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School of Biomedical Sciences, University of Leeds, Leeds LS2 9NL, United Kingdom
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The effects of acidosis on the action potential, resting potential, L-type Ca2+ (ICa), inward rectifier potassium (IK1), delayed rectifier potassium (IK), steady-state (ISS), and inwardly rectifying chloride (ICl,ir) currents of rat subepicardial (Epi) and subendocardial (Endo) ventricular myocytes were investigated using the patch-clamp technique. Action potential duration was shorter in Epi than in Endo cells. Acidosis (extracellular pH decreased from 7.4 to 6.5) depolarized the resting membrane potential and prolonged the time for 50% repolarization of the action potential in Epi and Endo cells, although the prolongation was larger in Endo cells. At control pH, ICa, IK1, and ISS were not significantly different in Epi and Endo cells, but IK was larger in Epi cells. Acidosis did not alter ICa, IK1, or IK but decreased ISS; this decrease was larger in Endo cells. It is suggested that the acidosis-induced decrease in ISS underlies the prolongation of the action potential. ICl,ir at control pH was Cd2+ sensitive but 4,4'-disothiocyanato-stilbene-2,2'-disulfonic acid resistant. Acidosis increased ICl,ir; it is suggested that the acidosis-induced increase in ICl,ir underlies the depolarization of the resting membrane potential.
action potential; chloride current; potassium current; perforated patch
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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CARDIAC MUSCLE can become acidic in pathological conditions such as coronary artery disease and some systemic disorders. However, these conditions differ because in coronary artery disease (e.g., myocardial infarction) acidosis only occurs in the ischemic region, whereas during systemic disorders (e.g., diabetes and respiratory depression) acidosis occurs homogeneously.
Acidosis alters action potential configuration (3, 27, 43) so that localized acidosis will alter action potential dispersion, which could be proarrhythmic. Homogeneous acidosis may also change action potential dispersion because the shape of the cardiac action potential is different in different regions of the heart (1, 41) as a consequence of regional differences in channel expression. Thus acidosis-induced changes in the currents carried by these channels may lead to heterogeneous changes in action potential configuration and hence dispersion.
Regional variation in the response of action potential configuration to acidosis might also explain the different acidosis-induced changes in action potential configuration reported in previous studies (3, 27, 43), which did not discriminate the region from which the cells were obtained. There are, however, other possible explanations for these differences. These include the severity and type of acidosis, and the recording conditions used [for example, L-type Ca2+ current (ICa) is decreased by acidosis when intracellular Ca2+ is buffered, as during whole cell recording, but not during perforated patch recording; 8, 15, 18, 26, 27].
The present study was designed, therefore, to investigate the electrophysiological response to acidosis of cells from different regions of the heart recorded under standard conditions. Preliminary results have been presented to the Physiological Society (25).
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Cell isolation. Male Wistar rats (~220-250 g) were stunned and then killed by cervical dislocation. The heart was rapidly removed, and cells were isolated by collagenase digestion of the Langendorff-perfused heart as described previously (26), with the following variation: after 7-min perfusion of the heart with collagenase-containing solution, small pieces of subepicardial (Epi) and subendocardial (Endo) muscle (<1 mm and <2 mm thick, respectively) were dissected from the left ventricular free wall. These were incubated separately in collagenase-containing isolation solution plus 1% bovine serum albumin for 5 min. The tissue was then filtered and the filtrate centrifuged; the supernatant was removed, and the cells were resuspended in isolation solution containing 0.75 mM Ca2+ and stored at room temperature. This procedure was repeated four to five times with the remaining tissue.
Experimental set up.
An aliquot of cells was transferred to the experimental chamber, which
was mounted on the stage of an inverted microscope (Diaphot TMD, Nikon;
Tokyo, Japan) and continuously perfused with the experimental solutions
(see Solutions and chemicals). The perforated patch-clamp
technique was used for most of the experiments in this paper, as
described previously (26). Electrodes (2-4 M
) were
used; the tip of the electrode was filled with pipette solution (see
Solutions and chemicals) and back filled with pipette solution containing 200-400 µg/ml amphotericin B. The pipette solution contained 1 mM CaCl2 to ensure that accidental
rupture of the membrane resulted in cell death. After electrical access was obtained and the capacity transients became constant, series resistance was compensated, and measurements were made. Changing between control and acid solutions while the electrode tip was in the
bath solution was used to ensure the absence of artifactual changes of
potential. Voltage and current signals were filtered at 1 kHz (low
pass) and digitized at 2 kHz; all experiments were performed at room temperature.
Measurement of action potentials. Action potentials were measured in current clamp configuration. Current pulses of 4-6 ms, sufficient to trigger an action potential, were injected every 2 s.
Measurement of ICa.
Holding potential was set to 
40 mV to inactivate Na+
current (INa), and a 200-ms depolarizing pulse
to 0 mV was applied every 2 s. Current-voltage relationships were
obtained using a series of test pulses between 30 and +50 mV. The
amplitude of the current was measured as the difference between peak
inward current and current remaining at the end of pulses. KCl in the
pipette solution and perfusate was replaced with CsCl to block
K+ currents.
