© 2000 American Heart Association, Inc.
UltraRapid Communications |
Possible Mechanism(s) of Arachidonic Acid–Induced Intracellular Acidosis in Rat Cardiac Myocytes
From the Institutes of Physiology (M.-L.W., C.-C.C.) and Pharmacology (M.-J.S.), College of Medicine, National Taiwan University, Taipei, Taiwan.
Correspondence to Drs Mei-Lin Wu and Ming-Ja Su, Institutes of Physiology and Pharmacology, College of Medicine, National Taiwan University, No. 1, Sec. 1, Jen-Ai Rd, Taipei, Taiwan.
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Abstract |
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Abstract—Arachidonic acid (AA) and other nonesterified fatty acids (FAs) have been shown to exert harmful effects during cardiac ischemia. By continuously measuring intracellular pH (pHi) changes in neonatal and adult cardiac myocytes, we have found, for the first time, that 10 µmol/L AA induces a substantial intracellular acidosis (0.3 to 0.4 pH units). We have ruled out the possibilities that the AA-induced acidosis is caused by (1) inhibition or stimulation of the pHi regulators, (2) protein kinase C activation or the generation of AA metabolites or free radicals, or (3) activation of NADPH oxidase or an inward H+ current. The AA-induced acidosis fits to a simple diffusion mechanism, as proposed by Kamp and Hamilton (flip-flop model) for artificial phospholipid bilayers. The important properties found in the cardiac myocyte are that (1) the initial rate of acid flux (JH) increases with the AA concentration (2 to 50 µmol/L), (2) FAs with a -COOH group (eg, AA, oleic acid, and linoleic acid) induce intracellular acidification, but FAs with a -COOCH3 group (eg, AA methyl ester) have little effect on the pHi, (3) tetradecylamine (FA amine) induces intracellular alkalosis, and, most importantly, (4) both the AA- and tetradecylamine-induced pHi changes can be reversed by 0.3% BSA. Because a low concentration of AA (10 µmol/L) can induce a substantial acidosis, the possible involvement of the FA-evoked acidosis in the negative inotropic effect during cardiac ischemia is discussed. The full text of this article is available at http://www.circresaha.org.
Key Words: arachidonic acid • intracellular acidosis • ventricular myocytes
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Introduction |
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Arachidonic acid (AA) is one of the most important nonesterified fatty acids (FAs), because both it and its metabolites have been shown to play both physiological and pathophysiological roles in a variety of cells.1 2 In addition to AA, another two nonesterified FAs, oleic acid and linoleic acid, are also released in considerable amounts from membrane phospholipids after 1 to 3 hours of cardiac ischemia.3 4 At least four types of ion channels in the heart are modulated by AA and have been suggested to be involved in ischemic-induced arrhythmias: (1) ATP-sensitive K+ channel (IK.ATP), (2) AA-activated K+ channel (IK.AA), (3) L-type Ca2+ channel (ICa), and (4) gap junctional channel, with the IK.ATP5 and junctional channel6 7 being inhibited, and the ICa8 9 and IK.AA5 10 activated, by 10 to 30 µmol/L AA.
Intracellular acidosis is also involved in many important physiological and pathophysiological functions. For example, lowering the intracellular pH (pHi) has a negative inotropic effect on the heart. At least three mechanisms have been suggested for this effect of the pHi on the active tension: An increase in [H+]i may (1) inhibit the ICa,11 12 13 ; (2) markedly reduce the sensitivity of the contractile elements to [Ca2+]i12 14 15 ; or (3) reduce Cai release from, and/or interfere with its uptake by, the sarcoplasmic reticulum (SR).12 14 15 16
There are only two reports of FAs evoking significant intracellular
acidosis in living cells, the cell types involved being pancreatic
ß-cells and adipocytes,17 18 whereas a few other studies
have been performed in artificial phospholipid
vesicles.19 20 21 The mechanism proposed by Kamp and
Hamilton17 18 19 20 21 22 for FA-induced acidosis, known as the
"flip-flop" model and involving simple diffusion, rather than
carrier-mediated transport, of free FAs across the lipid bilayer, is
based on the following evidence. Because FAs have very high oil/water
partition coefficients, almost all added FA molecules bind to the lipid
bilayer.22 The apparent pKs for FAs in both leaflets are
high (
7.6, using 13C NMR measurement) and
independent of FA chain length over the range of 8 to 26
carbons.22 23 One possible explanation is that the charged
surface of the membrane affects the ionization of FAs, increasing the
apparent pK and the formation of the nonionized form.22 At
an extravesicular pH of 7.4, bound FA in the outer leaflet consists of
almost equal amounts of FA-H and the ionized form,
FA- (Henderson-Hasselbalch equation).
