Hypoxia Alters the Sensitivity of the L-Type Ca2+ Channel to α-Adrenergic Receptor Stimulation in the Presence of β-Adrenergic Receptor Stimulation
Abstract—The effects of α-adrenergic receptor (α-AR) stimulation alone and the effects in the presence of β-adrenergic receptor (β-AR) stimulation were examined on L-type Ca2+ currents (ICa-L) in the absence and presence of hypoxia. The α-AR agonist methoxamine either had no effect or had a slight inhibitory effect on basal ICa-L in the absence and presence of hypoxia. Hypoxia significantly decreased the K0.5 for activation of ICa-L by norepinephrine from 79.8±6.6 to 13.3±0.7 nmol/L. To determine whether hypoxia specifically altered the sensitivity of the channel to α-AR stimulation, cells were exposed to increasing concentrations of methoxamine in the presence of 100 nmol/L isoproterenol (Iso). In the absence of hypoxia, methoxamine inhibited the Iso-activated ICa-L in a concentration-dependent manner with an EC50 of 86.9±9.9 μmol/L. However, in the presence of hypoxia, the EC50 for inhibition of ICa-L by methoxamine was significantly increased to 266.7±10.8 μmol/L. Methoxamine had little effect on ICa-L activated by forskolin or histamine in the absence or presence of hypoxia. In addition, inhibition of protein kinase C by bisindolylmaleimide 1 or protein kinase C β peptide inhibitor had no effect on the methoxamine-induced antagonism of ICa-L in the absence or presence of hypoxia. The tyrosine kinase inhibitor genistein attenuated the methoxamine response in nonhypoxic cells only. However, during hypoxia it was attenuated with the phospholipase A2 inhibitors mepacrine and indomethacin. These findings represent a novel regulation of the L-type Ca2+ channel by the phospholipase A2 pathway and illustrate the complexity of regulation of the channel under hypoxic conditions.
It is well recognized that cardiac Ca2+ channel function is regulated by sympathoadrenergic stimulation. Activation of β-adrenergic receptors (β-AR) results in an increase in peak inward current and a slowing of inactivation of the L-type Ca2+ channel (ICa-L) via protein kinase A–dependent phosphorylation of the channel.1
Although the functional existence of cardiac α-adrenergic receptors (α-ARs) has long been recognized, the effects of α-AR stimulation on ICa-L are still not fully understood. Even less work has been undertaken investigating the effects of α-AR stimulation in the presence of β-AR stimulation. However, among the few studies reported, there is good consensus that α-AR stimulation inhibits β-AR–activated responses. This has been reported for L-type Ca2+ channels,2 3 4 5 for the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel,6 and in studies on cardiac contractile function.7 What is not clear is how α-AR stimulation mediates the inhibition of β-AR responses. Two candidates to date include a possible involvement of protein kinase C (PKC)4 or tyrosine kinase.5
During episodes of hypoxia, the effects of sympathetic stimulation on ion channel function become more marked as excessive release of catecholamines is associated with a generalized sympathicoadrenal activation.8 Results from this laboratory have previously reported that hypoxia can increase the sensitivity of ICa-L to β-AR stimulation. This occurs via the activation of a C2 region–containing isoform of PKC.9 However, it is not known what effect hypoxia has on the response of ICa-L to α-AR stimulation. Myocardial ischemia is known to cause activation of a number of second-messenger pathways in the heart, including the products of phospholipase metabolism. The activation of phospholipase A2 (PLA2) is critical for the generation of bioactive lipids such as arachidonic acid and its metabolites.10 An accumulation of lysophosphatidylcholine and arachidonic acid has been found in ischemic myocardium and has been associated with reperfusion arrhythmias.11 In addition, hypoxia can induce the translocation and activation of phosphatidylinositol-specific PKC isoforms in the heart.12 It is likely therefore that sympathoadrenergic regulation of cardiac Ca2+ channels during hypoxia is more complex than the effects reported under nonhypoxic conditions.
The aims of this study were to characterize the effects of α-AR stimulation on basal ICa-L in the presence and absence of acute hypoxia and the effects of hypoxia on α-AR stimulation in the presence of β-AR stimulation. α-AR stimulation had little effect on basal ICa-L in the absence of hypoxia. Similar effects were recorded in the presence of hypoxia. However, hypoxia did regulate α-AR responses distinctly differently from the effects reported under nonhypoxic conditions. Consistent with previous results, the α-AR agonist methoxamine inhibited ICa-L activated by isoproterenol (Iso) via a tyrosine kinase-dependent mechanism in nonhypoxic cells. However, in the presence of hypoxia, the sensitivity of the Iso-activated current to methoxamine was significantly decreased and the mechanism involved PLA2. These results represent a unique regulation of L-type Ca2+ channels and provide significant insight into the regulation of ion channels under hypoxic conditions.