Measurement of inward rectifier K+ current.
Holding potential was set to 40 mV to inactivate
INa. The current-voltage relationship was
obtained using a series of 500-ms test pulses from 120 to +20 mV;
pulses were applied every 5 s. The current was measured at 500 ms
(7). CdCl2 (0.1 mM) was added to the perfusate
to block ICa.
Measurement of delayed rectifier K+ and steady-state
currents.
Depolarization induces two components of outward current in rat
ventricular myocytes (2): transient outward current
(ITO) and sustained current, which in turn
consists of two components (35): one of which, the delayed
rectifier K+ current (IK), shows
steady-state inactivation, whereas the other, steady-state current
(ISS), does not (7, 19). To evoke
IK and ISS, holding
potential was set to 80 mV and a series of 500-ms test pulses to
voltages between 40 and +40 mV was applied; each test pulse was
preceded by a 40-ms prepulse to 40 mV to inactivate INa, and pulses were applied every 2 s.
IK was calculated by subtracting ISS from the current at the end of the pulse
(7): ISS alone was evoked using
same the protocol except that the holding potential was 20 mV and a
prepulse was not used (at this holding potential IK and INa are
inactivated; Refs. 7 and 35). 4-Aminopyridine (4-AP) was
not used because of its inhibitory effects on inward rectifier
K+ current (IK1) (9),
IK (35), and
ISS (35). However, even in the
absence of 4-AP, ITO is inactivated within the
pulse duration so that the current at 500 ms will be unaffected
(4). CdCl2 (0.5 mM) was included in the
perfusate to block ICa.
Measurement of inwardly rectifying Cl
current.
Conventional whole cell (ruptured) patch clamp was used to measure
Cl currents (ICl). A 3 M KCl-agar
bridge-AgCl pellet was used as the bath electrode. Holding potential
was set to 40 mV. Two-second test pulses to voltages between 120
and +40 mV (in 20-mV increments) followed by 400-ms pulses to +40 mV
were applied; pulses were applied every 10 s (10).
Current was measured at 2 s.
Solutions and chemicals. The solutions used for cell isolation were as described previously (26). The solution used during the experiments contained (in mM) 108 NaCl, 5 KCl, 1 Na2HPO4 · 12H2O, 1 MgSO4 · 7H2O, 20 sodium acetate, 10 glucose, 1 CaCl2, and 10 HEPES, with 5 U/l insulin; pH adjusted to 7.40 or 6.50 using NaOH. The pipette solution contained (in mM) 130 KCl, 10 NaCl, 1.4 MgCl2 · 6H2O, 1 CaCl2, and 5 HEPES; pH adjusted to 7.10 using KOH. A 50 mg/ml stock solution of amphotericin-B was made in dimethylsulfoxide immediately before the experiments; this was diluted into the pipette solution before use.
The perfusate during ICl measurement contained (in mM) 120 TEACl, 10 CsCl, 1 MgCl2 · 6H2O, 10 HEPES, 2 BaCl2, 10 glucose, 1 CaCl2, 2 4-AP, and 0.01 nifedipine; pH adjusted to 7.4 or 6.5 using TEAOH. The pipette solution contained (in mM) 110 CsCl, 20 TEACl, 5 MgATP, 5 EGTA, and 5 HEPES; pH adjusted to 7.1 using CsOH. The osmolality of the perfusate and the pipette solution for ICl measurement was 270 ± 5 mosmol/kgH2O. A 10 mM stock solution of nifedipine and a 100 mM stock solution of 4,4'-disothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) were made with DMSO and kept in light-resistant containers. A 10 mM stock solution of strophanthidin was made in ethanol. All stock solutions were diluted into the perfusate immediately before use. Addition of DIDS (final concentration, 0.1 mM) did not alter the pH of the solution.Statistical analysis. Data are expressed as means ± SE for n cells. Paired or unpaired t-tests (two-tailed) were performed as appropriate. When an unpaired t-test was used, the variances were tested by the F-test. Statistical significance was taken as P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Effect of acidosis on action potentials in Epi and Endo cells.
Figure 1 shows action potentials recorded
from representative Epi (A) and Endo (B) cells at
control pH and during acidosis. At control pH, resting membrane
potential and the amplitude of the action potential were not
significantly different in Epi and Endo cells. However, the times for
25, 50, and 90% repolarization of the action potential
(APD25, APD50, and APD90,
respectively) were significantly longer in Endo cells than in Epi cells
(Fig. 1 and Table 1).
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80 mV.
Cell capacitance was 136 ± 4 pF in Epi (n = 23)
and 145 ± 7 in Endo cells (n = 23); these values
are not statistically different.
Effect of acidosis on ICa in Epi and Endo cells.
Figure 2A shows original
records of ICa elicited in representative Epi
(left) and Endo (right) cells at control pH and
during acidosis by depolarizing pulses from 40 to 0 mV. Figure
2B shows that the mean ICa
density-voltage relationships were not significantly different in Epi
and Endo cells: maximum current density was 3.1 ± 0.3 pA/pF
(n = 7) in Epi and 2.8 ± 0.5 pA/pF
(n = 7) in Endo cells (not significant, NS).