FA- diffuses slowly (T1/2
of minutes) and consequently cyclic transport of
H+ does not occur in bilayers. However, 50% of
FA-H diffuses rapidly (T1/2 of seconds) from the
outer to the inner leaflet ("flip"),19 22 where half
dissociates into FA- (and
H+) (see Figure 3
of References
19 or 22 ). For example, because
almost all FAs bind to the membrane, the addition of 20 nmol of FA in 2
mL of solution results in the donation of 5 nmol of
H+ (25% of the total FA), causing intravesicular
acidosis.19 To achieve ionization equilibrium in both
leaflets, 50% of the FA- in the outer leaflet
forms 25% FA-H and 25% of
FA-.19 22 On addition of BSA, the
FA-H leaves the outer leaflet to bind to BSA and is replaced by FA-H
diffusing from the inner leaflet ("flop"), resulting in transport
of H+ out of the vesicle.
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In contrast, in macrophages and neutrophils, the addition of
5 to 10 µmol/L AA induces a marked
alkalosis.24 25 This is explained by AA-mediated
activation of NADPH oxidase in the membrane, which catalyzes the
transmembrane movement of electrons from cytosolic NADPH to molecular
oxygen, resulting in the appearance of extracellular
O2-· and leading to the
depolarization of the membrane potential and the activation of an
outward-rectifying proton conductance (Hi efflux
or pHi increase), which has been confirmed by
patch-clamp experiments.24 25 26 27
In the present study, we have shown, for the first time in cardiac
cells, that a profound intracellular acidosis (0.3 to 0.4 pH units),
rather than alkalosis, is induced by the addition of 10 µmol/L
AA and other nonesterified FAs. Because the pHi
has a great influence on cardiac function and because AA is one of the
major nonesterified FAs that accumulate in ischemic tissue, the
possible mechanism(s) for this intriguing AA-induced acidosis has been
investigated in detail. In addition, the possible
pathophysiological role of AA-induced acidosis
during cardiac ischemia is discussed.
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Materials and Methods |
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Cell Preparation
Neonatal cardiomyocyte cultures were prepared from 2- to 3-day-old Wistar rats, as described by Borg et al.28 The rats were purchased from the animal center of National Taiwan University, College of Medicine. Briefly, rats were killed by cervical dislocation and then decapitated. Two ventricles were dissected and dissociated by mechanical chopping, then digested by 0.169% trypsin and 0.085% collagenase in Hanks solution. Cells were cultured in F-10 (Gibco), supplemented with 10% FCS, 10% horse serum, and 1% penicillin-streptomycin. Four- to 5-day-old cultured cells were used in the present study. The method for freshly isolated adult cardiac myocytes was described in our previous work.29
Chemicals and Solutions
Unless otherwise stated, all chemicals were purchased from Sigma
and all experiments performed at 37°C in nominally
HCO3-free, HEPES-buffered solution, consisting of
(in mmol/L) NaCl 118, KCl 4.5, MgCl2 1.0,
CaCl2 2.0, glucose 10, and HEPES 10 (pH adjusted
to 7.4 at 37°C with NaOH). The Cl-free, bicarbonate-free solution
contained (in mmol/L) sodium gluconate 118, potassium gluconate
4.5, MgSO4 4.0, glucose 10, and HEPES 10; 10
mmol/L calcium gluconate was added to compensate for
Ca2+ and Mg2+ chelation by
gluconate, and their respective concentrations were increased 4- and
5-fold.30
Measurement of Intracellular pH
The pHi was measured using
microfluorometry as previously described.29 31 32 In
brief, cells were loaded for 15 to 20 minutes at room temperature with
5 µmol/L BCECF AM (Molecular Probes) and were excited
alternately using 490- and 440-nm wavelength light. The following
equation was used to convert the fluorescence ratio (490/440)
into the pHi:
pHi=pKa+log[(Rmax-R)/(R-Rmin)]+log(F440min/F440max),
where R is the ratio of the 510-nm fluorescence at 490-nm
excitation over that at 440-nm excitation, Rmax
and Rmin are the maximum and minimum ratio values
from the data curve (data not shown), and the pKa
(-log of dissociation constant) is 7.16.
F440min/F440max is the
ratio of fluorescence measured at 440 nm of
Rmin and Rmax.