Materials and Methods
Isolation of Ventricular Myocytes
Cells were isolated as previously described.9 13 Briefly, hearts of adult guinea pigs were subject to retrograde coronary perfusion for 5 minutes with a Krebs Henseleit buffer (KHB), which 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). The heart was then perfused with Ca2+-free KHB for an additional 5 minutes after which collagenase B (Boehringer Mannheim) was added. After 30 to 45 minutes of digestion, the ventricles were cut down and minced in a solution 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 then gently filtered, and cells were allowed to settle in this solution before use.
Induction of Hypoxia and Data Acquisition
Currents were measured in 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). The solution was made hypoxic by bubbling with 100% nitrogen as previously described.9 All hypoxia experiments were performed at ≈17 mm Hg oxygen tension as determined by an oxygen-sensitive probe.9 Membrane currents were recorded using the whole-cell configuration of the patch-clamp technique. Some experiments were performed using the perforated patch technique (see the expanded Materials and Methods section in the online data supplement). Microelectrodes with tip diameters of 3 to 5 μm and resistances of 0.5 to 1.5 MΩ 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).
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. The interface between the intracellular and extracellular solutions at the tip of the patch pipette produced a junction potential of ≈5 mV. The data were not compensated for this offset. 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 was 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 post hoc test (Minitab).
An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.
Effects of α-AR Stimulation on Basal ICa-L
Myocytes were exposed to the α-AR agonist methoxamine in the presence and absence of hypoxia. In the absence of hypoxia, methoxamine either had no effect or caused a slight decrease in basal ICa-L that was reversed on washout of the drug with control Tyrode’s solution (Figure 1A⇓). In 6 cells, 25 μmol/L methoxamine inhibited the current 8.3±5.4% (P<0.05), whereas 100 μmol/L methoxamine only caused a further 1.7±0.5% (NS) decrease in the basal current. It has been previously reported that exposing the cardiac L-type Ca2+ channel or a subunit of the channel to hypoxia results in a reversible decrease in basal ICa-L.9 14 15 16 In the present study, a similar inhibition of basal ICa-L was recorded in the presence of hypoxia. In 5 cells, switching the external solution from one containing room oxygen tension (≈150 mm Hg) to a hypoxic solution (≈17 mm Hg) inhibited the basal current 24.9±7.2% (P<0.05). Subsequent exposure to 25 μmol/L methoxamine in the continued presence of hypoxia resulted in a further decrease (11.0±2.7%; P<0.05) in basal ICa-L. Exposure to 100 μmol/L methoxamine did not alter the current any further.
Cells were also exposed to 20 μmol/L phenylephrine (PE) in the presence of the β-AR antagonist propanolol (10 μmol/L). Because PE binds both β-AR and α-AR, simultaneously exposing cells to the β-AR antagonist propanolol ensures that only the effects of α-AR stimulation remain. In the absence of hypoxia, PE and propanolol decreased basal ICa-L 10.1±3.8% (n=4). Similarly in the presence of hypoxia, PE and propanolol caused a 7.0±3.4% inhibition of basal ICa-L (n=3).
Exposure of cells to methoxamine resulted in a slight decrease in the amplitude of ICa-L at positive potentials without shifting the current-voltage (I-V) relationship (Figure 1C⇑). A similar effect of methoxamine on the I-V relationship was recorded during hypoxia (Figure 1D⇑). Overall, these results indicate that α-AR stimulation can slightly inhibit basal ICa-L independent of the surrounding oxygen conditions. Hypoxia did not prevent the inhibition of basal ICa-L by methoxamine or PE in the presence of propanolol.
Hypoxia Alters the Sensitivity of ICa-L to Norepinephrine (NE)
To determine what effect, if any, hypoxia may have on the activation of ICa-L in the presence of both α-AR and β-AR stimulation, cells were exposed to increasing concentrations of NE and the concentration dependence of ICa-L on NE was determined. In the absence of hypoxia, the K0.5 for activation of the current was 79.8±6.6 nmol/L (Figure 2⇓). However, when cells were exposed to hypoxia, the K0.5 for activation of ICa-L was significantly decreased to 13.3±0.7 nmol/L. Because hypoxia can increase the sensitivity of ICa-L to Iso in the absence of α-AR stimulation,9 these data are consistent with the idea that the increase in sensitivity of ICa-L to NE was the result of an increase in the sensitivity of the channel to the β-AR component of the NE response.