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Effect of acidosis on IK1 in Epi and Endo cells.
Figure 3A shows original
traces of the current elicited by 500-ms test pulses from a holding
potential of 40 mV in representative Epi (left) and Endo
(right) cells; only currents elicited by test pulses between
120 and 40 mV are shown for clarity. Figure 3B shows
that the current density-voltage relationship of the sustained current
(i.e., current at the end of the pulse) was not significantly different
in Epi and Endo cells. The current elicited by test pulses negative to
40 mV is dominated by IK1 (22, 37,
44) and will be presented here. The current elicited by test
potentials positive to 40 mV also contains
ISS, which may explain the changes in the
current observed at more depolarized potentials (see below). Current
density at 120 mV was 3.83 ± 0.48 pA/pF (n = 9) in Epi cells and 3.53 ± 0.29 pA/pF (n = 6)
in Endo cells (NS).
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120 mV in Epi cells was 3.99 ± 0.51 pA/pF at control pH and 3.70 ± 0.34 pA/pF during acidosis
(NS, n = 8). The corresponding values in Endo cells
were 3.53 ± 0.29 pA/pF in control and 3.75 ± 0.61 pA/pF
in acidosis (NS, n = 6).
Thus it appears unlikely that IK1 underlies the
difference in action potential duration in Epi and Endo cells, or the
acidosis-induced change in the action potential or resting potential.
Effect of acidosis on IK and ISS in Epi and
Endo cells.
Figure 4 shows original traces of the
total outward current (left), ISS
(middle), and IK (right)
in representative Epi (top) and Endo (bottom)
cells. Test pulses of 500 ms to potentials between 40 and +40 mV were
first applied from a holding potential of 80 mV to evoke total
outward current (left). Pulses to the same test potentials
were then applied from a holding potential of 20 mV to inactivate
IK and ITO
(44) and evoke ISS alone (7; Fig.
4, middle). ISS was then subtracted
from the total outward current to obtain ITO
plus IK (right). However,
ITO inactivates within the pulse duration
(4) so that the current at the end of the pulse represents
IK. Note, however, that
ITO was present in Epi cells but not in Endo
cells. Figures 5A and
6A show mean current
density-voltage relationships for IK and
ISS, respectively, of Epi and Endo cells.
IK was significantly larger in Epi cells than in
Endo cells at most potentials positive to 10 mV (Fig. 5A):
IK current density at +40 mV was 2.18 ± 0.20 pA/pF (n = 7) in Epi cells and 1.68 ± 0.12 pA/pF in Endo cells (n = 7; P < 0.05). In contrast, ISS was not significantly different
in Epi and Endo cells (Fig. 6A): ISS
current density at 20 mV was 0.79 ± 0.06 pA/pF
(n = 7) in Epi cells and 0.83 ± 0.05 pA/pF
(n = 10) in Endo cells (NS).
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40 to +40 mV) in
Endo cells: acidosis decreased ISS density at
20 mV from 0.82 ± 0.07 to 0.66 ± 0.04 pA/pF
(P < 0.05, n = 6) in Epi cells and
from 0.83 ± 0.04 to 0.61 ± 0.04 pA/pF (P < 0.01, n = 6) in Endo cells.
When the protocol used to monitor ISS was
repeated with K+ replaced by Cs+, the current
was inhibited and acidosis had no significant effect (Fig.
6B). Thus it appears that ISS is
carried predominantly by K+, and the acidosis-sensitive
component is carried completely by K+. Because acidosis had
no effect on the Cs+-insensitive current, it has not been
subtracted from the other currents shown in Fig. 6.
It appears likely, therefore, that the lower density of
IK in Endo cells contributes to the longer
action potential observed in these cells, whereas the more pronounced
inhibition of ISS by acidosis in Endo cells will
contribute to the more marked prolongation of the action potential
produced by acidosis in these cells.
Effect of acidosis on ICl in ventricular cells.
A novel inwardly rectifying Cl current
(ICl,ir) has recently been described in cardiac
myocytes (10). This current is sensitive to
Cd2+ and resistant to DIDS, which, with its inward
rectification, distinguishes it from Cl currents
previously described in the heart. ICl,ir is
thought to be carried by ClC-2 chloride channels, which, when expressed in Xenopus oocytes, are sensitive to pH
(21). To investigate whether this current might
contribute to the response to acidosis, particularly the depolarization
of the resting membrane potential observed in Epi and Endo cells (Table
1), we have investigated whether acidosis alters
ICl,ir in ventricular myocytes. Because the
effect of acidosis on resting membrane potential is not significantly different in Epi and Endo cells (see Table 1), these
experiments were performed on myocytes isolated from the whole
ventricle, using K+- and Na+-free, nifedipine-
and Cs+-containing solutions (see METHODS) to
avoid contamination by currents carried by K+,
Na+, and Ca2+, and possible complications due
to effects of Cd2+ and DIDS on currents carried by these
ions (e.g., 40).
