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(Circulation Research. 2000;87:1164.)
© 2000 American Heart Association, Inc.

Cellular Biology

Hypoxia Increases the Sensitivity of the L-Type Ca²⁺ Current to ß-Adrenergic Receptor Stimulation via a C2 Region–Containing Protein Kinase C Isoform

Livia C. Hool

From the Department of Physiology, The University of Western Australia, Nedlands, Western Australia.

Correspondence to Dr Livia C. Hool, Department of Physiology, The University of Western Australia, Hackett Drive, Nedlands, WA, 6907, Australia. E-mail lhool{at}cyllene.uwa.edu.au

	Abstract

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Abstract—The effects of hypoxia on the L-type Ca²⁺ current (I_Ca-L) in the absence and presence of the ß-adrenergic receptor agonist isoproterenol (Iso) were examined. Exposing guinea pig ventricular myocytes to hypoxia alone resulted in a reversible inhibition of basal I_Ca-L. When cells were exposed to Iso in the presence of hypoxia, the K_0.5 for activation of I_Ca-L by Iso was significantly decreased from 5.3±0.7 to 1.6±0.1 nmol/L. The membrane-impermeant thiol-specific oxidizing compound 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) attenuated the inhibition of basal I_Ca-L by hypoxia 81.3±9.4% but had no effect on the increase in sensitivity of I_Ca-L to Iso. In addition, DTT mimicked the effects of hypoxia on basal I_Ca-L and the increase in sensitivity to Iso. Neither the inhibitors of guanylate cyclase LY-83583 or methylene blue nor the NO synthase inhibitor N^G-monomethyl-L-arginine monoacetate had any effect on the basal inhibition of I_Ca-L or the decrease in K_0.5 for activation of I_Ca-L by Iso during hypoxia. However, the protein kinase C (PKC) inhibitors bisindolylmaleimide I and Gö 7874 significantly attenuated the increase in sensitivity of I_Ca-L to Iso. More specifically, the response was attenuated when cells were dialyzed with a peptide inhibitor of the C2 region–containing classical PKC isoforms. The same effect was not observed with the PKC peptide inhibitor. These results suggest that hypoxia regulates I_Ca-L through the following 2 distinct mechanisms: direct inhibition of basal I_Ca-L and an indirect effect on the sensitivity of the channel to ß-adrenergic receptor stimulation that is mediated through a classical PKC isoform.

Key Words: hypoxia • ß-adrenergic receptor • L-type Ca²⁺ channels • nitric oxide • protein kinase C

	Introduction

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The effects of lowering oxygen tension (PO₂) on cellular function have been studied for some years. It is no exception that ion channel function can be modulated with alterations in oxygen tension. Recent reports have proposed that cardiac ion channel function may be modulated by redox status. In 1997, Fearon et al¹ demonstrated that the recombinant _1C subunit of the human cardiac L-type Ca²⁺ channel can be inhibited by hypoxia, and it was suggested that the effects were due to an alteration in the redox status of the channel. More direct support for the regulation of L-type Ca²⁺ channels by sarcolemma thiol redox state has been demonstrated in native channels in ferret ventricular myocytes.² It was found that thiol-specific oxidizing compounds and S-nitrosylating compounds activated the L-type Ca²⁺ current, whereas thiol-specific reducing compounds inhibited the current. They did not examine the effects of hypoxia. Nevertheless, for both cardiac and noncardiac ion channels, considerable evidence now suggests that hypoxia can directly influence ion channel function through an intrinsic redox sensor that is either part of the channel itself or closely associated with the channel.^{3 4}

The cell undergoes a number of biochemical changes during hypoxia that can also directly or indirectly regulate channel function.⁵ Some of these include changes in protein kinase/phosphodiesterase activities secondary to an increase in NO production. Campbell et al² observed that the NO donor 3-morpholinosydronimine (SIN-1) produced biphasic effects on L-type Ca²⁺ channels, which included a direct S-nitrosylation/oxidation-mediated stimulation and an indirect inhibition of the channel mediated through cGMP. In addition, a number of studies have reported that hypoxia can increase the translocation of protein kinase C (PKC) in the heart, although the functional significance of the redistribution of specific isoforms during hypoxia is still being determined.^{6 7} It is not unreasonable, therefore, to think that Ca²⁺ channel function may be regulated in a number of ways during hypoxia in addition to the more direct effects previously reported.

