© 1995 American Heart Association, Inc.
Articles |
Oxygen-Derived Free Radical Stress Activates Nonselective Cation Current in Guinea Pig Ventricular Myocytes
Role of Sulfhydryl Groups
From the Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary (Canada).
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Abstract |
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Abstract Oxygen-derived free radicals (O-Rs) cause alterations in cardiac electrical activity, including sustained depolarization, which may contribute to arrhythmic activity in reperfusion after ischemia. The ionic current(s) and cellular mechanism(s) underlying the sustained depolarization are not well defined. We used the whole-cell variant of the patch-clamp technique to study sustained depolarization in guinea pig ventricular myocytes during the extracellular application of O-Rs (generating system: dihydroxyfumaric acid, 3 to 6 mmol/L; FeCl3/ADP, 0.05:0.5 mmol/L). Myocytes superfused with O-Rs (pipette EGTA, 0.1 mmol/L) showed (1) sustained depolarization to between -40 and -10 mV, (2) oscillations in membrane potential, and (3) triggered activity. The depolarization resulted from an increase in quasi–steady state difference current reversing at
-18 mV, and the oscillations were
due to transient inward current. The latter were inhibited with
ryanodine (10 µmol/L) or high pipette EGTA (5 mmol/L), but the steady
state current was unaffected. Nonselective cation current
(INSC) (recorded with Cs+,
Li+, and Mg2+ replacing
K+, Na+, and
Ca2+, respectively; 20 mmol/L
tetraethylammonium chloride [TEA] and 5 mmol/L BAPTA in the pipette
solution and 10 mmol/L TEA, 10 µmol/L tetrodotoxin, and 10 µmol/L
nicardipine in the bath solution) was activated by O-Rs; the increase
in current was unaffected by preventing changes in
[Ca2+]i but was inhibited with
dithiothreitol. Oxidizing agents (diamide and thimerosal) or caffeine
(pipette EGTA, 0.1 mmol/L) produced a similar increase in membrane
conductance. INSC activated with O-Rs, oxidizing agents, or
caffeine was sensitive to SK&F 96365. O-R treatment was without effect
when INSC was already activated with caffeine. The data
suggest that (1) extracellular O-Rs activate a
Ca2+-sensitive INSC in the absence of
changes in [Ca2+]i, (2)
oxidative modification of extracellular sulfhydryl groups may be
involved, and (3) this mechanism is different from the
Ca2+-dependent activation of INSC by
intracellular O-Rs, indicating that O-Rs may alter ion channel activity
by differential mechanisms, depending on the compartment, extracellular
or intracellular, in which they are present.
Key Words: whole-cell steady state current • ventricular myocytes • oxygen-derived free radicals • nonselective cation current • sulfhydryl groups
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Oxygen-derived free radicals (O-Rs) are implicated in myocardial injury during reperfusion after a period of ischemia.1 A burst of O-Rs2 is thought to contribute to reflow arrhythmogenesis3 4 5 6 and contractile dysfunction (stunning).7 Several studies reported alterations in electrical activity in the heart due to exogenous O-R stress, leading to abnormal activity and arrhythmias.8 9 10 11 12 13 14 However, the specific ionic conductances involved and the subcellular changes responsible for altered channel activity during O-R stress remain poorly defined. Furthermore, it is not evident whether the effects of O-Rs in the extracellular compartment are necessarily identical to those induced by reactive species of oxygen in the intracellular compartment.
Theoretically, cardiac membrane currents could be affected by O-R stress as a result of (1) lipid peroxidation and changes in membrane fluidity, (2) direct oxidation of channel proteins or associated regulatory subunits,15 16 or (3) a change in a cytoplasmic regulatory factor, eg, [Ca2+]i.17 18 19 Several studies implicate a rise in [Ca2+]i as an important factor in causing changes in membrane currents during O-R stress.10 11 12 20 For example, sustained depolarization has been observed in intact heart preparations and single isolated myocytes20 21 22 23 ; we found that this change was associated with the activation of a nonselective cation current (INSC) due to an increase in [Ca2+]i resulting from abnormal Ca2+ handling by the sarcoplasmic reticulum (SR).20 Intracellular Ca2+ chelation by inclusion of 5 mmol/L EGTA in the whole-cell voltage-clamp patch pipette solution or pretreatment with ryanodine (10 µmol/L) inhibited the change in INSC and sustained depolarization.20
Lipid peroxidation was shown to occur in intact preparations with demonstrable changes in electrical activity.21 However, there is as yet no direct evidence showing alterations in a specific membrane conductance as a result of lipid peroxidation. On the other hand, direct protein oxidation was implicated in studies in which mechanical dysfunction induced by O-R stress was either reversed or prevented by sulfhydryl group reducing agents, such as N-acetylcysteine24 and dithiothreitol (DTT).15 25 Moreover, alterations in the activities of some enzymes and ion transporters were reported to result from O-R–induced oxidative modification of sulfhydryl groups on the proteins. For example, inactivation of the sarcolemmal Ca2+ pump,26 Na+,K+-ATPase,27 and SR Ca2+-ATPase28 29 by O-R stress could be reversed by DTT. Significantly, non–O-R–mediated sulfhydryl group oxidizing agents, such as diamide, were also shown to depress Ca2+- and Na+-ATPase activity.26 27 Direct sulfhydryl modification of a sarcolemmal ion conductance in the heart by O-R leading to abnormal electrical activity has not been previously described.