120 mV increased from 4.7 ± 1.0 pA/pF at control pH to 7.5 ± 1.5 pA/pF during acidosis
(n = 8, P < 0.01) but did not
significantly alter the time constants of activation. The effects of
acidosis were completely reversible (Fig. 7A).
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]pip) was decreased from 130 to 20 mM
by replacing CsCl with equimolar Cs-aspartate. This decreased current
and caused a negative shift in the reversal potential (Fig.
7B): the current at 120 mV decreased from 4.3 ± 0.7 pA/pF (n = 17) at 130 mM
[Cl]pip to 0.4 ± 0.1 pA/pF
(n = 5, P < 0.01) at 20 mM
[Cl]pip. Reversal potential, after
correction for liquid junction potential, increased from 6 mV
(predicted Cl equilibrium potential, 1 mV) at 130 mM
[Cl]pip to 36 mV (49 mV) at 20 mM
[Cl]pip (Fig. 7B). Reducing
[Cl]pip to 20 mM also reduced the
acidosis-induced increase in membrane current: at 120 mV, the
acidosis-induced change was 2.8 ± 0.6 pA/pF (n = 8) at 130 mM [Cl]pip and 0.2 ± 0.1 pA/pF (n = 5) at 20 mM
[Cl]pip (P < 0.01, Fig.
7C).
Effect of CdCl2 and DIDS on ICl,ir in
response to acidosis.
The original traces in Fig. 8,
A and B, show the effect of 0.1 mM
CdCl2 and 0.1 mM DIDS, respectively, on
ICl,ir, the response to acidosis in the presence
of these agents, and the subsequent recovery from acidosis in their
continuing presence.
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120 mV was 4.3 ± 1.0 pA/pF in control, 1.0 ± 0.2 pA/pF in the presence of CdCl2 (P < 0.01 vs. control), and 5.3 ± 0.8 pA/pF during acidosis in the
presence of CdCl2 (P < 0.001 vs. in the
presence of CdCl2, NS vs. control, n = 8).
In contrast, DIDS had no significant effect on basal current (Fig. 8,
B and D); however, in the presence of DIDS, the
effect of acidosis on the current was markedly reduced (Fig. 8,
B and D): the current at 120 mV was 4.9 ± 1.3 pA/pF in control, 5.3 ± 1.1 pA/pF in the presence of
DIDS (NS vs. control), and 6.4 ± 1.4 pA/pF during acidosis in
the presence of DIDS (P < 0.05 vs. in the presence of
DIDS, n = 6).
Figure 8E shows the mean current density-voltage
relationships for the acidosis-induced current (i.e., the increase in
current during acidosis) in the absence of inhibitors and in the
presence of CdCl2 or DIDS, showing that the
acidosis-sensitive component is resistant to CdCl2 but
sensitive to DIDS: acidosis-induced current at 120 mV is 2.8 ± 0.6 pA/pF in control (n = 8), 4.3 ± 1.0 pA/pF in the presence of CdCl2 (n = 8, NS
vs. control), and 1.1 ± 0.2 pA/pF in the presence of DIDS
(n = 6, P < 0.05 vs. control).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In the present study, acidosis was produced by decreasing the pH of the perfusate (i.e., extracellular pH) from 7.4 to 6.5. Intracellular pH (pHi) decreases by 0.5 units in response to this maneuver and reaches a new steady state within 3 min (14). The presence of a perforated patch electrode containing 5 mM HEPES does not affect the change of pHi (not shown). Measurements were made after a 5-min exposure to the acid solution (16), when both intracellular and extracellular pH had decreased. This can occur in vivo, for example, during ischemia when both extracellular and pHi decrease as a consequence of metabolite accumulation, and the decrease of pHi in the present study (0.5 pH units) is similar to that reported during global ischemia (0.8 pH units; 12). Acidosis alters the intracellular environment, increasing intracellular Ca2+ concentration ([Ca2+]i) (13), intracellular Na+ concentration ([Na+]i) (13), calcium-calmodulin-dependent kinase (CaMKII) activity via the increase in [Ca2+]i (14), and inhibiting protein phosphatase activity (32). We used the perforated patch-clamp technique to minimize disruption to these normal responses to investigate the physiological response to acidosis.
The methods used to record the currents measured in the present study have been described previously. Pharmacological K+ channel inhibitors were not used because many such inhibitors affect more than one channel (35). However, a depolarized holding potential is known to inhibit ITO and IK (44), allowing measurement of ISS (7; consistent with the noninactivating current shown in Fig. 4). It has also previously been shown that the current at the end of a 200-ms depolarizing pulse is insensitive to 4-AP, and thus that ITO inactivates during the pulse (4) so that the remaining current consists of ISS and IK. The protocol used to monitor IK1 has also been described previously (e.g., 7).
Regional differences in the effect of acidosis on the action potential. Action potential configuration is different in Epi and Endo cells in many species (for review see Ref. 1), and the rat action potential shows similar regional differences to other species, being shorter in Epi cells than in Endo cells (Fig. 1; Refs. 7 and 9). This appears to be due mainly to the higher density of ITO in Epi cells (6, 7, 9, 16, 44), although the present study shows that IK, which was larger in Epi cells, may also contribute to the shorter action potential.