Hypoxia Alters the Sensitivity of ICa-L to α-AR Stimulation
The increase in sensitivity of ICa-L to NE may also be due to an effect of hypoxia on the α-AR component of the response. To determine what effect hypoxia may have on α-AR stimulation, cells were exposed to increasing concentrations of an α-AR agonist in the presence of a constant concentration of a β-AR agonist. In the absence of hypoxia, cells were first exposed to 100 nmol/L Iso followed by Iso plus increasing concentrations of methoxamine. Consistent with previous reports, methoxamine inhibited the Iso-activated current.5 6 Exposure to 100 μmol/L methoxamine resulted in approximately half-maximal inhibition of ICa-L, whereas 300 μmol/L methoxamine completely inhibited the Iso-activated current (Figure 3A⇓). In the absence of hypoxia, the EC50 for inhibition of ICa-L by methoxamine was 86.9±9.9 μmol/L (Figure 4⇓). However, in the presence of hypoxia, 100 μmol/L methoxamine had little effect on the Iso-activated current, whereas 300 μmol/L methoxamine produced approximately half-maximal inhibition (Figure 3B⇓). This time methoxamine inhibited the Iso-activated current with an EC50 of 266.7±10.8 μmol/L (P<0.01; Figure 4⇓).
To ensure that the effects of methoxamine on the Iso-activated current were mediated via the α-AR, cells were incubated in the α1-AR antagonist prazosin for at least 1 hour before measurement of currents. In the absence of hypoxia, 2 μmol/L prazosin significantly attenuated the methoxamine response. Cells were sequentially exposed to 30, 100, and 300 μmol/L methoxamine in the presence of 100 nmol/L Iso and prazosin. Under these conditions, methoxamine inhibited the Iso-activated current 2.0±1.4%, 7.1±4.3%, and 16.9±9.1% (n=5). These values are significantly (P<0.05) lower than the inhibition recorded in the absence of prazosin (see Figures 3A⇑ and 4⇑). Similar results were recorded in the presence of hypoxia. Cells exposed to 100, 300, and 500 μmol/L methoxamine in the presence of Iso and prazosin resulted in 3.1±2.1%, 26.2±3.2%, and 32.4±8.4% inhibition of the Iso-activated current (n=5). Again, exposure to prazosin significantly attenuated the methoxamine response. These results strongly suggest that the effects of methoxamine on Iso-activated ICa-L are mediated via the α1-AR both under conditions of room oxygen tension and during hypoxia.
Figures 3C⇑ and 3D⇑ illustrate the effects of Iso and methoxamine 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.1 17 Consistent with previously published data, 100 nmol/L Iso shifted the peak current ≈9.3±0.9 mV in the negative direction relative to control in the absence of hypoxia. The addition of 100 μmol/L methoxamine resulted in a similar shift (−8.3±0.4 mV) and a decrease in the peak current (Figure 3C⇑). Previous studies have shown that hypoxia does not induce a shift in the I-V relationship for the native L-type Ca2+ channel.9 The effects of methoxamine and Iso in the presence of hypoxia on the I-V relationship were compared therefore with the effects of hypoxia alone. In the presence of hypoxia, Iso shifted the I-V relationship −9.9±3.5 mV relative to hypoxia. Exposure of cells to 300 μmol/L methoxamine and Iso resulted in a decrease in the peak current and a shift of −6.7±2.5 mV relative to hypoxia (Figure 3D⇑). These results suggest that the effects of methoxamine on the I-V relationship are similar under conditions of room oxygen tension and hypoxia.