O-Rs are generated through several different reaction pathways in the intracellular and extracellular compartments of cardiac myocytes.30 Some O-Rs can readily cross cell membranes; eg, superoxide radical will permeate anion channels, and the reactive oxygen metabolite H2O2 is membrane permeant. Other O-Rs are extremely reactive; eg, hydroxyl radical has reaction rates on the order of 0.1 to 10x109 mol-1 · s-1 with most cellular constituents, such as amino acids, sugars, and phospholipids.31 32 For this reason, they are thought to be too reactive to diffuse very far from their sites of generation.31 32 Therefore, it is conceivable that some O-Rs may exert their effects largely within the compartment in which they are generated.8 Phospholipids as well as sulfhydryl groups on proteins within the sarcolemma show differential distribution, and regulatory molecules influencing channel activity are generally confined within one compartment. Therefore, the effects of O-R stress on ion channels or the mechanism involved may vary, depending on the cellular compartment in which the radicals are generated. However, as yet, the only data in support of this hypothesis come from limited experiments on frog myocytes with photoilluminated rose bengal.33 Some differences in action potential configuration were evident, but the underlying changes in ionic conductances were not assessed. No studies involving mammalian myocytes have as yet explored the possibility of differential influences of extracellular versus intracellular O-R stress in myocytes from the same species by use of an identical O-R–generating system and recording conditions.
In the present study, we sought to determine the effects of extracellular O-R stress on membrane potential and steady state membrane currents in guinea pig ventricular myocytes and to identify the mechanism(s) responsible for the changes in ion channel activity. To permit comparisons with our previous experiments involving intracellular O-R stress20 and to probe for differential effects of O-Rs related to the cellular compartment in which the reactive species were generated, we used an identical generating system and recording conditions. The present study focuses on the mechanism by which extracellular O-Rs activate nonselective cation current and contribute to sustained depolarization. Changes in other currents are not addressed in detail.
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Materials and Methods |
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Cell Isolation
Guinea pigs (250 to 450 g) were anesthetized with CO2 and then killed by cervical dislocation. Their hearts were quickly removed and placed in well-aerated ice-cold Krebs-Henseleit solution (K-H) containing (mmol/L) NaCl 120, NaHCO3 25, KCl 4.8, NaH2PO4 1.2, MgSO4 1.2, glucose 11, taurine 13, and CaCl2 1.8 (pH 7.4 with 95% O2/5% CO2) before mounting on a Langendorff apparatus. The atria were removed during perfusion in a retrograde fashion with K-H (37°C) at a constant pressure for a stabilization period of 10 to 15 minutes. Single ventricular myocytes were isolated by using a modified version of an enzymatic dispersion technique34 ; ventricles were initially perfused with nominally Ca2+-free modified MEM-Joklik (GIBCO BRL, Life Technologies Inc) solution for 10 minutes before perfusing in a recirculating manner at a rate of 14 to 15 mL/min with collagenase (74.52 U/mL, Worthington Biochemical Corp), hyaluronidase (0.6 mg/mL, Sigma Chemical Co), and trypsin inhibitor (0.2 mg/mL, Sigma) added to 50 µmol/L Ca2+-modified MEM-Joklik solution for 20 to 30 minutes. Both ventricles were then removed, placed in 50 µmol/L Ca2+–containing MEM-Joklik solution, and cut into small pieces (2 mm3). After a final wash in 50 µmol/L Ca2+–containing MEM-Joklik solution, the tissues were stored in medium 199 at room temperature, and single myocytes were obtained when needed by gentle trituration.
Whole-Cell Recordings
Isolated guinea pig ventricular myocytes were placed in a
recording chamber and superfused with the bath solution at a flow rate
of 1.8 mL/min (the time required for effective changes in the bath
solution was 15 seconds, based on the time required for a stable
shift in resting membrane potential with a jump from 4.8 to 9.6 mmol/L
[K+]o). Only rod-shaped myocytes that
had clear and distinct striations and demonstrated marked shortening
and relaxation upon stimulation were used. All experiments were
performed at 22°C.
The whole-cell configuration of the patch-clamp
technique35 was used for voltage- and current-clamp
recordings with a model 8900 patch-clamp amplifier (Dagan Corp). Patch
pipettes were pulled with a model P-87 puller (Sutter Instruments) and
fire-polished to a final resistance of 0.5 M
when filled with a
control pipette solution. The pipette tip was positioned above the
cell, and the pipette potential and capacitance were nullified. After
the patch membrane was ruptured, the series resistance (1.0 to 5.0
M) and cell capacitance were compensated. Membrane voltage and
whole-cell currents (filtered at 1 kHz) were recorded directly to hard
disk via an A/D convertor (TL-1-125 LabMaster, Axon Instruments)
interfaced with an IBM-clone computer running
PCLAMP software (Axon Instruments). Data
analysis was performed with PCLAMP
(CLAMPFIT). Since the pipette potentials were nulled in
external solution, current-clamp tracings and voltage-clamp protocols
were corrected for junction potential in the different experimental
conditions used. An average value for junction potential was determined
for several pipettes in each recording condition by nulling in the
pipette solution before the pipettes were transferred to the external
solution and the pipette potential was recorded. Junction potential
values of +10.0±1.8 mV (n=16 pipettes) for the potassium gluconate
pipette/NaCl bath and -7.8±0.4 mV (n=18 pipettes) for the CsCl
pipette/NaCl or LiCl bath were obtained.