The effects of ß-adrenergic receptor (ß-AR) stimulation on L-type Ca²⁺ channel function under nonhypoxic conditions have been well documented.⁸ It is pertinent that during episodes of hypoxia, a generalized sympathicoadrenal activation is accompanied by an excessive release of local catecholamines in the heart.⁹ Despite this, the effects of ß-AR stimulation on cardiac L-type Ca²⁺ conductance during hypoxia have not been investigated.

The aims of this study were to determine the effects of ß-AR stimulation on native L-type Ca²⁺ channels during acute hypoxia, using the whole-cell configuration of the patch-clamp technique to enable the identification of the intracellular mechanisms involved. Consistent with effects on the recombinant _1C subunit,^{1 10} exposing guinea pig ventricular myocytes to hypoxia resulted in a decrease in basal L-type Ca²⁺ conductance (I_Ca-L). When cells were also exposed to the ß-AR agonist isoproterenol (Iso), hypoxia significantly increased the sensitivity of the channel to ß-AR stimulation. Inhibitor peptides were used to investigate the role of classical and novel PKC isoforms in the response. The PKCß peptide is derived from the C2 region of PKCß and can prevent the translocation and activation of PKC, PKCß₁, PKCß₂, and PKC but not PKC. The increase in sensitivity to ß-AR stimulation could be attenuated with the PKCß peptide but not the PKC peptide. These data provide evidence for dual regulation of L-type Ca²⁺ channels during hypoxia, including a direct effect on basal I_Ca-L and an indirect effect involving a classical PKC isoform in the presence of ß-AR stimulation.

	Materials and Methods

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Cell Isolation
Ventricular myocytes were isolated using a modification of the collagenase dissociation method.^{11 12} Adult Tricolor guinea pig hearts were subjected to coronary perfusion via the aorta on a Langendorff apparatus with a Krebs-Henseleit buffer (KHB). The KHB contained (in mmol/L) NaCl 120, KCl 4.8, CaCl₂ 1.5, MgSO₄ 2.2, NaH₂PO₄ 1.2, NaHCO₃ 25, and glucose 11 (pH maintained at 7.35). After 5 minutes of perfusion with this solution, the heart was then perfused with Ca²⁺-free KHB for an additional 5 minutes. Collagenase B (Boehringer Mannheim) was then added to achieve a final concentration of 0.27 mg/mL. After 30 to 45 minutes of digestion, the ventricles were cut down and minced in a high-K⁺ KHB containing (in mmol/L) potassium glutamate 110, KCl 25, KH₂PO₄ 10, MgSO₄ 2, taurine 20, creatine 5, EGTA 0.5, HEPES 5, and glucose 20 (pH adjusted to 7.4 with KOH). The minced tissue was gently filtered through a 1-mm mesh, and cells were allowed to settle in the high K⁺ KHB solution in an Erlenmeyer flask at 37°C. Just before use, an aliquot of cells was reintroduced to Ca²⁺ slowly by adding some of the control extracellular modified Tyrode’s solution that contained (in mmol/L) NaCl 140, CsCl 5.4, CaCl₂ 2.5, MgCl₂ 0.5, HEPES 5.5, and glucose 11 (pH adjusted to 7.4 with NaOH).

Data Acquisition and Analysis
Membrane currents were recorded using the whole-cell configuration of the patch-clamp technique.¹³ The solution in the pipettes contained (in mmol/L) CsCl 115, HEPES 10, EGTA 10, tetraethylammonium chloride 20, MgATP 5, Tris-GTP 0.1, phosphocreatine 10, and CaCl₂ 1 (pH adjusted to 7.05 at 37°C with CsOH).