Measurement of Intracellular Ca2+
The [Ca2+]i was
measured using fura-2 AM as previously described.33 34
Measurement of Intracellular Na+
The [Na+]i was
measured using SBFI-AM. The ratio of 510-nm emission at the excitation
wavelengths, respectively, of 340 and 380 nm for a small group of cells
was calculated and converted to the
[Na+]i by in vivo
calibration.35 36
Electrophysiology
The whole-cell configuration of the patch-clamp technique
was used to record ionic currents in adult ventricular
myocytes.37 38 During measurement of the proton current,
possible contamination by either K+,
Na+, Ca2+, or
Cl- currents was prevented by bathing the cell
in Ca-free, Mg-containing cesium aspartate, HEPES (100
mmol/L)-buffered solution (pHo 7.4), and
internally dialyzed with cesium aspartate HEPES (100
mmol/L)-buffered solution (pHi 7.1). Membrane
currents were determined by 3-second depolarizing or hyperpolarizing
steps from the holding potential of -40 mV to potential levels between
+60 and -120 mV (see Figure 4).
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Statistical Methods
All data are expressed as the mean±SEM for n experiments.
Statistical comparison was by the paired or nonpaired Student’s
t test, with a value of P<0.05 being considered
statistically significant.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
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Results |
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The resting pHi in neonatal and adult cardiac myocytes was found to be 7.13±0.01 (n=43) and 7.29±0.02 (n=37), respectively. Because it is easier to work with neonatal cells continuously perfused on coverslips, rather than adult cells in suspension, most of the work was performed using neonatal cells.
Figure 1A shows an example of the
intracellular acidosis evoked by continuous perfusion of neonatal
cardiac myocytes with 2 to 30 µmol/L AA. Table 1 shows the mean values using
concentrations of 2 to 50 µmol/L AA. The addition of 2
µmol/L AA resulted in only a small transient
pHi decrease (Table 1), and little, or no,
pHi decrease was seen when the concentration of
AA was lower than 2 µmol/L. At 10 µmol/L AA, the evoked
acidosis was -0.42±0.02 pH unit (n=30, Table 1), and this was
completely reversed after removal of AA (Figure 1C, n=7). The
maximum intracellular acidosis (-0.46±0.03 pH units, n=9) was seen
using 20 to 30 µmol/L AA, although there was no statistical
difference (P>0.05) between the results using AA
concentrations between 10 and 50 µmol/L (Table 1). A
linear (nonsaturating) increase in the initial rate of acid flux,
JH (=the intrinsic buffering power,
ßi, x
pHi/min, Figure 1B), was noted when 2 to 50 µmol/L
[AA]o was added at the same resting
pHi of 7.1 (at pHi 7.1,
ßi=14 mmol/L,
ßi=-40.0
pHi+298.2.29
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For technical reasons, adult ventricular myocytes were used
in an unperfused suspension. In a control experiment, the addition of 1
mL of 10 µmol/L AA to neonatal or adult ventricular
myocytes resulted in no significant difference in the responses of
these two cell types, the values being -0.30±0.01 pH units for
neonatal myocytes (n=4) and -0.29±0.04 pH units for adult myocytes
(n=8, P>0.05; see Figure 1D
).
A higher concentration of AA (20 µmol/L) has been shown to
increase the resting
[Ca2+]i in adult
ventricular myocytes.39 If a similar effect
were to occur in our system, the acidosis evoked by 10 µmol/L AA
could be due to changes in
[Ca2+]i, because it has
been shown in cardiac Purkinje fibers that a rise in
[Ca2+]i results in
acidosis, due to competition between Cai and
Hi for their common binding sites.40
Moreover, the [Ca2+]i can
also be increased if the
[Na+]i increases
(Nai exchange for Cao). We
therefore measured the basal
[Ca2+]i and
[Na+]i (shown,
respectively, in Figures 2A and 2B) and
found that neither changed on addition of 10 µmol/L AA. Because
the intracellular acidosis was seen when Nao was
totally removed (Na-free/Cao-containing medium;
data not shown), probably due to a rise in the
[Ca2+]i
(Nai exchange for Cao). To
prevent the masking of AA-induced acidosis when Na-free conditions were
used, the medium was also made Ca-free (Na-/Ca-free medium; see Figure 2C and Table 2).