Solutions
The exogenous free radical–generating system consisted of
dihydroxyfumaric acid (3 to 6 mmol/L) and FeCl3/ADP
(0.05:0.5 mmol/L), which were added to the bath solution (pH readjusted
to 7.4 with NaOH). To record action potentials and whole-cell net
membrane current, the bath solution contained (mmol/L) NaCl 120,
NaHCO3 3.6, KCl 4.8, NaH2PO4 1.2,
MgSO4 1.2, glucose 11, CaCl2 1.8, and HEPES 5
(pH 7.4 with NaOH), and the pipette solution contained (mmol/L)
potassium gluconate 130, KCl 10, MgCl2 1,
Na2ATP 5, EGTA 0.1, and HEPES 5 (pH 7.2 with KOH). In some
experiments, Ca2+ chelation in the intracellular
compartment was increased by including 5 mmol/L EGTA or BAPTA in the
pipette solution. To isolate INSC, the whole-cell pipette
solution contained (mmol/L) CsCl 140, MgCl2 1,
Na2ATP 5, tetraethylammonium chloride (TEA) 20, EGTA 0.1 or
5, and HEPES 5 (pH 7.2 with CsOH), and the bath solution contained
(mmol/L) NaCl 120, NaHCO3 3.6,
NaH2PO4 1.2, CsCl 4.8, CaCl2 1.8,
MgSO4 1.2, HEPES 5, TEA 10, BaCl2 0.2,
nicardipine 10 µmol/L, and tetrodotoxin (TTX) 10 µmol/L (pH 7.4
with NaOH; equilibrium potential of Cl-
[ECl], +3.9 mV). In several experiments,
Na+-Ca2+ exchange current was also
blocked by equimolar replacement of Na+ and
Ca2+ in the bath with Li+ and
Mg2+ (pH adjusted to 7.4 with LiOH), respectively,
and 5 mmol/L BAPTA in the pipette. Under these conditions,
ECl was shifted from -60.4 to +3.1 mV and
K+, Na+, and L-type
Ca2+ currents were suppressed. In a limited number
of experiments, K+ and Na+ in the bath and
pipette were completely replaced with TEA, and Cl- was
maintained (pH 7.4 and 7.2 with TEA-OH, respectively).
Drugs
MEM-Joklik solution and medium 199 were purchased from GIBCO.
Nicardipine (10 µmol/L) was purchased from Research Biochemicals Inc.
Ryanodine (10 µmol/L) and SK&F 96365 (100 µmol/L) were purchased
from Calbiochem Corp. All other chemicals were purchased from
Sigma.
Data Analysis
Data were expressed as mean±SEM. Statistical analysis was
performed by using paired or unpaired Student's t test for
single comparisons and ANOVA followed Dunnett's test for multiple
comparisons as indicated in text.
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Results |
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The whole-cell recording technique was used to monitor the time-dependent changes in electrical activity and net quasi–steady state membrane currents of isolated guinea pig ventricular myocytes during exposure to O-Rs. A low concentration of EGTA (0.1 mmol/L) was included in the pipette solution in an attempt to preserve [Ca2+]i fluctuations and contractions upon electrical stimulation. Experiments were conducted at 22°C to slow the progression of changes in electrical activity during exposure to O-Rs and to provide time to record the underlying alterations in membrane current by switching from current-clamp to voltage-clamp mode. Fig 1A
shows that O-R stress depolarized resting
membrane potential, depressed plateau voltage, prolonged action
potential duration (tracing b), and caused low- amplitude oscillations
in membrane potential (tracing c) compared with control conditions
(tracing a). After the initial depolarization, myocytes failed to
repolarize after an action potential and showed a sustained
depolarization to between -40 and -10 mV (tracing d). These changes
were observed in an additional 17 myocytes, and on average, the
sustained depolarization occurred after 4.8±0.9 minutes of O-R
exposure and did not show any reversal for more than an additional 15
minutes (as opposed to the transient period of sustained depolarization
during intracellular O-R stress20 ). In 14 of 18 myocytes,
delayed afterdepolarizations (DADs) and triggered activity occurred
before the period of sustained depolarization. These findings are
similar to those reported for canine myocytes impaled with conventional
microelectrodes and exposed to a similar generating
system.36
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To determine the change in membrane conductance underlying the
sustained depolarization, a ramp protocol was applied to determine the
quasi–steady state current-voltage (I-V) relation between -130 and
+30 mV (8-s ramp from a holding potential of -85 mV; ramp rate, 20
mV/s). Fig 1B
shows the changes in net quasi–steady state current
during extracellular O-R stress. Under control conditions, the
quasi–steady state I-V relation displayed a typical N-shaped
appearance, and in most cells the current had two reversal potentials
with a positive slope conductance (reversal potential
[Er]), corresponding to stable membrane potentials at
rest (-75 to -80 mV) and during the plateau (+10 mV) (eg, Fig 1B).
Compared with a control recording (tracing a), those obtained during
the sustained depolarization (tracings b and c) had the following
changes: (1) a positive shift in the more negative Er and a
negative shift in the more positive Er of the I-V relation
so that the two Er moved together with time (eg, Fig 2B) and only a single Er was present at
-30 to -10 mV, when stable depolarization was observed (eg, Figs 1B and 3B) (average values for Er are given in Table 1; only the more negative value for Er under
control conditions is indicated to contrast the difference between
normal resting potential and sustained depolarization during O-R
stress), (2) a negative shift in the holding current at -85 mV, and
(3) inward oscillations of membrane current or transient inward current
(Iti), observed upon stepping back to -85 mV at the end of
the ramp. These changes in steady-state current showed no evidence of
reversal for an additional 15 minutes of recording.
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To determine the change in steady state current(s) responsible for the
change in steady state I-V relation, the O-R–sensitive difference
current was calculated by digital subtraction of tracings recorded
before from those obtained during O-R stress (eg, Fig 1C
represents tracing c minus tracing a of Fig 1B). The difference
current possessed an Er of -28.4 mV in this myocyte.
Similar results were obtained from an additional five myocytes, and
average values for Er of the difference current are given
in Table 1. Since the current was recorded in the absence of blockers,
it could represent changes in one or more ionic
conductances.