Solutions were made hypoxic by bubbling the reservoir leading to the bath with 100% nitrogen and using a combination of stainless steel (Alltech) and silastic tubing (Cole-Parmer) for the delivery of solutions. For additional information on equipment used please see the online-only data supplement (available at http://www.circresaha.org). All experiments were performed at 37°C. Once the whole-cell configuration was achieved, the holding potential was set at -80 mV. Na⁺ channels and T-type Ca²⁺ channels were inactivated by applying a 50-ms prepulse to -30 mV immediately before each test pulse. The time course of changes in Ca²⁺ conductance were monitored by applying a 75-ms test pulse to 0 mV once every 10 seconds.

Results are reported as mean±SE. Statistical comparisons of responses were made between groups of cells using 1-way ANOVA and the Tukey posthoc test (Minitab).

Peptide Synthesis
The PKC peptides ßC2-4 (SLNPEWNET; PKCß; amino acids 218 to 286) and V1-2 (EAVSLKPT; PKC [14–21])¹⁴ were synthesized and purified in the Protein Facility, Biochemistry Department, The University of Western Australia. All peptides were >86% pure.

An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.

	Results

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Hypoxia Inhibits Native L-Type Ca²⁺ Channels in Guinea Pig Ventricular Myocytes
To examine the effect of hypoxia on I_Ca-L, membrane currents were measured first in cells superfused with Tyrode’s solution at room oxygen tension (150 mm Hg) and then in cells exposed to Tyrode’s solution made hypoxic to a PO₂ of 17 mm Hg. Exposure to hypoxia resulted in a decrease in basal I_Ca-L that could be reversed on restoring the bath O₂ to room oxygen tension by switching to control solution (Figure 1A). In 56 cells, exposing cells to a PO₂ of 17 mm Hg reversibly inhibited basal I_Ca-L 22.1±1.4%. This represented a decrease in current density from 4.9±0.6 to 3.8±0.5 pA/pF. To determine whether the effect of hypoxia on I_Ca-L was dependent on the level of PO₂, I_Ca-L was measured at various levels of hypoxia. In cells exposed to Tyrode’s solution made hypoxic to 80 mm Hg oxygen tension, I_Ca-L was inhibited 16.9±1.0% (n=4). Cells were then exposed to extracellular solution made hypoxic to a level of 5 mm Hg, and I_Ca-L was inhibited 21.1±2.6% (n=4). It appeared that the inhibition of I_Ca-L was dependent on the level of PO₂; however, inhibition appeared to saturate by 17 mm Hg.

View larger version (17K): [in this window] [in a new window]   Figure 1. Figure 1. Hypoxia inhibits basal ICa-L. A, Time course of changes in membrane current recorded during exposure to hypoxic Tyrode’s solution (PO2 of 17 mm Hg). a, b, and c refer to time points at which membrane currents (shown in Figure 1B) were recorded. B, Membrane currents recorded at time points in protocol illustrated in panel A. C, Mean±SE of current-voltage (I-V) relationships measured in 9 cells during voltage steps from -60 mV to +80 mV. I-Vs were recorded in 2 µmol/L nisoldipine (nisol) to indicate that calcium currents recorded were L-type.

Exposure of cells to hypoxia has been reported to induce a small shift in the current-voltage (I-V) relationship for the recombinant _1C subunit of the L-type Ca²⁺ channel.¹ In the present study, inhibition by hypoxia was most obvious as a reduction in the amplitude of I_Ca-L at -10, 0, and +10 mV, and no apparent shift in the I-V relationship was observed (n=9; Figure 1C).