Previous studies of the effect of acidosis on the action potential have given variable results, some showing prolongation and some shortening of the action potential (e.g., 3, 27). In the present study, acidosis prolonged APD50; this change was larger in Endo than in Epi cells. The possible mechanisms are considered below.Regional differences in the effect of acidosis on ICa. The ICa density-voltage relationship of Epi cells and Endo cells was not significantly different, consistent with previous studies in the rat (6, 9), cat (24), and guinea pig (30).
Acidosis did not alter the current density-voltage relationship of ICa in Epi or Endo cells. This is consistent with previous reports using the perforated patch-recording technique in the rat ventricle (8, 15, 26) and suggests that a change in ICa is unlikely to underlie the change in APD during acidosis.The possible role of Na2+/Ca2+ exchange in the response to acidosis. Previous work has shown that Na+/Ca2+ exchange current (INa/Ca) affects the slow repolarization phase in the rat ventricle and that inhibition of the exchanger shortens the APD (36). Because Na+/Ca2+ exchange is inhibited during acidosis (15, 26, 39), this would be expected to shorten rather than prolong the action potential. In addition, Na+/Ca2+ exchange expression appears to be the same in Epi and Endo cells (31), making it unlikely that the exchanger can account for the regional differences in the response to acidosis, although it has been reported that exchange current is smaller in Endo cells (34), which, if the exchanger were involved, might be expected to make the response to acidosis less pronounced in these cells.
The possible role of ITO in the response to acidosis.
ITO is greater in Epi cells than in Endo cells
(Fig. 4; Refs. 6, 7, and 9);
this is the major factor underlying the difference in action potential
configuration between Epi and Endo cells. The effect of acidosis on
ITO has been investigated previously in the same
conditions as those used in the present study by Hulme and Orchard
(16), who showed that acidosis does not alter
ITO when the holding potential is negative to
60 mV, so that changes in ITO would not
contribute to the effect of acidosis on the action potential.
Regional differences in the effect of acidosis on IK1. The present study showed no regional differences in IK1. Previous studies have given variable results: some studies have shown no regional differences between Epi and Endo cells from rats (7, 9, 37), guinea pigs (30), and dogs, (29), whereas others have shown that IK1 is greater in Endo cells than in Epi cells (6, 44). IK1 in all of these studies was measured using ruptured patch whole cell recording; because IK1 is sensitive to extracellular Na+ concentration ([Na+]o) and [Ca2+]i, it is possible that the recording conditions may contribute to the observed differences.
Previous studies of the effect of acidosis on IK1 have shown little or no effect during pH changes of the magnitude used in the present study (11, 19, 43). The present data are, therefore, consistent with previous studies and with the idea that Kir2.1/ Kir2.2 underlie IK1 in the rat ventricle because Kir2.1 is insensitive to pH (45). These data also suggest that acidosis-induced changes in IK1 do not play a significant role in the response to acidosis.Regional differences in the effect of acidosis on IK. Casis et al. (7) reported no regional differences in IK between Epi and Endo cells. However, Yao et al. (44) reported that IK is larger in Epi than in Endo cells, consistent with the present data. It is possible that the use of 4-AP by Casis et al. (7) might have affected IK. The higher density of IK in Epi cells, in conjunction with the higher density of ITO (16), will contribute to the faster repolarization in Epi cells.
However, acidosis had no significant effect on IK either in Epi or Endo cells, consistent with Xu and Rozanski (43), who showed that a 1.2-unit decrease in the pH of the pipette solution in whole cell configuration does not alter IK in rat ventricular cells and making it unlikely that IK contributes to the acidosis-induced changes in the action potential.Regional differences in the effect of acidosis on ISS. Several investigators have described a sustained current that is not inactivated even at more positive holding potentials in rat ventricular myocytes (7, 20, 35), and there appears to be no regional difference in the distribution of this current (Ref. 7 and this study). When K+ was replaced with Cs+, ISS was largely suppressed (compare Fig. 6, A and B), and acidosis did not alter the current (Fig. 6B), indicating that the measured current is carried predominantly by K+ and that the acidosis-sensitive increase in current is carried by K+.
Acidosis decreased ISS in Epi and Endo cells, although the inhibition was greater in Endo cells (Fig. 6). This current appears to contribute to the repolarization of rat ventricular myocytes (35) so that the acidosis-induced decrease in ISS might explain the prolongation of the action potential observed during acidosis and the greater prolongation observed in Endo cells. Scamps (35) suggested that ISS is carried by Kv1.2 or Kv2.1; more recently James et al. (20) suggested that ISS is carried by TBAK-1/TASK-1, which is inhibited by acidosis (23), consistent with the present data. One potential problem with this hypothesis is that acidosis inhibits ISS throughout the voltage range studied, but the effect of acidosis on the action potential is most apparent at APD50. It seems possible, however, that the early and late phases of the action potential are dominated by other larger currents so that the effect of ISS on APD will be less apparent at these times.Effect of acidosis in ICl,ir.