We previously found that intracellular O-R stress caused a transient
period of sustained depolarization in guinea pig ventricular myocytes
due to activation of Ca2+-sensitive
INSC. The change in conductance was attributed to elevated
[Ca2+]i due to mishandling of
Ca2+ by the SR; ryanodine pretreatment or
intracellular Ca2+ chelation with high EGTA (5
mmol/L) inhibited the alterations in membrane potential and
current.20 The change in the shape of the I-V relation and
the average value of Er for the difference current (-18
mV) for the present data were similar to those values previously
reported by us to occur during intracellular O-R stress20
and by others to occur during elevations in
[Ca2+]i37 38 and were
attributed to the activation of a Ca2+-sensitive
nonselective cation conductance (INSC). For this reason, we
determined whether similar alterations in
[Ca2+]i were involved in the response
to extracellular O-R stress.
Myocytes were pretreated with 10 µmol/L ryanodine39 for
30 minutes before O-R stress, and in a second set of separate
experiments, the concentration of EGTA in the pipette was increased to
5 mmol/L to chelate intracellular Ca2+. Comparison
of Fig 2A with Fig 1A shows that qualitatively similar changes in
electrical activity were obtained despite pretreatment with ryanodine,
depolarization, prolongation of action potential duration, and
depression in plateau amplitude followed by a period of sustained
depolarization. Under voltage-clamp conditions, the positive shift in
the negative Er of the control steady state I-V relation
and the negative shift in the holding current were unaffected by
ryanodine (Fig 2). However, we never observed DADs, oscillations in
membrane potential, or Iti after pretreatment with
ryanodine. The difference current (Fig 2C), obtained by digital
subtraction of control from O-R tracings, had an I-V relation that
reversed at -24.3 mV in the myocyte shown and, on average, was not
statistically different from that obtained in the absence of ryanodine
(Table 1). These data exclude abnormal Ca2+ release
from SR as a requirement for sustained depolarization and the change in
steady state difference current during extracellular O-R stress.
Whether the change induced by extracellular O-R stress was due to
an increase in [Ca2+]i derived from a
Ca2+ source other than the SR was explored by
dialyzing myocytes with a higher concentration of EGTA (5 mmol/L). Fig 3 shows that 5 mmol/L EGTA failed to suppress the
changes in action potential configuration, resting potential, and
steady state current induced by O-Rs. The average value for
Er of the steady state current from four myocytes is shown
in Table 1 and was not statistically different from that obtained with
low EGTA in the pipette solution. However, as with the ryanodine
experiments, myocytes dialyzed with 5 mmol/L EGTA failed to exhibit
DADs, low-amplitude oscillations in membrane potential, or
Iti. These data suggest that the sustained depolarization
and change in steady state current during extracellular O-R stress did
not require an increase in
[Ca2+]i.
To determine whether INSC was activated and the role of
[Ca2+]i in the change in conductance,
the previous experiments with ryanodine and EGTA were repeated by using
recording conditions to isolate the current. Nicardipine (10 µmol/L)
and TTX (10 µmol/L) were used to block L-type Ca2+ and
Na+ currents, respectively. K+ currents were
inhibited by adding 10 mmol/L TEA and 0.2 mmol/L BaCl2 to
the bath solution and 20 mmol/L TEA to the pipette solution and by
replacing K+ in both solutions with an equimolar
concentration of Cs+. Under these conditions,
ECl was shifted to +3.9 mV from a value of -60.4 mV for
the potassium gluconate pipette and NaCl bath solutions. Fig 4 shows results from a myocyte pretreated with ryanodine
(10 µmol/L). O-R stress caused the activation of a steady state
current, which reversed at +1.6 mV in this myocyte. Average values for
Er of the difference current from five myocytes pretreated
with ryanodine are given in Table 2. Fig 5 shows similar changes in the steady state I-V relation
in a myocyte dialyzed with a pipette solution containing 5 mmol/L EGTA.
The average value for Er of steady state current in four
myocytes was not statistically different from that obtained from
myocytes dialyzed with 0.1 mmol/L EGTA (Table 2). The effects of
extracellular O-R on INSC after pretreatment with ryanodine
and high EGTA were therefore consistent with the net whole-cell current
data. However, we were concerned that a localized increase in
[Ca2+]i in the subsarcolemmal space,
resulting from Ca2+ entry through
Na+-Ca2+ exchange,40 might
have occurred. This increase in
[Ca2+]i might have activated
INSC because of an inadequate intracellular
Ca2+ chelation by EGTA. We sought to rule this out
by (1) dialyzing myocytes with 5 mmol/L BAPTA, a
Ca2+ chelator known to have a higher
Ca2+ binding affinity than EGTA,41 and
(2) equimolar substitution of Na+ and
Ca2+ with Li+ and
Mg2+, respectively, to block the exchanger.
ECl was +3.1 mV under these conditions. Fig 6 shows that a myocyte superfused with O-R under these
conditions still exhibited a positive shift in Er (panel
A). The Er of the difference current was -0.7 mV in this
myocyte, and similar results were obtained in another seven myocytes.
Average values for Er are given in Table 2. On average,
membrane conductance before O-R treatment was low (0.31±0.05 nS) and
showed zero current at -38 mV. A positive shift in Er
to -3 mV, due to the activation of a difference current with an
Er of +3 mV, was observed after 5.9±1.5 minutes of
exposure to O-R. This latency was not statistically different from that
required for (1) the change in Er with high EGTA and both
Ca2+ and Na+ in the bath solution or (2)
the sustained depolarization in current-clamp recordings
(P>.05, by ANOVA). This same recording condition was used
in all subsequent experiments unless specified otherwise.
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Three sets of control experiments were performed to discount a role for
(1) altered Cl- current, (2) nonspecific leak current, or
(3) seal resistance in the change in membrane current during O-R
exposure. First, Li+ and Cs+ in the bath and
pipette were completely replaced with TEA, but Cl-
concentrations were maintained. Under these conditions, no change in
membrane current was observed within 10 to 15 minutes of exposure to
O-Rs (n=3; data not shown). Second, whether there was any change in
membrane conductance with time in the absence of O-R was determined.
