Myxothiazol significantly increased the sensitivity of ICa-L to Iso (Figure 4A). Exposure to 3 and 10 nmol/L Iso resulted in currents that were 76.8±3.7% and 93.0±2.7%, respectively, of the currents recorded in the presence of 1 µmol/L Iso (n=8). These currents were similar to the currents recorded when cells were dialyzed with catalase (Figure 1A) and when the cells were exposed to hypoxia in the absence of catalase.4 To determine whether the effect of myxothiazol was a nonspecific effect of the drug on ICa-L or the ß-AR pathway, cells were dialyzed with myxothiazol and exposed to H2O2 (8.8 to 88 µmol/L) for at least 5 minutes before currents were recorded in the presence of Iso. Under these conditions, exposure to 3 and 10 nmol/L Iso resulted in currents that were 31.8±12.0% and 72.2±9.6%, respectively, of the currents recorded in the presence of 1 µmol/L Iso (n=5). These currents were significantly smaller than the currents recorded in cells dialyzed with myxothiazol and not exposed to H2O2 (P<0.01). These results suggest that the source of generation of H2O2 is the mitochondria. To further support these data, cells were dialyzed with FCCP (1.4 µmol/L), an uncoupler of electron transport in the mitochondria. Consistent with the results obtained when cells were dialyzed with myxothiazol, exposure to FCCP and 3 and 10 nmol/L Iso resulted in currents that were similar in magnitude to currents recorded in cells dialyzed with catalase (n=5, Figure 5A). When 5 cells were dialyzed with FCCP and exposed to 8.8 µmol/L H2O2 before Iso (Figure 5B), currents recorded during exposure to 3 and 10 nmol/L Iso and normalized to 1 µmol/L Iso in the same cell were significantly smaller than normalized currents recorded during 3 and 10 nmol/L Iso in cells dialyzed with FCCP and not exposed to H2O2. Figure 6A summarizes the results.
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H2O2 Attenuates the Effect of Hypoxia
To examine whether the effect of hypoxia on the sensitivity of ICa-L to Iso is mediated by alterations in H2O2 levels, cells were exposed to H2O2 first and then to hypoxia and Iso, and changes in ICa-L were recorded. H2O2 alone had very little effect on basal ICa-L. In 6 cells, currents recorded after exposure to 8.8 µmol/L H2O2 were 1.2±2.7% larger than currents recorded in the absence of H2O2 (P=NS). Prior exposure of cells to H2O2 attenuated the inhibition of basal ICa-L by hypoxia (Figure 6B). Currents recorded during hypoxia were 0.6±1.7% less than the currents recorded during exposure of cells to H2O2. This is consistent with previous data from this laboratory demonstrating that exposure of cells to the oxidizing compound DTNB prevented the effect of hypoxia on basal ICa-L.4 In the presence of hypoxia after exposure to H2O2, 3 and 10 nmol/L Iso elicited currents that were 51.8±9.3% and 87.4±6.6%, respectively, of the currents recorded during exposure to 1 µmol/L Iso. These data were significantly (P<0.05) smaller than currents recorded during hypoxia and not exposed to H2O2. In addition, exposure of cells to H2O2 did not alter currents already enhanced by 1 µmol/L Iso (a maximally stimulating concentration of the agonist). The current density recorded during exposure to 1 µmol/L Iso in room oxygen (10.2±1.5 pA/pF, n=7) was not significantly different from the current density recorded during hypoxia (10.0±0.8 pA/pF, n=8) or hypoxia and H2O2 (12.3±2.5 pA/pF, n=6).