Hypoxia Increases the Sensitivity of I_Ca-L to ß-AR Stimulation
The effect of hypoxia on I_Ca-L in the presence of a ß-AR agonist, Iso, was examined. For all further hypoxia studies, cells were exposed to a PO₂ of 17 mm Hg. In the absence of hypoxia, exposure of myocytes to 1 nmol/L Iso produced a subthreshold response, and the current was maximally stimulated in the presence of 100 nmol/L (Figure 2A). The K_0.5 for activation of the Ca²⁺ current in the absence of hypoxia was 5.3±0.7 nmol/L (Figure 3A). When cells were exposed to hypoxia alone, as before, basal I_Ca-L was inhibited (Figure 2B). This time the threshold concentration for producing a response to Iso was 0.1 to 1 nmol/L. The concentration of Iso that produced a half-maximal activation (K_0.5) of the Ca²⁺ current was 1.6±0.1 nmol/L, and the current was maximally stimulated with 10 nmol/L Iso (Figure 3A). The increase in sensitivity to Iso was not due to a decrease in the rate of oxidative degradation of the ß-AR agonist during bubbling with nitrogen (see online-only data supplement available at http://www.circresaha.org). In addition, hypoxia did not alter the response to a maximally stimulating concentration of Iso (see online-only data supplement). These findings demonstrate that exposing myocytes to hypoxia resulted in a significant (P<0.001) increase in the sensitivity of I_Ca-L to activation by ß-AR stimulation.

View larger version (20K): [in this window] [in a new window]   Figure 2. Figure 2. Hypoxia increases the sensitivity of ICa-L to ß-AR stimulation. A, Time course of changes in membrane current from a cell exposed to increasing concentrations of Iso in the absence of hypoxia, including membrane currents recorded (inset). a through e refer to time points at which membrane currents (shown in inset) were recorded. B, Representative experiment from a cell exposed to increasing concentrations of Iso in the presence of hypoxia including membrane currents recorded (inset). a through f refer to time points at which membrane currents (shown in inset) were recorded.

View larger version (17K): [in this window] [in a new window]   Figure 3. Figure 3. A, Concentration dependence of Iso activation of ICa-L in the absence (n=5 to 11 at each data point) and presence (n=5 to 10 at each data point) of hypoxia. Exposure to 10 µmol/L Iso represented a supramaximally stimulating concentration of agonist. Ca2+ conductance (GCa) measured at each concentration of Iso was normalized to GCa measured in the presence of 1 µmol/L Iso in the same cell. Data were fit to a logistic equation using a nonlinear least-squares curve-fitting routine (SigmaPlot, SPSS Inc.). B, Mean±SE of I-V relationships recorded in 9 cells during exposure to 10 nmol/L Iso in the absence (triangles) and presence of hypoxia (circles) and 10 nmol/L Iso during hypoxia in the presence of 2 µmol/L nisoldipine.

Figure 3B illustrates the effects of Iso on the I-V relationship for the channel in the absence and presence of hypoxia. It is well documented that Iso shifts the peak current in a negative direction.¹⁵ Consistent with previously published data, 10 nmol/L Iso alone shifted the peak current 10.8±2.0 mV in the negative direction relative to the peak current recorded in control (no hypoxia) or hypoxia alone (n=6). However, in 9 cells, exposure to 10 nmol/L Iso in the presence of hypoxia increased the magnitude of the peak current without further shifting the I-V relationship (-11.7±1.7 mV). This was not statistically different from the potential at which the peak current was recorded in Iso alone. The increase in magnitude of the current recorded during Iso in the presence of hypoxia remained prominent at more positive potentials up to +50 mV (Figure 3B).

To examine the possibility that hypoxia may be acting at the level of the ß-AR, experiments were performed to determine the effect of hypoxia on I_Ca-L activated by histamine. Apart from binding H₂-histaminergic receptors, histamine activates I_Ca-L through the same cAMP-dependent pathway used by ß-adrenergic agonists. In the absence of hypoxia, 30 nmol/L histamine produced a response that was 9.0±3.4% of the current elicited by 3 µmol/L histamine, a maximally stimulating concentration of the agonist (n=6, Figure 4A). Exposure to 30 nmol/L histamine in the presence of hypoxia, however, resulted in a current that was 27.3±3.6% of the current elicited by 3 µmol/L histamine (n=7; Figure 4B). This was a statistically significant increase in the magnitude of the response. Similar results were recorded with forskolin (see online-only data supplement). These results suggest that hypoxia was increasing the sensitivity of the channel to ß-AR stimulation by acting at a level downstream from the ß-AR.