Representative recordings are shown in Fig 7; in
three myocytes, no change was observed within 29.0±3.6 minutes, a
considerably longer time period compared with that required for the
changes induced by O-R exposure. Finally, Fig 7B shows a lack of change
in recordings obtained in the cell-attached configuration during a
20-mV test pulse from -67 mV before and after O-R stress. On average,
seal resistance was 12.9±1.5 G before and 11.5±1.2 G after
25.5±2.5 minutes of O-R stress in four myocytes. These values were not
statistically different (P>.05, by paired Student's
t test).
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Given the absence of any role for
[Ca2+]i in the activation of
INSC due to extracellular O-Rs, the possibility of direct
oxidative modification was tested with the sulfhydryl group reducing
agent, DTT.15 26 27 28 29 42 DTT was used in two different ways:
(1) myocytes were pretreated with DTT (1 mmol/L) for 30 minutes and
then exposed to O-Rs, or (2) myocytes were exposed to O-Rs and then to
DTT. Fig 8 shows that O-R stress failed to activate
INSC in a myocyte pretreated with DTT, and similar results
were obtained in three other myocytes. In all cases, the time over
which the current failed to change was much longer (25±1.1 minutes)
than that required for the effects of O-Rs. Fig 9 shows
that DTT in the continued presence of O-Rs inhibited the changes in
INSC; the positive shift in Er for the steady
state current and the negative shift in the holding current were both
reversed. The average value of Er for the difference
current after DTT (-42.3±3.4 mV, n=4) was significantly different
from that during (P<.05, by ANOVA followed by Dunnett's
test) but not different from that before O-R stress (control and O-R
values in Table 3, P>.05 by ANOVA).
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If an oxidation of sulfhydryl groups was involved in the response to
O-Rs, then similar changes in membrane conductance would be expected
for non–O-R–oxidizing agents, such as diamide43 44 or
thimerosal.45 46 Indeed, diamide (0.5 mmol/L) induced
changes in the steady state I-V relation identical to those of O-R
stress (Fig 10). The difference current activated by
diamide possessed an Er of +5.1 mV in the myocyte shown
(Fig 10B), and an average value is given in Table 3. The positive shift
in Er due to diamide was reversed after superfusion with
DTT (1 mmol/L) in the continued presence of diamide (Fig 10). Similar
results were obtained from an additional five myocytes. After DTT,
Er returned to -45.3±5.2 mV, which was not significantly
different (P>.05, by ANOVA) from control conditions but
different (P<.05, by ANOVA followed by Dunnett's test)
from conditions in the presence of diamide (values for control and
diamide are shown in Table 3). The average value of Er
(Table 3) and the time required (3.9±0.8 minutes) for activation of
the diamidesensitive current were not statistically different from
values obtained after extracellular O-R exposure
(P>.05, by ANOVA). Diamide was without effect on membrane
conductance when Li+ and Cs+ were completely
replaced with TEA but Cl- was maintained in the bath and
pipette (n=3; data not shown).
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Fig 11 shows representative recordings of the
effects of the hydrophilic membrane-impermeant sulfhydryl group oxidant
thimerosal (5 µmol/L). An increase in conductance was observed that
was due to a difference current with an Er of +2.8 mV in
the myocyte shown (Fig 11B). A similar increase in current was observed
in seven additional myocytes, and average values for Er in
control conditions and in the presence of thimerosal are given in Table 3. On average, the shift in Er was observed after 3.5±0.9
minutes of treatment. This value was not statistically different from
that required for sustained depolarization and the shift in
Er during O-R stress (P>.05, by ANOVA). Fig 11
also shows that DTT (1 mmol/L) inhibited the current in the continued
presence of thimerosal (Fig 11A), and pretreatment with DTT prevented
subsequent activation of INSC by thimerosal (Fig 11C).
Similar results were obtained in two myocytes exposed to DTT after
thimerosal and in four myocytes pretreated with DTT. No change in
membrane current was observed for 13.3±1.9 minutes during thimerosal
exposure after DTT pretreatment; this time was much longer than that
required for thimerosal to affect INSC.
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Some variability in the current activated by O-Rs, diamide, and
thimerosal was evident. INSC in several (three of eight
O-R–treated, two of six diamide-treated, and six of eight
thimerosal-treated) myocytes showed evidence of voltage dependence.
This varied from the slight outward rectification as shown in Fig 6B to
the marked voltage dependence and relaxation of current after the ramp
in Fig 11B. Myocytes pretreated with ryanodine (five of six) or
dialyzed with 5 mmol/L EGTA (two of six) also demonstrated slight
voltage dependence during O-R stress.
Fig 12 shows representative examples of the
effect of SK&F 96365 (100 µmol/L), an inhibitor of receptor-operated
cation channels,47 and Ca2+-activated
nonselective cation channels48 on INSC
activated by O-Rs, diamide, and thimerosal. This compound inhibited the
effects of O-Rs or the oxidants regardless of whether the membrane
current exhibited voltage dependence (Fig 12A and 12C) or was largely
ohmic (Fig 12B). Similar results were obtained from an additional five,
three, and three myocytes, and on average, current at +48 mV decreased
by 95.6±3.5%, 92.1±4.5%, and 81.2±8.9% with SK&F 96365 during
O-R, diamide, and thimerosal treatment, respectively.
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Fig 13A demonstrates that SK&F 96365 also
inhibited the Ca2+-sensitive INSC
activated after treatment with caffeine (10 mmol/L) and release of
intracellular Ca2+ stores. The recording conditions
were unchanged from previous experiments, except that the cells were
dialyzed with 0.1 EGTA to permit changes in
[Ca2+]i. Caffeine with 0.1 mmol/L
pipette EGTA caused an increase in membrane current (with an average
delay of 0.8±0.3 minutes [n=10]) due to activation of a difference
current with an Er of +2 mV in the myocyte shown. The
average value for Er during caffeine was not statistically
different from that following O-R, diamide, or thimerosal treatment
(Table 3). Outward rectification was observed in 4 of 10 myocytes when
the current was partially activated (Fig 13A and 13B), but marked
voltage dependence, as displayed in Fig 11, was not seen during
caffeine treatment.