The present study showed a marked increase in
ICl,ir during acidosis. This increase in inward
current may contribute to the prolongation of the action potential,
although the small amplitude of the current at more depolarized
potentials when [Cl]pip was in the
physiological range (20 mM) suggests that its contribution would be
very small. It could, however, underlie the acidosis-induced
depolarization of the resting membrane potential. Such depolarization
has been a consistent observation (for review see Ref. 33)
and appears to be the same in atrial and ventricular cells and in Epi
and Endo cells, although the mechanism has remained obscure:
acidosis-induced depolarization occurs following inhibition of
Ca2+-sensitive currents (including
INa/Ca), ICa,
K+ currents, and Na+ pump current. The
amplitude of the current at 80 mV is sufficient to account for the
depolarization: injection of 40 pA current at 80 mV induced a
4.0 ± 0.8 mV (n = 3) depolarization (not shown), giving a cell input resistance of 100 ± 20 M
(n = 3), within the normal range for rat ventricular
myocytes (20-200 M, Ref. 42). Thus the small (28 pA) acidosis-induced inward shift of ICl,ir at
80 mV would be expected to cause a 2.8-mV depolarization, consistent
with the 3-mV depolarization observed during acidosis.
current, Ca2+-activated
Cl current, or swelling-induced Cl current,
because these do not show inward rectification (for review see Ref.
17). However, ICl,ir shows marked
similarities to the current carried by ClC-2 channels. These channels
have been found in atrial and ventricular cells (5, 10);
the Cl current carried by these channels shows inward
rectification (10, 21), is of comparable magnitude to that
recorded in the present study (10), is sensitive to
Cd2+ and resistant to DIDS at control pH, and is increased
by acidosis when expressed in Xenopus oocytes
(21). Thus it appears that ICl,ir
is carried by ClC-2.
An interesting observation is that the acidosis-induced increase of
current has a different pharmacology from that of the current observed
at control pH, showing Cd2+ insensitivity and DIDS
sensitivity. In the case of Cd2+, the acid-induced current
in the presence of Cd2+ is not significantly different from
that in control (Fig. 8E); if anything, it is slightly
larger. If Cd2+ and H+ were competing for a
common binding site, as suggested for other channels, it might be
expected that this current would be smaller in the presence of
Cd2+. However, it appears that the stimulatory effect of
acidosis on ICl,ir is independent of the
inhibitory effect of Cd2+ and thus that Cd2+
and H+ are acting at different sites or that acidosis is
altering the concentration of the active form of Cd2+. The
observation that ICl,ir is sensitive to
Cd2+ at control pH but that acidosis still increases this
current in the presence of Cd2+ is germane to the
measurements of K+ currents in the present study, in which
Cd2+ was present in the bathing solution (see
METHODS). However, ICl,ir is small
at physiological levels of intracellular [Cl] and at
the range of potentials used in the measurement of K+
currents (e.g., Fig. 7B), particularly compared with the
K+ currents being measured. Thus although
activation of ICl,ir may have occurred in these
experiments, the effect is likely to be very small; the observation
that two of the three K+ currents measured did not change
during acidosis supports this idea.
There are a number of possible explanation for why DIDS partially
inhibited the acidosis-induced increase of
ICl,ir. First, it is possible that
ICl,ir is already maximal. This is unlikely because DIDS had no effect on current at control pH (Fig.
8D) and acidosis could increase the current from this level
in the absence of DIDS (Fig. 7B). Second, DIDS may bind to a
pH-sensitive domain of the channel, although this is unlikely because
DIDS only inhibited the acid-induced increase of current, rather than all ICl,ir. Third, protonation of DIDS may alter
its effectiveness. Finally, acidosis may increase
ICl,ir, not by acting on the channel, but by
causing a DIDS-sensitive increase of intracellular
[Cl], although the possible mechanism is unclear: the
Cl/OH exchange (or
H+-Cl coinflux) mechanism described by Sun et
al. (38) is DIDS insensitive, whereas the DIDS-sensitive
Na+-HCO
| |
ACKNOWLEDGEMENTS |
|---|
This work was supported by the British Heart Foundation and Wellcome Trust. K. Komukai also thanks the Uehara Memorial Foundation.
| |
FOOTNOTES |
|---|
Address for reprint requests and other correspondence: C. Orchard, School of Biomedical Sciences, Univ. of Leeds, Leeds LS2 9NL, UK (E-mail: c.h.orchard{at}leeds.ac.uk).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published March 28, 2002;10.1152/ajpheart.01042.2001
Received 29 November 2001; accepted in final form 22 March 2002.
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REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Antzelevitch, C,
Sicouri S,
Litovsky SH,
Lukas A,
Krishman SC,
Diego JMD,
Gintant GA,
and
Liu DW.
Heterogeneity within the ventricular wall. electrophysiology and pharmacology of epicardial, endocardial, and M cells.
Circ Res
69:
1427-1449,
1991
2.
Apkon, M,
and
Nerbonne JM.
Characterization of two distinct depolarization-activated K+ currents in isolated adult rat ventricular myocytes.