View larger version (15K): [in this window] [in a new window]   Figure 4. Figure 4. Hypoxia increases the sensitivity of ICa-L to activation by the H2-receptor agonist histamine. A, Time course of changes in membrane current from a cell exposed to a threshold concentration of histamine (30 nmol/L) and a maximally stimulating concentration of histamine (3 µmol/L) in the absence of hypoxia. B, Representative experiment from a cell exposed to 30 nmol/L histamine and 3 µmol/L histamine in the presence of hypoxia.

Basal Inhibition of I_Ca-L and the Altered ß-AR Sensitivity During Hypoxia Are Mediated Through Distinct Mechanisms
To examine whether the effects of hypoxia on basal I_Ca-L and the increased sensitivity of I_Ca-L to Iso may involve the reduction of extracellular thiol groups, cells were exposed to hypoxia in the presence of the thiol-specific oxidizing agent 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) and Iso. Figure 5A illustrates the protocol in a typical experiment. Hypoxia alone inhibited basal I_Ca-L on average 24.5±3.8% (n=5). However, subsequent application of 200 µmol/L DTNB significantly attenuated the inhibition of basal I_Ca-L by 81.3±9.4%. In the continued presence of hypoxia and DTNB, 3 nmol/L Iso activated a current that was 73.1±7.0% of the current elicited by 1 µmol/L Iso, and 10 nmol/L Iso activated a current that was 90.0±5.2% of the current elicited by 1 µmol/L Iso. This was not statistically significantly different from the currents activated by 3 and 10 nmol/L Iso during hypoxia in the absence of DTNB (70.9±10.7, n=8; 98.0±2.0%, n=5, respectively). DTNB is relatively membrane impermeant.^{16 17} These data imply that oxidation of thiol groups on the extracellular segment of the channel can reverse the effects of hypoxia on basal I_Ca-L, suggesting that basal inhibition is a result of the reduction of thiol groups on the channel or an intrinsic protein associated with the channel. If this were true, one could predict that in the presence of DTNB, hypoxia should not alter I_Ca-L. To test this, 6 cells were exposed first to DTNB and then to DTNB in the presence of hypoxia. Interestingly, exposing cells to DTNB alone resulted in a 26.3±2.2% decrease in basal I_Ca-L. However, when cells were exposed to DTNB in the presence of hypoxia, there was very little further change in current (0.9±0.5% decrease in current). These results support the idea that the inhibition of basal I_Ca-L involves the modification of thiol groups on or near the channel. In addition, the increase in sensitivity of I_Ca-L to Iso during hypoxia was unchanged in the presence of DTNB, suggesting that the mechanism for this effect was different from the mechanism involved in basal inhibition of the current by hypoxia.

View larger version (16K): [in this window] [in a new window]   Figure 5. Figure 5. A, Thiol-specific oxidizing compound DTNB reverses the effects of hypoxia on basal ICa-L, without altering the increased sensitivity of ICa-L to Iso. A, Time course of changes in membrane current from a cell exposed to hypoxia, 200 µmol/L DTNB, and increasing concentrations of Iso as indicated. B, DTT mimics the effects of hypoxia on basal ICa-L and the increase in sensitivity of ICa-L to Iso. Shown is a representative experiment from a cell exposed to 1 mmol/L DTT and increasing concentrations of Iso as indicated.