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The effect of SK&F 96365 (100 µmol/L) on the caffeine-induced
conductance was determined by applying the drug after a stable maximal
increase in current due to caffeine was observed. In the continued
presence of caffeine, SK&F 96365 caused a marked decrease in current in
the representative data shown in Fig 13. Similar results were
obtained in an additional three myocytes. On average, the current at
+48 mV declined by 92.4±2.5% by 3 to 4 minutes (n=4) of treatment
with SK&F 96365. Fig 13C shows that in the absence of SK&F 96365, the
caffeine-sensitive current declined only slightly over 16.5 minutes.
Similar results were obtained in two other myocytes. On average, the
current at +48 mV declined from its peak value by 10.5±1.9% (n=3) by
10 minutes, which was significantly less than that caused by SK&F 96365
(P<.05, by unpaired Student's t test). This
slight decline may reflect a slow decrease in
[Ca2+]i due to Ca2+
extrusion in the face of a net release of Ca2+ from
the stores, the absence of Na+-Ca2+
exchange with Li+ in the bath, and perhaps altered
Ca2+ sequestration due to the nonphysiological ionic
constituents (Cs+ and Li+) of the recording
solutions. That the change in current due to caffeine depended on
increased [Ca2+]i was tested by
dialyzing myocytes with 5 mmol/L BAPTA. In three myocytes, caffeine had
no effect on INSC when applied in the presence of 5 mmol/L
internal BAPTA (data not shown).
In a final series of experiments, the question of whether the current
affected by O-R was the same or a different conductance from the
Ca2+-sensitive INSC activated during
caffeine treatment was tested. The O-R–generating system was applied
in the presence of caffeine after the Ca2+-sensitive
INSC activated by caffeine treatment had reached a stable
peak. Fig 14 shows that once INSC was
activated by caffeine (10 mmol/L), subsequent exposure to O-Rs did not
further enhance the membrane current. Similar data were obtained from
two other myocytes. Interestingly, we did not observe any decline in
the current over >15 minutes in the presence of O-Rs and caffeine.
This finding was different from the 10% decline observed with
caffeine alone. After 15 minutes, the outward current at +48 mV had
only declined by 0.5±1.6% (n=3) of its peak value in response to
caffeine alone. It is possible that O-Rs affected the channels so that
they remained open despite a decline in
[Ca2+]i. Alternatively, we cannot rule
out the possibility that O-Rs depressed Ca2+
extrusion or sequestration, so that no change in
[Ca2+]i and INSC occurred
with time.
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|
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study reports the novel findings that (1) oxidative stress due to O-Rs generated in the bath solution activated a nonselective cation current in isolated guinea pig ventricular myocytes by modifying sulfhydryl groups, independent of changes in [Ca2+]i, and (2) the cardiac nonselective cation current activated by O-Rs, sulfhydryl group oxidizing agents, or caffeine is inhibited by SK&F 96365, a blocker of receptor-operated channels and Ca2+-activated INSC in other cell types.47 48 The data indicate that extracellular O-R stress may oxidize extracellular sulfhydryl groups, leading to an increase in Ca2+-activated INSC in the absence of a change in [Ca2+]i. We previously reported that a Ca2+-dependent mechanism was involved in the activation of Ca2+-sensitive INSC during intracellular O-R stress in similar myocytes under identical recording conditions.20 Therefore, the present data also provide the first indication that the mechanism by which an ionic conductance in mammalian cardiac myocytes is affected by O-R stress may vary depending on the compartment in which the radical species are present.
Membrane potential of guinea pig ventricular myocytes exposed to
O-Rs generated in the bath solution showed sustained depolarization to
between -40 and -10 mV. This depolarization was accompanied by the
activation of a quasi–steady state difference current with an
Er (-18 mV) different from the equilibrium potential of
any single ion under the recording conditions used. Similar changes in
membrane conductance, attributed to Ca2+-activated
INSC, were previously observed after elevations in
[Ca2+]i due to (1) intracellular O-R
stress,20 (2) intracellular Ca2+
injection via a microelectrode,37 or (3) dialysis with a
patch pipette solution containing micromolar
Ca2+.38 Er values for the
difference currents in these previous studies were
-19.9±0.7,20 -22±11,37 and -14±0.6
mV,38 respectively. To monitor changes in INSC
in the present study, we used the following recording conditions:
(1) Na+, Ca2+, and
K+ conductances were suppressed with TTX, nicardipine,
BaCl2, and K+ replacement with
Cs+. (2) The Na+-Ca2+
exchanger was inhibited by buffering
[Ca2+]i to a low level with 5 mmol/L
BAPTA in the pipette solution, and Na+ and
Ca2+ in the bath solution were replaced with
Li+ and Mg2+.49 50 51 Membrane
conductance under these conditions was very low, and zero current
occurred at -35 mV. The ionic basis of the residual conductance is
unknown, but it may arise from a combination of seal leak,
INSC, and Cl- current and/or K+
current not completely suppressed by Cs+ and
Ba2+ in the pipette and bath, respectively. Within
5.9±1.5 minutes of exposure to extracellular O-R stress, membrane
conductance increased substantially, and zero current occurred at
-3 mV (Tables 2 and 3). This latency was not different from the
4.8±0.9 minutes required for the sustained depolarization in
current-clamp recordings, indicating that the alteration in membrane
conductance observed with Cs+-Li+ recording
conditions contributes to the change in membrane potential.