J Gen Physiol
97:
973-1011,
1991
3.
Bethell, HW,
Vandenberg JI,
Smith GA,
and
Grace AA.
Changes in ventricular repolarization during acidosis and low-flow ischaemia.
Am J Physiol Heart Circ Physiol
275:
H551-H561,
1998
4.
Bogdanov, KY,
Spurgeon HA,
Vinogradova TM,
and
Lakatta EG.
Modulation of the transient outward current in adult rat ventricular myocytes by polyunsaturated fatty acids.
Am J Physiol Heart Circ Physiol
274:
H571-H579,
1998
5.
Britton, FC,
Hatton WJ,
Rossow CF,
Duan D,
Hume JR,
and
Horowitz B.
Molecular distribution of volume-regulated chloride channels (CLC-2 and ClC-3) in cardiac tissues.
Am J Physiol Heart Circ Physiol
279:
H2225-H2233,
2000
6.
Bryant, S,
Shipsey SJ,
and
Hart G.
Normal regional distribution of membrane current density in rat left ventricle is altered in catecholamine-induced hypertrophy.
Cardiovasc Res
42:
391-400,
1999
7.
Casis, O,
Iriarte M,
Gallego M,
and
Sanchez-Chapula JA.
Differences in regional distribution of K+ current densities in rat ventricle.
Life Sci
63:
391-400,
1998[Web of Science][Medline].
8.
Choi, HS,
Trafford AW,
Orchard CH,
and
Eisner DA.
The effect of acidosis on systolic Ca and sarcoplasmic reticulum calcium content in isolated rat ventricular myocytes.
J Physiol
529:
661-668,
2000
9.
Clark, RB,
Bouchard RA,
Salinas-Stefanon E,
Sanchez-Chapula J,
and
Giles WR.
Heterogeneity of action potential waveforms and potassium currents in rat ventricle.
Cardiovasc Res
27:
1795-1799,
1993
10.
Duan, D,
Ye L,
Britton F,
Horowitz B,
and
Hume JR.
A novel anionic inward rectifier in native cardiac myocytes.
Circ Res
86:
e63-e71,
2000
11.
Findlay, I.
Effects of pH upon the inhibition by sulphonylurea drugs of ATP-sensitive K+ channels in cardiac muscle.
J Pharmacol Exp Ther
262:
71-79,
1992
12.
Garlick, PB,
Radda GK,
and
Seeley PJ.
Studies of acidosis in the ischaemic heart by phosphorus nuclear magnetic resonance.
Biochem J
184:
547-554,
1979[Web of Science][Medline].
13.
Harrison, SM,
Frampton JE,
McCall E,
Boyett MR,
and
Orchard CH.
Contraction and intracellular Ca2+, Na+, and H+ during acidosis in rat ventricular myocytes.
Am J Physiol Cell Physiol
262:
C348-C357,
1992
14.
Hulme, JT,
Colyer J,
and
Orchard CH.
Acidosis alters the phosphorylation of Ser16 and Thr17 of phospholamban in rat cardiac muscle.
Pflügers Arch
434:
475-483,
1997[Web of Science][Medline].
15.
Hulme, JT,
and
Orchard CH.
Effect of acidosis on Ca2+ uptake and release by the sarcoplasmic reticulum of intact rat ventricular myocytes.
Am J Physiol Heart Circ Physiol
275:
H977-H987,
1998
16.
Hulme, JT,
and
Orchard CH.
Effect of acidosis on transient outward potassium current in isolated rat ventricular myocytes.
Am J Physiol Heart Circ Physiol
278:
H50-H59,
2000
17.
Hume, JR,
Duan D,
Collier ML,
Yamazaki J,
and
Horowitz B.
Anion transport in heart.
Physiol Rev
80:
30-81,
2000.
18.
Irisawa, H,
and
Sato R.
Intra- and Extracellular actions of proton on the calcium current of isolated guinea pig ventricular cells.
Circ Res
59:
348-355,
1986
19.
Ito, H,
Vereecke J,
and
Carmeliet E.
Intracellular protons inhibit inward rectifier K+ channel of guinea-pig ventricular cell membrane.
Pflügers Arch
422:
280-286,
1992[Web of Science][Medline].
20.
James, AF,
Ramsey JE,
Reynolds AM,
Hendry BM,
and
Shattock MJ.
Effects of endothelin-1 on K+ currents from rat ventricular myocytes.
Biochem Biophys Res Commun
284:
1048-1055,
2001[Web of Science][Medline].
21.
Jordt, SE,
and
Jentsch TJ.
Molecular dissection of gating in the CIC-2 chloride channel.
EMBO J
16:
1582-1592,
1997[Web of Science][Medline].
22.
Kilborns, MJ,
and
Fedida D.
A study of the developmental changes in outward currents of rat ventricular myocytes.
J Physiol
430:
37-60,
1990
23.
Kim, Y,
Bang H,
and
Kim D.
TBAK-1 and TASK-1, two-pore K+ channel subunits: kinetic properties and expression in rat heart.
Am J Physiol Heart Circ Physiol
277:
H1669-H1678,
1999
24.