These results, however, do not preclude the possibility that the mechanisms of both effects may involve the reduction of critical thiol groups. If this were true, then exposing cells to a thiol-specific reducing agent would be expected to reproduce either or both effects. To test this, cells were exposed to the membrane-permeant DTT (1 mmol/L) alone followed by DTT in the presence of 3 and 10 nmol/L Iso. Figure 5B illustrates the effects of DTT. In 6 cells, DTT alone inhibited basal I_Ca-L 24.4±4.5%. This was similar to the effects of hypoxia on basal I_Ca-L (22.1±1.4%, n=56). Subsequent exposure to 3 and 10 nmol/L Iso in the continued presence of DTT resulted in currents that were on average 71.4±14.9% and 97.6±1.9% of the current elicited by 1 µmol/L Iso. This was similar to the currents activated by 3 and 10 nmol/L Iso during hypoxia (see Figure 3). Exposure to DTT appeared to mimic the effects of hypoxia on basal I_Ca-L and the altered sensitivity to Iso. Similar effects were obtained when cells were exposed to DTT and histamine (see online-only data supplement). These data indicate that the inhibition of basal I_Ca-L and the increase in sensitivity of I_Ca-L to Iso during hypoxia may involve the reduction of thiol groups. Because DTNB did not alter the increase in sensitivity of I_Ca-L to Iso, it is likely that the sites of the mechanisms for the effects differ.

The Increase in Sensitivity of I_Ca-L to ß-AR Stimulation Does Not Involve a NO-Dependent Mechanism
A possible role for NO in the increase in sensitivity of I_Ca-L to Iso during hypoxia was investigated. Three sets of experiments were performed to determine whether cGMP or NO synthase was involved. Neither LY-83583 (20 µmol/L), methylene blue (25 µmol/L), nor N^G-monomethyl-L-arginine monoacetate (L-NMMA; 100 µmol/L) had any effect on the increase in sensitivity of I_Ca-L to Iso during hypoxia (see Figures 6A and 6B), strongly suggesting that the mechanism does not involve a NO-dependent process.

View larger version (21K): [in this window] [in a new window]   Figure 6. Figure 6. Inhibition of guanylate cyclase or NO synthase does not alter the increase in sensitivity of ICa-L to Iso during hypoxia. A, Time course of changes in membrane current recorded from a cell dialyzed with the guanylate cyclase inhibitor LY-83583 (20 µmol/L) and increasing concentrations of Iso in the presence of hypoxia. B, Summary of the effects of LY-83583 (H+LY), methylene blue (H+M) and L-NMMA (H+LN) on the magnitude of the Ca2+ current activated by 3 and 10 nmol/L Iso in the presence of hypoxia. Responses to 3 and 10 nmol/L Iso were normalized to 1 µmol/L Iso in the same cell. Data are mean±SE of 5 experiments, except the column representing the response to 3 nmol/L Iso in the presence of hypoxia alone (H) where the mean±SE of 8 experiments is shown. *Statistical significance (P<0.05) between control (Con; cells not exposed to hypoxia or inhibitors) and all other groups within each concentration of Iso.

Inhibition of PKC Attenuates the Increase in Sensitivity of I_Ca-L to Iso
A possible role for PKC in the increase in sensitivity of I_Ca-L to Iso during hypoxia was examined. The first group of experiments involved the use of pharmacological inhibitors. Cells were superfused with the highly specific PKC inhibitor bisindolylmaleimide (Bis) I at a concentration of 300 nmol/L, followed by hypoxia and increasing concentrations of Iso. Exposure to 3 and 10 nmol/L Iso in the presence of Bis I and hypoxia resulted in a significant decrease (P<0.05) in the magnitude of the response to the ß-AR agonist compared with the response during hypoxia in the absence of Bis I (Figure 7). However, exposure to the inactive analogue Bis V using the same protocol had no effect on the increase in sensitivity of I_Ca-L to Iso. In addition, in the absence of hypoxia, Bis I did not alter the response of I_Ca-L to Iso. In 5 cells, 3 and 10 nmol/L Iso activated I_Ca-L 39.7±3.0% and 67.2±3.5% of the current activated by 1 µmol/L Iso. This was not statistically different from the currents recorded in cells in the absence of Bis I (Figure 3). A second PKC inhibitor, Gö 7874, was applied at a concentration of 300 nmol/L. This alternative PKC inhibitor also significantly attenuated the Iso response during hypoxia (Figure 7).