Sustained depolarization during extracellular O-R stress cannot be explained by (1) activation of a Cl- conductance, (2) a rise in a nonspecific membrane leak, or (3) a change in seal resistance. We did not observe any change in membrane conductance during O-R or diamide treatment when Na+ and K+ in the bath and pipette were replaced with TEA but Cl- concentration was unchanged. Similarly, no change in membrane current occurred with time in untreated myocytes, nor did O-R stress change the seal resistance of cell-attached patches. Furthermore, the effects of O-Rs, diamide, and thimerosal were inhibited by DTT or SK&F 96365; neither would be expected to alter seal resistance or depress a nonspecific membrane leak.
We do not attribute the change in conductance to altered ionic selectivity of another conductance, eg, a K+ current, or of a nonselective cation conductance. Alterations in the major K+ currents in cardiac myocytes carried by inward rectifier, delayed rectifier, and ATP-sensitive K+ channels occur during O-R stress, but the data do not suggest that these conductances are converted into nonselective cation channels; increased or depressed conductance was observed in the absence of simultaneous changes in INSC.8
Tarr et al52 recently concluded that a rise in leak
current in frog atrial myocytes due to O-Rs from photoilluminated rose
bengal resulted from a change in selective permeability of nonselective
cation channels or from alterations in multiple channels. With
Na+ and K+ in the bath and pipette solutions,
Er for leak current showed an initial negative shift toward
the K+ reversal potential, followed by a positive shift to
0 mV. To account for the negative shift in Er,
the authors postulated that either (1) INSC was initially
more permeable to K+ over Na+, and O-R
caused a biphasic change in selectivity, with K+ permeation
rising first and then Na+ permeation, or (2) a separate
K+ conductance was initially enhanced, followed by an
increase in INSC. We never observed a negative shift in
Er during O-R stress with Na+ and
K+ or Cs+ and Li+ in the bath and
pipette solutions. This implies that the early enhanced K+
permeation noted by Tarr et al does not occur in guinea pig ventricular
myocytes exposed to O-Rs from dihydroxyfumaric acid and
FeCl3/ADP.
We attribute the increase in membrane conductance and positive shift in
Er during extracellular O-R stress to the activation of
Ca2+-sensitive INSC on the basis of the
following observations. First, during O-R stress with Cs+
and Li+ in the bath and pipette solutions, Er
for quasi–steady state whole-cell current showed a positive shift from
-38 to -3 mV. The Er of the O-R–sensitive difference
current was +3 mV, a value close to that expected (-3.7 mV) for a
nonselective cation conductance under our recording conditions based on
the Goldman-Hodgkin-Katz equation53 and assuming equal
permeation of Li+ and Cs+ in the channel and no
accumulation of the permeant ions on either side of the membrane. The
slight difference between the two values may result from a combination
of slight differences in (1) permeation of Li+ and
Cs+ and/or (2) the expected and actual concentrations of
ions along the inner and outer surfaces of the membrane. The disparity
between the Er for the difference current in net whole-cell
recording conditions (-18 mV) and in the presence of blockers to
isolate INSC (+3 mV) may reflect the contribution of
changes in additional conductances in the net current recordings.
Second, the change in conductance during extracellular O-R stress or sulfhydryl group oxidizing agent treatment was sensitive to SK&F 96365.47 48 This compound was originally reported to inhibit receptor-operated and L-type Ca2+ channels47 but has since been shown to affect Ca2+-activated nonselective cation channels, eg, channels activated by elevations in [Ca2+]i due to thapsigargin and cyclopiazonic acid.48 Effects on L-type Ca2+ channels were not a concern in the present experiments, since this conductance was suppressed with nicardipine and substitution of Ca2+ with Mg2+. Third, preliminary observations indicate that INSC activated by extracellular O-R stress is suppressed by external Ni2+ and Gd3+ (5 and 0.1 mmol/L, respectively; R. Jabr, E.A. Aiello, and W.C. Cole, unpublished observations, 1994), well-known ionic inhibitors of nonselective cation channels. Finally, caffeine treatment to elicit SR Ca2+ store release54 was shown to cause an identical increase in membrane conductance due to Ca2+-activated INSC under conditions similar to those in the oxidant experiments but only when intracellular Ca2+ chelation was low (0.1 mmol/L EGTA versus 5 mmol/L BAPTA). INSC activated by caffeine had a similar Er and sensitivity to SK&F 96365 compared with the conductance activated by O-R stress. Importantly, extracellular O-R stress was without an additive effect on membrane conductance when the Ca2+-sensitive INSC was already activated by caffeine. Taken together, these data suggest that the conductance affected by O-Rs and the sulfhydryl group oxidizing agents was the Ca2+-activated INSC.
INSC activated by O-R stress, diamide, thimerosal, or
caffeine exhibited variability in voltage dependence, including outward
rectification and relaxation of current on stepping back to a negative
holding potential after a ramp protocol. The variations in difference
current recordings obtained from net current data (Figs 1 through 3)
may reflect slight variations in other conductances in some myocytes
during extracellular O-R stress. However, this cannot explain the
rectification observed under recording conditions modified to monitor
INSC (Figs 4 through 6 and 9 through 14). It is unlikely
that the voltage dependence was related to oxidative modification,
since several myocytes exhibited rectification at early times after
treatment with caffeine. Interestingly, INSC activated by
intracellular Ca2+ injections in guinea pig myocytes
displayed less rectification than the current present at resting
[Ca2+]i, as revealed by EGTA
injections.37 It is possible that the voltage dependence
of Ca2+-activated INSC may be a property
that is manifest only when the channels are activated at lower
[Ca2+]i, as would be expected
in our experiments with 5 mmol/L BAPTA in the pipette solution and no
extracellular Ca2+. Interestingly, Hill et
al55 described a Ca2+-activated
nonselective channel in reconstituted canine ventricular sarcolemmal
vesicle that showed voltage dependence at low (<100 nmol/L)
Ca2+. Further experiments are required to resolve
this issue.