Previous data have shown that the effect of hypoxia can be attenuated when cells are dialyzed with ßPKC peptide inhibitor, which prevents the translocation and binding of the ßPKC isoform to its receptor for activated C kinase.17 We examined whether the effect of catalase, ie, that of increasing the sensitivity of ICa-L to Iso, was mediated via the ßPKC isoform. Cells were dialyzed with catalase (500 U/mL pipette solution) and 100 to 500 nmol/L ßPKC peptide inhibitor. Under these conditions, 3 and 10 nmol/L Iso elicited currents that were 61.2±9.3% and 81.0±7.7%, respectively, of currents recorded during 1 µmol/L Iso (n=6, Figure 7A). These currents were significantly (P<0.05) smaller than currents recorded during dialysis of cells with catalase alone (Figures 1A and 2). To examine a possible role for the 
isoform and any nonspecific effects of the peptide inhibitors, cells were dialyzed with 500 nmol/L PKC peptide inhibitor and catalase. Three and 10 nmol/L Iso elicited currents that were 91.0±7.2% and 98.8±1.2%, respectively, of currents re- corded in the presence of 1 µmol/L Iso. These currents were similar to currents recorded when cells were dialyzed with catalase alone (Figure 1A) and significantly larger (P<0.05) than currents recorded when cells were dialyzed with the ßPKC peptide and catalase (Figure 7A). These data further support a possible role for H2O2 in the effects of hypoxia on the sensitivity of ICa-L to Iso. They suggest that a decrease in the levels of H2O2 mediates an increase in the sensitivity of ICa-L to Iso that involves the ßPKC isoform.
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Hypoxia Causes a Decrease in the Generation of Cellular H2O2
Our data suggest that hypoxia is associated with a decrease in the cellular level of H2O2. To confirm this, we measured the generation of H2O2 using the fluorescence indicator CM-H2DCFDA. Figure 8A illustrates the protocol used. Data acquisition began 30 minutes after loading the cell with CM-H2DCFDA (see Materials and Methods). In 5 cells exposed only to room oxygen, no difference in the generation of H2O2 was measured between 30 and 90 minutes after the addition of CM-H2DCFDA. In another set of experiments, the cells were gassed with humidified nitrogen and oxygen, which resulted in a PO2 of 15 mm Hg. Exposure to hypoxia resulted in a significant decrease in the generation of H2O2 compared with the rate measured during exposure to room oxygen (0.004±0.0021 versus 0.012±0.0026 arbitrary units, respectively [n=6], P<0.05; Figure 8A). We then expressed the rate of generation of H2O2 as the slope of signal measured between 70 and 90 minutes over the slope of signal measured between 30 and 50 minutes for normoxia only and normoxia followed by hypoxia. Figure 8B summarizes the results. These data indicate that hypoxia causes a significant decrease in the generation of H2O2 in quiescent adult guinea pig ventricular myocytes.
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Discussion |
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The results of the present study demonstrate for the first time that H2O2 regulates the sensitivity of cardiac L-type Ca2+ channels to ß-AR stimulation. When cells were perfused with pipette solution containing catalase (which specifically catalyzes the breakdown of H2O2 to water and oxygen), a significant increase in the sensitivity of ICa-L to Iso was recorded (Figures 1 and 2). Similarly, a significant increase in the sensitivity of ICa-L to Iso has been recorded during hypoxia.4 We are proposing that the effect of hypoxia on the channel is a result of changes in cellular H2O2 levels. In addition, the response could be mimicked when mitochondrial function was inhibited with myxothiazol or FCCP (Figures 4, 5, and 6A), suggesting that the source of production of H2O2 is the mitochondria. Intracellular ATP concentration was kept constant at 5 mmol/L, and intracellular pH was buffered at 7.05, indicating that the effect was not secondary to a compromise of energy metabolism. In addition, both the effect of catalase and the effect of hypoxia could be attenuated when cells were preexposed to H2O2. This occurred even when H2O2 was omitted from solutions containing Iso to minimize oxidation of the ß-AR agonist by H2O2. These results suggest that during normoxia, elevated levels of H2O2 inhibit ß-AR responsiveness of the L-type Ca2+ channel. Consistent with this concept, we have demonstrated that hypoxia is associated with a decrease in cellular H2O2 levels in cardiac myocytes, assessed using the fluorescence indicator CM-H2DCFDA. Previous studies support a decrease in H2O2 levels in response to low O2.10–13