View larger version (19K): [in this window] [in a new window]   Figure 7. Figure 7. Summary of the effects of pharmacological and peptide inhibitors of PKC on the increase in sensitivity of ICa-L to Iso during hypoxia. Peak inward currents recorded in response to 3 and 10 nmol/L Iso were normalized to 1 µmol/L Iso in the same cell. Data are mean±SE of the number of cells as indicated in parentheses. *Statistical significance (P<0.05) when compared with hypoxia alone (H) within each concentration of Iso. A, Effects of Bis I (+Bis I), Bis V (+Bis V), Gö 7874 (+Go), PKCß peptide (+ßPKC), and PKC peptide (+PKC) on the normalized mean inward current recorded during exposure to 3 nmol/L Iso and hypoxia. B, Effects of Bis I, Bis V, Gö 7874, PKCß peptide, and PKC peptide on the normalized mean inward current recorded during exposure to 10 nmol/L Iso and hypoxia.

To determine which isoforms of PKC may be involved in the hypoxic responses, 100 nmol/L PKCß peptide inhibitor or 100 nmol/L PKC peptide inhibitor was added to the pipette solution in separate sets of experiments. Cells were dialyzed with the PKCß peptide and then exposed to hypoxia followed by 3 and 10 nmol/L Iso. The PKCß peptide significantly attenuated the increase in sensitivity of I_Ca-L to Iso (Figures 7 and 8A). To determine a possible role for the isoform and any nonspecific effect of the peptide inhibitors on L-type Ca²⁺ conductance, cells were dialyzed with 100 nmol/L PKC peptide and exposed to increasing concentrations of Iso in the presence of hypoxia. Contrary to the effect of PKCß peptide inhibitor, intracellular dialysis of cells with PKC peptide did not alter the increase in sensitivity of I_Ca-L to Iso (Figures 7 and 8B). These results strongly suggest that the activation of PKC is involved in the altered ß-adrenergic responses of I_Ca-L during hypoxia, implicating a classical isoform of PKC.

View larger version (15K): [in this window] [in a new window]   Figure 8. Figure 8. The PKCß peptide attenuates the increase in sensitivity of ICa-L to Iso, whereas the PKC peptide does not. Shown is time course of changes in membrane current from a cell dialyzed with 100 nmol/L PKCß peptide and exposed to hypoxia and increasing concentrations of Iso as indicated (A) and 100 nmol/L PKC peptide and exposed to hypoxia and increasing concentrations of Iso as indicated (B).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The results of the present study describe the effects of lowering PO₂ on L-type Ca²⁺ channel function in guinea pig ventricular myocytes. Specifically, the effects of hypoxia alone on native channel function and the effects of hypoxia in the presence of ß-AR stimulation were investigated. Consistent with previously reported results, hypoxia alone caused a reversible inhibition of basal I_Ca-L.^{1 10 18} The effects of hypoxia on ion channel function have been proposed to involve a redox modulation of channel activity.^{3 4} The results of the present study support this hypothesis, because the inhibition of I_Ca-L in response to hypoxia could be reversed with the membrane-impermeant oxidizing compound DTNB (see Figure 5A). In addition, the inhibition could be mimicked with the thiol-specific reducing agent DTT (see Figure 5B). Similar results were observed in native ferret L-type Ca²⁺ channels in which DTT inhibited the channel and subsequent application of DTNB reversed the effects of DTT.² The presence of channel auxiliary subunits (and their cysteine residues) may be a necessary requirement for these responses, given that DTT has no effect or increases the current produced by the recombinant ₁ subunit of the L-type Ca²⁺ channel.^{10 19}

The effects of hypoxia on the sensitivity of I_Ca-L to Iso, histamine, and forskolin identify a novel regulation of the L-type Ca²⁺ channel by ß-AR stimulation involving the second messenger PKC. Because hypoxia also increased the sensitivity of the channel to activation by histamine and forskolin, it would appear that the effects involve the cAMP pathway downstream from the ß-AR. There is good evidence that guinea pig ventricular myocytes express a constitutive NO synthase.^{20 21} Despite the documented involvement of NO in altered myocardial function associated with hypoxia,^{22 23} neither inhibition of NO synthase nor guanylate cyclase had any effect on the basal inhibition of I_Ca-L or the increase in sensitivity to ß-AR stimulation. These data would appear to exclude the involvement of NO in the increased ß-adrenergic responses in this study.