Effect of Methoxamine on ICa-L Activated by Forskolin
Previous studies on the CFTR Cl− channel and the L-type Ca2+ channel have suggested that the mechanism for the methoxamine-induced inhibition lies close to the β-AR, given that methoxamine could not inhibit currents activated by forskolin or histamine.5 6 To determine whether the methoxamine-induced inhibition of ICa-L during hypoxia might be due to a direct effect on the L-type Ca2+ channel or an indirect effect on the second-messenger pathway involved in the channel activation, cells were exposed to the adenylate cyclase agonist forskolin and methoxamine. Methoxamine had little effect on ICa-L activated by 3 μmol/L forskolin. In the absence of hypoxia, forskolin and 100 μmol/L methoxamine activated a current that was 97.2±1.3% of the current activated by forskolin alone. Cells were also exposed to forskolin in the presence of 300 and 500 μmol/L methoxamine. Under these conditions, forskolin and methoxamine activated currents that were 85.1±3.2% and 76.3±4.0% of the current activated by forskolin alone (Figure 5A⇓; n=5). This slight inhibition was significantly less than the effects of the same concentrations of methoxamine on the Iso-activated current (Figure 4⇑). Similar results were recorded in the presence of hypoxia. Methoxamine and forskolin (100 μmol/L) activated a current that was 95.8±3.4% of the current produced in the presence of forskolin alone. In addition, 300 and 500 μmol/L methoxamine activated ICa-L 89.6±5.5% and 78.6±8.1% of the current activated by forskolin alone (Figure 5B⇓; n=5). These results were similar to the effects of methoxamine on forskolin-activated currents in the absence of hypoxia. These data suggest that the effects of methoxamine under room oxygen tension and during hypoxia are mediated “upstream” from adenylate cyclase in the cAMP cascade.
It was also found that methoxamine had little effect on ICa-L activated by the H2-histaminergic receptor agonist histamine (3 μmol/L) both in the absence and presence of hypoxia (see the online data supplement). Consistent with previously reported data, this suggests that methoxamine may be acting close to or at the level of the β-AR under both conditions.
Methoxamine-Induced Inhibition Does Not Involve PKC
Previous studies in rat cardiac myocytes under nonhypoxic conditions have suggested that α-AR inhibition of β-AR–activated ICa-L involves PKC, specifically a C2 region–containing PKC isoform.4 Possible roles for PKC in the methoxamine-mediated inhibition under room oxygen tension and during hypoxia were examined. First, in the absence of hypoxia, cells were exposed to the highly specific inhibitor of PKC, bisindolylmaleimide 1 (Bis 1), at a concentration of 300 nmol/L before exposure to 100 nmol/L Iso followed by Iso plus methoxamine. In 4 cells, Bis 1 had no effect on basal ICa-L (Figure 6A⇓). In the continued presence of Bis 1, 100 μmol/L methoxamine inhibited the Iso-activated current 34.7±9.1% followed by a 77.4±7.3% inhibition during exposure to 300 μmol/L methoxamine. These results are similar to the effects of methoxamine in the absence of Bis 1 under nonhypoxic conditions (Figure 4⇑), suggesting that PKC does not play a role in the inhibitory response. These data are consistent with reports on the effects of methoxamine on the Iso-activated CFTR Cl− channel in guinea pig cardiac myocytes in which no evidence was found for an involvement of PKC.6
Next, a possible role for PKC under hypoxic conditions was examined. Cells were exposed to Bis 1 in the presence of hypoxia followed by 100 nmol/L Iso and methoxamine (Figure 6B⇑). In the presence of Bis 1 and hypoxia, 300 μmol/L methoxamine inhibited the Iso-activated current 60.3±11.1% and 500 μmol/L methoxamine inhibited the current 99.0±1.0% (n=5). These results are similar to the effects of methoxamine in the presence of hypoxia and in the absence of Bis 1 (Figure 4⇑). Similar results were recorded with cells dialyzed with 100 nmol/L PKCβ peptide inhibitor, which prevents the activation and translocation of C2 region–containing PKC isoforms18 (n=5; see online data supplement). Overall, these results appear to rule out a possible role for PKC in the methoxamine-mediated inhibition both under conditions of room oxygen tension and during hypoxia.
Genistein Attenuates the Methoxamine-Induced Inhibition Under Nonhypoxic Conditions Only
Recent studies have implicated a role for tyrosine kinase in the methoxamine-induced inhibition of Iso-activated L-type Ca2+ currents.5 Therefore, a possible role for tyrosine kinase in the inhibition of Iso-activated ICa-L was examined. First, the effect of the tyrosine kinase inhibitor genistein was examined on the methoxamine response in the absence of hypoxia. Cells were exposed to 50 μmol/L genistein before 100 nmol/L Iso followed by Iso plus methoxamine (Figure 7A⇓). Consistent with previous reports,13 genistein alone caused a 47.8±13.8% reduction in basal ICa. In the continued presence of genistein, methoxamine had little effect on the Iso-activated ICa-L. Methoxamine at 100 μmol/L and Iso activated a current that was 99.0±1.0% of the current activated by Iso alone; 300 μmol/L methoxamine activated a current that was 82.6±3.5% of the current activated by Iso alone (n=5). The effect of methoxamine in the presence of genistein was significantly attenuated compared with the effect in the absence of the tyrosine kinase inhibitor (Figures 3A⇑ and 4⇑).