Kimura, S,
Bassett AL,
Furukawa T,
Furukawa N,
and
Myerburg J.
Differences in the effect of metabolic inhibition on action potentials and calcium currents in endocardial and epicardial cells.
Circulation
84:
768-777,
1991
25.
Komukai, K,
Pascarel C,
and
Orchard CH.
Effect of acidosis on the action potential, ICa and background steady-state current in isolated sub-endocardial and sub-epicardial rat ventricular myocytes (Abstract).
J Physiol
526P:
97P,
2000.
26.
Komukai, K,
Pascarel C,
and
Orchard CH.
Compensatory role of CaMKII on ICa and SR function during acidosis in rat ventricular myocytes.
Pflügers Arch
442:
353-361,
2001[Web of Science][Medline].
27.
Kurachi, Y.
The effects of intracellular protons on the electrical activity of single ventricular cells.
Pflügers Arch
394:
264-270,
1982[Web of Science][Medline].
28.
Leem, CH,
Lagadic-Gossmann D,
and
Vaughan-Jones RD.
Characterization of intracellular pH regulation in the guinea-pig ventricular myocyte.
J Physiol
517:
159-180,
1999
29.
Liu, DW,
Gintant GA,
and
Antzelevitch CC.
Ionic bases for electrophysiological distinctions among epicardial, midmyocardial, and endocardial myocytes from the free wall of the canine left ventricle.
Circ Res
72:
671-687,
1993
30.
Main, MC,
Bryant SM,
and
Hart G.
Regional differences in action potential characteristics and membrane currents of guinea-pig left ventricular myocytes.
Exp Physiol
83:
747-761,
1998[Abstract].
31.
McDonald, RL,
Colyer J,
and
Harrison SM.
Expression of Na+-Ca2+ exchanger protein is lower in guinea-pig left ventricular mid-myocardial myocytes compared with surface myocytes (Abstract).
J Physiol
521P:
28P,
1999.
32.
Mundina-Weilenmann, C,
Vittone L,
Cingolani HE,
and
Orchard CH.
Effects of acidosis on phosphorylation of phospholamban and troponin I in rat cardiac muscle.
Am J Physiol Cell Physiol
270:
C107-C114,
1996
33.
Orchard, CH,
and
Cingolani HE.
Acidosis and arrhythmias in cardiac muscle.
Cardiovasc Res
28:
1312-1319,
1994
34.
Quinn, FR,
McIntosh MA,
Cobbe SM,
and
Smith GL.
Sodium-calcium exchange current density is higher in endocardial than epicardial cells (Abstract).
Biophys J
78:
224A,
2001.
35.
Scamps, F.
Characterization of a beta-adrenergically inhibited K+ current in rat cardiac ventricular cells.
J Physiol
491:
81-97,
1996
36.
Schouten, VJA,
and
ter Keurs HEDJ
The slow repolarization phase of the action potential in rat heart.
J Physiol
360:
13-25,
1985
37.
Shimoni, Y,
Severson D,
and
Giles W.
Thyroid status and diabetes modulate regional differences in potassium currents in rat ventricle.
J Physiol
488:
673-688,
1995
38.
Sun, B,
Leem CH,
and
Vaughan-Jones RD.
Novel chloride-dependent acid loader in the guinea-pig ventricular myocyte: part of a dual acid-loading mechanism.
J Physiol
495:
65-82,
1996
39.
Teracciano, CMN,
and
MacLeod KT.
Effects of acidosis on Na+/Ca2+ exchange and consequences for relaxation in guinea pig cardiac myocytes.
Am J Physiol Heart Circ Physiol
267:
H477-H487,
1994
40.
Wang, HS,
Dixon JE,
and
McKinnon D.
Unexpected and differential effects of Cl channel blockers on the Kv4.3 and Kv4.2 K+ channels.
Circ Res
81:
711-718,
1997
41.
Watanabe, T,
Delbridge LM,
Bustamante JO,
and
McDonald TF.
Heterogeneity of the action potential in isolated rat ventricular myocytes and tissue.
Circ Res
52:
280-290,
1983
42.
White, RL,
Spray DC,
Campos de Carvalho AC,
Wittenberg BA,
and
Bennett MVL
Some electrical and pharmacological properties of gap junctions between adult ventricular myocytes.
Am J Physiol Cell Physiol
249:
C447-C455,
1985
43.
Xu, Z,
and
Rozanski G.
Proton inhibition of transient potassium outward current in rat ventricular myocytes.
J Mol Cell Cardiol
29:
481-490,
1992.
44.
Yao, JA,
Jiang-M,
Fan JS,
Zhou YY,
and
Tseng GN.
Heterogeneous changes in K currents in rat ventricles three days after myocardial infarction.
Cardiovasc Res
44:
132-145,
1999
45.
Zhu, G,
Liu C,
Qu Z,
Chanchevalap S,
Xu H,
and
Jiang C.
CO2 inhibits specific inward rectifier K+ channels by decrease in intracellular and extracellular pH.
J Cell Physiol
183:
53-64,
2000[Web of Science][Medline].
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