The present study implies that the mechanism responsible for increased INSC and sustained depolarization during extracellular O-R stress is different from that which we previously reported for intracellular O-R stress.20 Direct comparison of the present data with our previous experiments involving intracellular O-R stress was possible because the same generating system, recording conditions, and myocytes were used, but the generating system was included in the bath rather than the pipette solution.20 Myocytes dialyzed with O-Rs showed three stages of change in electrical activity, including (1) slight depolarization due to a decline in inward rectifier K+ current, (2) increased action potential duration, depression of plateau amplitude, and a transient period of sustained depolarization, and (3) decline in action potential duration and hyperpolarization due to ATP-sensitive K+ current.20 We attributed the sustained depolarization of stage 2 to a transient increase in Ca2+-activated INSC because the shift in membrane potential was associated with a current that (1) had an Er that was not consistent with the equilibrium potential of any single ion and varied with changes in K+ and Na+ concentrations in the bath, (2) did not discriminate between K+, Na+, Cs+, or Li+, and (3) was prevented by pretreatment with ryanodine or dialysis with 5 mmol/L EGTA in the pipette solution.20 The latter observations suggested that mishandling of Ca2+ by the SR and transient elevation in [Ca2+]i was responsible for the change in INSC during intracellular O-R stress.20 However, as shown in the present study, the sustained depolarization and increase in INSC during extracellular O-R stress did not reverse with time, and alterations in [Ca2+]i did not appear to be involved. That a change in [Ca2+]i may not be required is controversial on the basis of previous studies; photoillumination of extracellular rose bengal was without effect on INSC in rabbit ventricular myocytes in the presence of 5 mmol/L pipette EGTA,12 but Tarr et al52 still observed increased leak current in frog atrial myocytes with an identical generating system in the absence of extracellular Ca2+.
We are confident that increased INSC during extracellular O-R stress does not require changes in [Ca2+]i on the basis of the following: We went to considerable lengths to eliminate [Ca2+]i as a factor by strongly chelating intracellular Ca2+, suppressing Ca2+ influx, replacing bath Ca2+, and/or depleting internal Ca2+ stores before exposure to O-Rs. The absence of low-amplitude oscillations in membrane potential, triggered activity, or delayed afterdepolarizations due to Iti during extracellular O-R stress after pretreatment with ryanodine or after dialysis with 5 mmol/L EGTA or BAPTA is consistent with adequate control of [Ca2+]i. Itis are thought to result from increased [Ca2+]i due to abnormal SR Ca2+ release and stimulation of forward mode Na+-Ca2+ exchange56 but may also involve Ca2+-activated INSC.55 57
Our data provide evidence that oxidative modification of extracellular sulfhydryl groups on the nonselective cation channel, or an associated regulatory protein, may be involved in the response to extracellular O-R stress. The activation of INSC by extracellular O-Rs was inhibited by the sulfhydryl group reducing agent DTT. Moreover, a DTT-sensitive increase in INSC was also induced by non–O-R–oxidizing agents. These data suggest that the changes in INSC during extracellular O-R stress are the result of oxidative modification of sulfhydryl group–containing amino acids and argue against a role for localized changes in the phospholipid milieu due to lipid peroxidation. A similar inhibition by DTT was previously reported for the effects of O-Rs and other oxidants on cardiac ion transporters, such as the sarcolemmal Ca2+-ATPase26 and Na+,K+-ATPase,27 the SR Ca2+-ATPase,28 29 and ATP-sensitive K+ channels in pancreatic cells.45 Whether the sulfhydryl groups are on the nonselective channel or on an associated regulatory protein is unresolved. However, the data suggest that the thiol groups affected by O-Rs are within the extracellular compartment; thimerosal, a hydrophilic and membrane-impermeant oxidizing agent,45 46 caused an increase in INSC similar to that produced by extracellular O-Rs. Thimerosal was previously shown to inhibit ATP-sensitive K+ channels in excised patches from pancreatic cells,45 but only when it was applied on the intracellular face and not when it was in the pipette solution, implying a modification of sulfhydryl groups in the cytoplasmic compartment.
In summary, the present study is the first to report the sensitivity of a cardiac sarcolemmal ion channel to direct oxidative stress and to indicate a differential mechanism of modulation of a membrane current depending on the compartment in which the O-Rs are generated. Our data agree with previous studies in which sulfhydryl group modifying agents were found to block abnormal electrical activity that was believed to arise as a result of an altered redox state due to O-Rs in reperfusion.58 59 N-Acetylcysteine reduced the incidence of arrhythmias caused by reperfusion in Langendorff-perfused rat hearts59 and canine hearts in vivo after coronary artery ligation.58 Further studies are required to assess (1) the role of direct oxidative modification of other membrane channels in abnormal electrical activity in hearts in response to elevated O-Rs or depressed endogenous scavenger levels, (2) the significance of oxidative modification of INSC to early reperfusion arrhythmias, and (3) the properties of the single nonselective cation channels affected by O-R stress.
|
Acknowledgments |
|---|
This study was supported by Medical Research Council of Canada grant MT-10569. Dr Jabr received a traineeship from the Canadian Heart Foundation and Dr Cole was a Medical Research Council of Canada Scholar. The authors express their thanks to Yi-Jing Chen and Rodel Padua for their excellent technical assistance in the isolation of cardiac myocytes.
|
Footnotes |
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Reprint requests to Dr W.C. Cole, Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, 3330 Hospital Dr, NW, Calgary, Alberta, Canada T2N 4N1.
Previously published as preliminary observations in abstract form (Biophys J. 1994;66:A434).
Received August 17, 1994; accepted January 18, 1995.
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