Next, the effect of genistein on the methoxamine response during hypoxia was examined. In contrast to the results recorded in the absence of hypoxia, genistein had little effect (Figure 7B⇑). Methoxamine at 300 and 500 μmol/L inhibited the current 57.4±5.2% and 91.3±4.3%, respectively (n=5). This was not significantly different from the effect of methoxamine on the Iso-activated current in the presence of hypoxia and the absence of genistein (Figures 3B⇑ and 4⇑). These results suggest that the mechanism for the inhibition by methoxamine during hypoxia is distinctly different from the mechanism in room oxygen tension.
Methoxamine Inhibits via a PLA2-Mediated Mechanism During Hypoxia
Hypoxia results in activation of PLA2 and accumulation of phospholipids in the heart. A possible role for PLA2 in the hypoxic response was determined. Cells were exposed to the PLA2 inhibitor mepacrine at a concentration of 3 μmol/L in the presence of hypoxia followed by 100 nmol/L Iso and Iso plus methoxamine. Consistent with previous reports,19 mepacrine inhibited basal ICa-L 52.6±18.8%. In 6 cells, mepacrine attenuated the effect of methoxamine on the Iso-activated current. Methoxamine at 300 μmol/L was able to inhibit the current 11.2±4.4%, and 500 μmol/L methoxamine inhibited the current 29.3±11.0%. This was significantly (P<0.05) less than the inhibition recorded in the absence of mepacrine during hypoxia (Figures 3B⇑ and 4⇑). It was also less than the inhibition recorded in the presence of mepacrine and the absence of hypoxia. Under these conditions, methoxamine at 100 μmol/L inhibited the Iso-activated current 38.1±6.2%, and 300 μmol/L methoxamine inhibited the current 69.4±8.6% (n=4). This was not significantly different from the effects of the same concentrations of methoxamine on the Iso-activated current in the absence of hypoxia and mepacrine (Figures 3A⇑ and 4⇑).
To further confirm a role for PLA2, cells were exposed to indomethacin (Indo) at a concentration of 200 μmol/L. Indo alone inhibited basal ICa-L 40.2±7.0% (Figure 8B⇓; n=6). In the continued presence of Indo and hypoxia, 300 μmol/L methoxamine inhibited the Iso-activated current 18.1±8.2% and 500 μmol/L methoxamine inhibited the current 32.3±11.0%. The inhibition in the presence of Indo and hypoxia was significantly less (P<0.05) than the inhibition recorded in the absence of Indo (Figures 3B⇑ and 4⇑). It was also less than the inhibition recorded in the presence of Indo and the absence of hypoxia (Figure 8A⇓). Under these conditions, 100 μmol/L methoxamine inhibited the Iso-activated current 35.3±3.3% and 300 μmol/L methoxamine inhibited the currents 68.9±9.5% (n=5). This was not significantly different from the effects of the same concentrations of methoxamine in the absence of Indo and hypoxia (Figures 3A⇑ and 4⇑).
The perforated patch method was used to determine whether the whole-cell mode of the patch-clamp technique was accurately identifying the intracellular messenger pathways involved in the methoxamine response. Perforated patches were achieved with pipettes containing amphotericin B at a concentration of 0.2 mg/mL pipette solution. Cells were then exposed to hypoxia, Indo (200 μmol/L), and Iso and methoxamine (300 and 500 μmol/L) sequentially as in previous experiments (Figure 8B⇑). During perforated patches, 300 μmol/L methoxamine inhibited the Iso-activated current only 25.2±8.4% and 500 μmol/L methoxamine inhibited the current 36.5±7.3% (n=5). These data were not significantly different from data from experiments in which the whole-cell mode was used (Figure 8B⇑). These results strongly implicate a role for PLA2 in the inhibition of Iso-activated L-type Ca2+ currents by methoxamine during hypoxia.
