Published online before print May 10, 2001, doi: 10.1161/hh1001.090841
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2001 American Heart Association, Inc.
Cellular Biology |
Hypoxia Alters the Sensitivity of the L-Type Ca2+ Channel to 
-Adrenergic Receptor Stimulation in the Presence of ß-Adrenergic Receptor Stimulation
From the Department of Physiology, The University of Western Australia, Crawley, Western Australia.
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
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Abstract—The effects of
-adrenergic receptor (-AR) stimulation alone and the
effects in the presence of ß-adrenergic receptor (ß-AR) stimulation
were examined on L-type Ca2+ currents
(ICa-L)
in the absence and presence of hypoxia. The -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 -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 ß 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.
Key Words: -adrenergic receptor • ß-adrenergic receptor • hypoxia • L-type Ca2+ channels • phospholipase A2
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
It is well recognized that cardiac Ca2+ channel function is regulated by sympathoadrenergic stimulation. Activation of ß-adrenergic receptors (ß-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 -adrenergic
receptors (-ARs) has long been recognized, the effects of -AR
stimulation on ICa-L
are still not fully understood. Even less work has been undertaken
investigating the effects of -AR stimulation in the presence of
ß-AR stimulation. However, among the few studies reported, there is
good consensus that -AR stimulation inhibits
ß-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 -AR stimulation mediates the inhibition of ß-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
ß-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
-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
-AR stimulation on basal
ICa-L in
the presence and absence of acute hypoxia and the effects of
hypoxia on -AR stimulation in the presence of ß-AR
stimulation. -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
-AR responses distinctly differently from the effects reported under
nonhypoxic conditions. Consistent with previous results, the
-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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 M
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.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Effects of
-AR Stimulation on Basal
ICa-LMyocytes were exposed to the
-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 ß-AR
antagonist propanolol (10 µmol/L). Because PE binds both
ß-AR and -AR, simultaneously exposing cells to the
ß-AR antagonist propanolol ensures that only the effects
of -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 -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 -AR and ß-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 -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 ß-AR component of the NE response.
|
Hypoxia Alters the Sensitivity of
ICa-L
to -AR Stimulation
The increase in sensitivity of
ICa-L to
NE may also be due to an effect of hypoxia on the -AR
component of the response. To determine what effect hypoxia may
have on -AR stimulation, cells were exposed to increasing
concentrations of an -AR agonist in the presence of a constant
concentration of a ß-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 -AR, cells were
incubated in the 1-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 1-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 ß-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 ß-AR under both conditions.
Methoxamine-Induced Inhibition Does
Not Involve PKC
Previous studies in rat cardiac myocytes under
nonhypoxic conditions have suggested that -AR inhibition of
ß-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 PKCß 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.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The results of this study describe the effects of
-AR stimulation on basal
ICa-L
and on ß-AR–activated
ICa-L in
guinea pig cardiac myocytes in the absence and presence of acute
hypoxia. The -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 -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 -AR
stimulation. Regardless of the effects of -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 -AR
stimulation.
The effect of -AR stimulation in the presence of ß-AR
stimulation was also examined. Consistent with previous
reports2 3 5 6
in the absence of hypoxia, -AR stimulation antagonized the
ß-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 -AR response. An involvement of PKC has been suggested in rat
ventricular
myocytes4 ; however, -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
-AR stimulation on second-messenger activation. In the present
study, -ARs were directly activated with the -AR agonist
methoxamine. The effects of methoxamine could also be
attenuated with the 1- 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 -AR antagonism of the ß-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 ß-AR component of the NE
response induced by
hypoxia9 without any
alteration in the -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 ß-AR, because methoxamine had
little effect on the forskolin- and histamine-activated
currents. There is no evidence to suggest that
PLA2 can directly modulate ß-AR function.
However, it has been shown that tyrosine kinase inhibition with
genistein can increase the sensitivity of
ICa-L to
ß-AR stimulation.13 This
suggests that basal tyrosine kinase activity exerts an
inhibitory effect on ß-AR responses. If
PLA2 or a metabolite of the pathway were to
exert a similar effect on the ß-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.
Functional Significance
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 ß-AR responses by -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 ß-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 -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.
|
Acknowledgments |
|---|
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.
|
Footnotes |
|---|
Original received December 18, 2000; revision received March 27, 2001; accepted March 29, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. McDonald TF, Pelzer S, Trautwein W, Pelzer DJ. Regulation and modulation of calcium channels in cardiac, skeletal, and smooth muscle cells. Physiol Rev. 1994;74:365–507.
2.
Chen L, El-Sherif
N, Boutjdir M. 1-Adrenergic activation
inhibits ß-adrenergic-stimulated unitary
Ca2+ currents in cardiac
ventricular myocytes. Circ
Res. 1996;79:184–193.
3.
Boutjdir M, Restivo
M, Wei Y, El-Sherif N. 1- and ß-Adrenergic
interactions on L-type calcium current in cardiac myocytes.
Pflügers Arch Eur J
Physiol. 1992;421:397–399.
4. Zhang ZH, Johnson JA, Chen L, El-Sherif N, Mochly-Rosen D, Boutjdir M. C2 region-derived peptides of ß-protein kinase C regulate cardiac Ca2+ channels. Circ Res. 1997;80:720–729.
5.
Belevych A,
Nulton-Persson A, Turc A, Harvey RD. -Adrenergic antagonism of the
cardiac L-type Ca2+ current response to
ß-adrenergic stimulation involves a tyrosine
phosphorylation-dependent mechanism.
Circulation. 2000;102(suppl
II):II-336. Abstract.
6.
Oleksa LM, Hool LC,
Harvey RD. 1-Adrenergic inhibition of the
ß-adrenergically activated Cl-
current in guinea pig ventricular myocytes.