The fact that DTT mimicked both the basal inhibition of I_Ca-L and the increase in sensitivity to ß-AR stimulation by hypoxia (Figure 5B) suggests that the mechanisms of both responses involve the reduction of thiol groups. Because the thiol-specific oxidizing agent DTNB had no effect on the increased sensitivity of I_Ca-L to Iso, it would appear that there are 2 potential redox sites modulating channel function. What is the likely candidate mediating the increase in sensitivity of the channel to ß-AR stimulation? The oxidation of critical cysteine residues of type I adenylate cyclase can inhibit stimulation of this enzyme by calcium and calmodulin.²⁴ It would seem plausible that the converse may increase enzyme activity. However, cardiac cells do not possess a type I adenylate cyclase. Additionally, although PKC can regulate the activity of adenylate cyclase, the isoforms of adenylate cyclase in cardiac muscle are also not affected by PKC.²⁵

Both pharmacological inhibitors of PKC, Bis I and Gö 7874, attenuated the increase in sensitivity of I_Ca-L to Iso. The strongest evidence for an involvement of PKC, however, is seen with the effects of the PKC peptide inhibitors (Figures 7 and 8). The C2-4 peptide derived from PKCß can block classical PKC isoforms.²⁶ These include PKC, PKCß₁, PKCß₂, and PKC and are calcium sensitive. Guinea pig adult heart expresses the , ß_2, , , and isoforms.⁶ The novel isoform PKC can be activated by diacylglycerol but not by calcium. Intracellular application of PKCß peptide inhibitor, which prevents the translocation and binding of the isoform to its receptor for activated C kinase,²⁶ attenuated the increase in sensitivity of I_Ca-L to Iso (Figure 8A). Dialysis of cells with PKC peptide inhibitor did not (Figure 8B). The involvement of PKC, in particular the PKCß isoform, in hypoxic responses has been described before.^{6 7 27} The results of this study strongly implicate a classical PKC isoform in the regulation of cardiac L-type Ca²⁺ channels during hypoxia. Although any one of the classical isoforms could be the mediator of the hypoxic response, the ß isoform appears to play a dominant role in many hypoxic and ischemic processes.

It is also possible that PKC may be facilitating protein kinase A (PKA)–dependent channel phosphorylation by directly stimulating PKA or decreasing phosphatase activity. Because there is no evidence to suggest that hypoxia can directly modify PKA or phosphatase activity, what is more likely is that the 2 mechanisms involve 1 or more sites on the channel with modifications of cysteine residues. For example, a cytosolic site of action could involve the modulation of the response of the channel to PKA and PKC. Some support for this argument comes from studies on the effects of PKC and PKA activation on epithelial and cardiac CFTR Cl^- channels. In these studies, exposure of cells to an agonist of 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.^{28 29 30 31} Like CFTR Cl^- channels, the L-type Ca²⁺ channel appears to possess 2 sites of phosphorylation by PKA, each conferring distinct modifications of channel behavior.^{32 33} This is not consistent with reports that argue that activation of PKC by phorbol esters inhibits basal I_Ca-L.³⁴ However, this response was not determined in the presence of hypoxia. What is possible is that hypoxia induces a translocation of a classical PKC isoform that does not by itself increase basal I_Ca-L, but in the presence of ß-AR stimulation results in a significant increase in current due to a modification in channel phosphorylation.

	Acknowledgments

 
This study was supported by the National Health and Medical Research Council of Australia. L.C.H. is a National Health and Medical Research Council Peter Doherty Fellow. The author thanks Associate Professor Barry Madsen for reading and critiquing the manuscript.

Received May 18, 2000; revision received October 12, 2000; accepted October 12, 2000.

	References

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