The results of this study describe the effects of α-AR stimulation on basal ICa-L and on β-AR–activated ICa-L in guinea pig cardiac myocytes in the absence and presence of acute hypoxia. The α-AR agonists methoxamine and PE (in the presence of propanolol) either had no effect or produced a slight inhibition of basal ICa-L in the absence of hypoxia. Conflicting results have been reported regarding the effects of α-AR stimulation on basal ICa-L. Either no change3 20 21 22 23 or an increase24 25 has been reported. Some reasons for this lack of clarity may lie in variability between species and the activation of selective subpopulations of second messengers (such as PKC isoforms) by α-AR stimulation. Regardless of the effects of α-AR stimulation on ICa-L in room oxygen tension, in the present study there was no difference between the effect of methoxamine in the absence or presence of hypoxia. This indicates that hypoxia does not affect the response of basal channel activity to α-AR stimulation.
The effect of α-AR stimulation in the presence of β-AR stimulation was also examined. Consistent with previous reports2 3 5 6 in the absence of hypoxia, α-AR stimulation antagonized the β-AR–activated current. The effect of methoxamine could not be attenuated by inhibition of PKC but by inhibition of tyrosine kinase with genistein. This is consistent with a recent report5 in which genistein antagonized the ability of methoxamine to inhibit Iso-activated L-type Ca2+ currents and the tyrosine phosphatase inhibitor pervanadate mimicked the α-AR response. An involvement of PKC has been suggested in rat ventricular myocytes4 ; however, α-AR activation was bypassed in these studies and a phorbol ester was used to examine the role of PKC on Iso-stimulated currents. The study may not have been representative of the specific effects of α-AR stimulation on second-messenger activation. In the present study, α-ARs were directly activated with the α-AR agonist methoxamine. The effects of methoxamine could also be attenuated with the α1- AR antagonist prazosin, implicating a specific receptor subtype in the response. In addition, a previous study in which PKC inhibitors were used to attenuate the effects of methoxamine on Iso-activated CFTR Cl− conductance concluded that PKC did not play a role in the response.6
The present study also characterized the effects of hypoxia on α-AR antagonism of the β-AR–stimulated ICa-L. In the presence of hypoxia, the sensitivity of ICa-L to NE (Figure 2⇑) was significantly increased. However, this could simply be explained by an increase in the β-AR component of the NE response induced by hypoxia9 without any alteration in the α-AR component. This was clarified by determining the concentration dependence of ICa-L (activated by 100 nmol/L Iso) on methoxamine (Figures 3⇑ and 4⇑). Interestingly, hypoxia decreased the sensitivity of the current to methoxamine. It did so through a PLA2-dependent mechanism that did not involve PKC or tyrosine kinase (Figures 6 through 8⇑⇑⇑). This represents a novel regulation of the L-type Ca2+ channel and is consistent with the results indicating that the effect was “upstream” from activation of adenylate cyclase and specific (or close) to the β-AR, because methoxamine had little effect on the forskolin- and histamine-activated currents. There is no evidence to suggest that PLA2 can directly modulate β-AR function. However, it has been shown that tyrosine kinase inhibition with genistein can increase the sensitivity of ICa-L to β-AR stimulation.13 This suggests that basal tyrosine kinase activity exerts an inhibitory effect on β-AR responses. If PLA2 or a metabolite of the pathway were to exert a similar effect on the β-AR in the presence of hypoxia (but with less efficacy than tyrosine kinase given that the sensitivity of the channel to methoxamine was significantly less during hypoxia than under nonhypoxic conditions), this might explain the inhibitory effect of methoxamine during hypoxia.
Because a marked accumulation of arachidonic acid has been found in the ischemic myocardium and associated with arrhythmias during hypoxia, the results of the present study may help to the explain some of the mechanisms involved in the induction of arrhythmias. The antagonism of β-AR responses by α-AR stimulation has been proposed as a control mechanism in the regulation of L-type Ca2+ channels during excessive sympathetic activation. The L-type Ca2+ channel plays an integral role in both excitation and contraction in the heart, and excessive exposure to β-AR stimulation results in a prolongation of the action potential leading to the trigger of early afterdepolarizations and arrhythmias. A decrease in the sensitivity of the channel to α-AR stimulation would exacerbate these effects. Further characterization of the role of phospholipids in this response may help to provide a better understanding of the arrhythmogenic mechanisms associated with hypoxia.
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.
Original received December 18, 2000; revision received March 27, 2001; accepted March 29, 2001.
- © 2001 American Heart Association, Inc.
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