Circ Res. 1996;78:1090–1099.
7.
Danziger RS, Sakai
M, Lakatta EG, Hansford RG. Interactive - and ß-adrenergic actions
of norepinephrine in rat cardiac myocytes.
J Mol Cell Cardiol. 1990;22:111–123.
8. Newton GE, Adelman AG, Lima VC, Seidelin PH, Schampaert E, Parker JD. Cardiac sympathetic activity in response to acute myocardial ischemia. Am J Physiol. 1997;272:H2079–H2084.
9. Hool LC. Hypoxia increases the sensitivity of the L-type Ca2+ current to ß-adrenergic receptor stimulation via a C2 region–containing protein kinase C isoform. Circ Res. 2000;87:1164–1171.
10.
Fedida D, Braun
AP, Giles WR. 1-Adrenoceptors in
myocardium: functional aspects and transmembrane signaling
mechanisms. Physiol Rev. 1993;73:469–487.
11. van der Vusse GJ, Glatz JF, Stam HC, Reneman RS. Fatty acid homeostasis in the normoxic and ischemic heart. Physiol Rev. 1992;72:881–940.
12. Takeishi Y, Jalili T, Ball NA, Walsh RA. Responses of cardiac protein kinase C isoforms to distinct pathological stimuli are differentially regulated. Circ Res. 1999;85:264–271.
13. Hool LC, Middleton LM, Harvey RD. Genistein increases the sensitivity of cardiac ion channels to ß-adrenergic receptor stimulation. Circ Res. 1998;83:33–42.
14.
Fearon IM, Palmer
AC, Balmforth AJ, Ball SG, Mikala G, Schwartz A, Peers C.
Hypoxia inhibits the recombinant 1C subunit of the human
cardiac L-type Ca2+ channel.
J Physiol. 1997;500:551–556.
15.
Fearon IM, Palmer
AC, Balmforth AJ, Ball SG, Varadi G, Peers C. Modulation of recombinant
human cardiac L-type Ca2+ channel 1C
subunits by redox agents and hypoxia.
J Physiol. 1999;514:629–637.
16. Fearon IM, Varadi G, Koch S, Isaacsohn I, Ball SG, Peers C. Splice variants reveal the region involved in oxygen sensing by recombinant human L-type Ca2+ channels. Circ Res. 2000;87:537–539.
17. Ono K, Trautwein W. Potentiation by cyclic GMP of ß-adrenergic effect on Ca2+ current in guinea-pig ventricular cells. J Physiol. 1991;443:387–404.
18. Ron D, Luo J, Mochly-Rosen D. C2 region-derived peptides inhibit translocation and function of ß protein kinase C in vivo. J Biol Chem. 1995;270:24180–24187.
19. Xiao YF, Zeind AJ, Kaushik V, Perreault-Micale CL, Morgan JP. Mechanism of suppression of cardiac L-type Ca2+ currents by the phospholipase A2 inhibitor mepacrine. Eur J Pharmacol. 2000;399:107–116.
20.
Apkon M, Nerbonne
JM. 1-Adrenergic agonists selectively
suppress voltage-dependent K+ current in rat
ventricular myocytes. Proc
Natl Acad Sci
U S A. 1988;85:8756–8760.
21. Hartmann HA, Mazzocca NJ, Kleiman RB, Houser SR. Effects of phenylephrine on calcium current and contractility of feline ventricular myocytes. Am J Physiol. 1988;255:H1173–H1180.
22.
Terzic A, Puceat
M, Clement O, Scamps F, Vassort G.
1-Adrenergic effects on intracellular pH and
calcium and on myofilaments in single rat cardiac cells.
J Physiol. 1992;447:275–292.
23. Ertl R, Jahnel U, Nawrath H, Carmeliet E, Vereecke J. Differential electrophysiologic and inotropic effects of phenylephrine in atrial and ventricular heart muscle preparations from rats. Naunyn-Schmiedebergs Arch Pharmacol. 1991;344:574–581.
24.
Zhang S, Hiraoka
M, Hirano Y. Effects of 1-adrenergic
stimulation on L-type Ca2+ current in rat
ventricular myocytes. J Mol
Cell Cardiol. 1998;30:1955–1965.
25.
Liu QY, Karpinski
E, Pang PK. L-channel modulation by -1 adrenoceptor activation in
neonatal rat ventricular cells: intracellular mechanisms.
J Pharmacol Exp Ther. 1900;271:944–951.
This article has been cited by other articles:
 |
|
 |
|
  H. M. Viola, P. G. Arthur, and L. C. Hool Transient Exposure to Hydrogen Peroxide Causes an Increase in Mitochondria-Derived Superoxide As a Result of Sustained Alteration in L-Type Ca2+ Channel Function in the Absence of Apoptosis in Ventricular Myocytes Circ. Res., April 13, 2007; 100(7): 1036 - 1044. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. C. Hool, C. A. Di Maria, H. M. Viola, and P. G. Arthur Role of NAD(P)H oxidase in the regulation of cardiac L-type Ca2+ channel function during acute hypoxia Cardiovasc Res, September 1, 2005; 67(4): 624 - 635. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. C. Hool Differential regulation of the slow and rapid components of guinea-pig cardiac delayed rectifier K+ channels by hypoxia J. Physiol., February 1, 2004; 554(3): 743 - 754. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. C. Hool and P. G. Arthur Decreasing Cellular Hydrogen Peroxide With Catalase Mimics the Effects of Hypoxia on the Sensitivity of the L-Type Ca2+ Channel to {beta}-Adrenergic Receptor Stimulation in Cardiac Myocytes Circ. Res., October 4, 2002; 91(7): 601 - 609. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||