A number of hypotheses have been proposed regarding the nature of the oxygen sensor(s) underlying hypoxic responses. It is well recognized that ion channels are responsive to changes in local oxygen levels, raising the possibility that the channel can function as the oxygen sensor or responds to changes imposed by a nearby oxygen sensor, such as NADPH-oxidase.15,18,19 Reversible alterations in channel function obtained in excised patches support this view.18 However, the mitochondria have also been considered as a potential oxygen sensor.15,16,19,20 Although the site of production of H2O2 in the mitochondria remains controversial, consistent with this hypothesis, oxygen or reactive oxygen species either directly interact with effector proteins or activate a signal transduction pathway.19 We have previously shown that hypoxia regulates ICa-L by direct inhibition of basal ICa-L and by an effect on the sensitivity of the channel to Iso that involves the ßPKC isoform.4 In support of a direct modulation of channel activity, exposure of cells to H2O2 alone had little effect on basal ICa-L and prevented the inhibition of basal ICa-L by hypoxia (Figure 6B). In addition, exposing the cells to catalase in extracellular solution only mimicked the effect of hypoxia on basal ICa-L. The magnitude of inhibition of basal ICa-L by extracellular catalase was not as great as that recorded during hypoxia alone,4 but this is not unexpected given that the cell is continuing to produce H2O2 that readily crosses the plasma membrane.8,9 This level of basal channel inhibition could also indicate that there is >1 redox site involved in the inhibition of basal ICa-L by hypoxia. Nevertheless, this is consistent with our previous results, which demonstrated that the oxidizing compound DTNB attenuates the effects of hypoxia4 and is also consistent with results in the ferret heart, in which DTNB reverses the effects of dithiothreitol.6
In the present study, we further characterize the mechanisms involved in the increased sensitivity of ICa-L to Iso during hypoxia and show that perfusing the myocytes with catalase results in a similar response. In addition, we have shown that inhibitors of the mitochondria mimic the hypoxic response, implicating the mitochondria as the source of production of H2O2. Our data indicate that catalase increases the sensitivity of the channel to Iso downstream from the ß-AR. This is because catalase also increases the sensitivity of the channel to forskolin, which directly activates adenylate cyclase. This observation is consistent with the effects of hypoxia on the sensitivity of the channel to forskolin and histamine (which binds the same cAMP-dependent pathway but via activation of H2-histaminergic receptors).4 Therefore, the effects of catalase and hypoxia are likely to be acting either on a downstream component of the cAMP pathway, PKC, or the channel itself. Previous studies support a modulation of the response of ion channel activity to PKA by PKC.21–24 In these reports, exposure of the cells to PKC elicited little or no current, but subsequent exposure to a ß-AR agonist in the continued presence of the PKC agonist resulted in a potentiated response. In fact, phosphorylation by PKC is a requirement for acute activation of some ion channels by PKA.22 We and others have previously proposed that hypoxia alters 
1 site on the L-type Ca2+ channel with modifications of cysteine residues4,5,25,26 and that one of these sites may involve the response of the channel to PKA and PKC. However, we cannot rule out the possibility that any of the effectors downstream from cAMP or PKC or a phosphodiesterase could be targets of H2O2.
The results of the present study suggest that a reduction in cellular H2O2 levels during hypoxia alters the response of the L-type Ca2+ channel to Iso. Redox modification of the channel may occur because of changes in H2O2 levels. This modification would then result in a potentiated response of the channel to a ß-AR agonist. These results support a role for H2O2 in modifying cellular responses and provide further insight into the effects of hypoxia on ion channels.
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Acknowledgments |
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This study was supported by the National Health and Medical Research Council of Australia. Dr Hool is a National Health and Medical Research Council Peter Doherty Fellow. The authors wish to thank James Lui for performing assays of H2O2 production using CM-H2DCFDA.
Received December 3, 2001; revision received August 8, 2002; accepted August 20, 2002.
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