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Possible involvement of inhibition or stimulation of the
pHi regulators, namely, the Na-H exchanger
(NHE),29 41 the Na-HCO3
cotransporter,42 and the Cl-OH exchanger,43
was then tested. The first two are acid extruders, whereas the third is
an acid loader (ie, alkaline extruder). After pretreatment with
Na-/Ca-free medium, in which the NHE is blocked, with DIDS, which
blocks the Na-HCO3 cotransporter, or with Cl-free
medium, in which the Cl-OH exchanger is blocked, no inhibition of the
acidosis induced by 10 µmol/L AA was seen (Figures 2C
through 2E and Table 2). In Cl-free medium, the basal
pHi was increased, probably due to a reverse mode
of the Cl-OH exchanger.43 The more pronounced AA-evoked
acidosis seen in Cl-free medium (Table 2) probably results from
the greater release of H+ by AA in the alkaline
cytoplasm (Henderson-Hasselbalch equation; see Discussion).
We also suggest that it is less likely that protein kinase C (PKC) and
AA metabolites are involved in the AA-induced acidosis. Pretreatment
with 1 µmol/L TPA (a PKC activator) or 100
µmol/L H7 (a PKC inhibitor) had little effect on the
AA-induced acidosis (Table 2). Inhibitors of the
three AA metabolic pathways, NDGA (5 µmol/L, a
5,12-lipoxygenase inhibitor),
indomethacin (30 µmol/L, a
cyclooxygenase inhibitor), or econazole
(30 µmol/L, a cytochrome P450 inhibitor), also had
no effect (Table 2). Because AA metabolism can
generate free radicals, the effect of the addition of three free
radical scavengers was tested. After pretreatment with either a mixture
of two scavengers, superoxide dismutase (SOD, an
O2-· scavenger) and
catalase (reduction of H2O2
into H2O) or U78517F (an
O2-·/OH·
scavenger), no inhibitory effect was seen (Table 2).
It has been suggested that AA activation of NADPH oxidase may produce a
large amount of intracellular protons.24 25 26 If this were
true in cardiac cells, activation of NADPH oxidase, which is expressed
in cardiac myocytes,45 could result in the AA-induced
acidosis. We therefore checked whether NADPH oxidase activation and
resynthesis of NADPH by the hexose monophosphate shunt were involved.
In the presence of a mixture of diphenyleneiodonium (DPI, 10
µmol/L, NADPH oxidase inhibitor)46 47 and
BiCNU (100 µmol/L, glutathione reductase
inhibitor),48 the AA-induced acidosis was not
affected (Table 2).
If the inward proton conductance was activated by AA at a
resting membrane potential more negative than the theoretical
H+ equilibrium potential
(EH=-18 mV, -18=60x[7.1 to 7.4], if
pHi/pHo=7.1/7.4), the
amplitude of the acidosis should be decreased by depolarization of the
membrane potential. Using a fluorescence measurement technique,
we tested this possibility in neonatal cardiac myocytes (Figure 3). Once the AA-induced acidosis had
stabilized at pHi 6.5 (Figure 3A),
depolarization of the membrane potential by 100 mmol/L KCl
resulted in a 72±5% (n=5) recovery from acidosis (-0.42±0.02 pH
unit, n=30), increasing to 78±6% (n=7) when 3 µmol/L
valinomycin (a K+ ionophore) was also added. The
marked KCl/valinomycin-induced pHi recovery was
seen only with the AA-induced acidosis, because it did not occur when
another two acid loading methods, addition of propionate/EIPA (Figure 3B) or the Na-free
medium/NH4+ rebound technique
(Figure 3C), were used (EIPA or Na-free medium was used to
inhibit possible acid extrusion via the NHE during intracellular acid
load). In both cases, the rates of pHi recovery
in the presence (R2 in Figures 3B and 3C)
or absence (R1 in Figures 3B and 3C) of
KCl/valinomycin were similar, respectively, 0.008±0.002 and
0.009±0.001 pH unit/min (P>0.05, paired t
test, n=5) when propionate/EIPA was used, 0.012±0.002 and 0.014±0.003
pH unit/min (P>0.05, paired t test, n=4) when
Na-free medium/NH4+ rebound was
used. From the results of the above experiments, it seems that changes
in membrane potential affect the amplitude of AA-induced acidosis. As
shown by Meech and Thomas,49
Zn2+ (1 mmol/L) blocks the proton
conductance pathway. However, this inhibitor was not used
in pHi measurement experiments, because it was
found to severely quench the signals at 490 and 440 nm (applying 1
mmol/L Zn2+ to BCECF free acid in the cell bath).
Instead, whole-cell patch-clamping of adult ventricular
myocytes (see Materials and Methods) was used to test the possibility
that AA activates an inward H+
current.
Typical families of currents were recorded before (Figure 4A) or after the addition of 30
µmol/L AA (Figure 4B) or 30 µmol/L AA/1 mmol/L
Zn2+ (Figure 4C) for 5 minutes. The
currents were generated by applying 9 successive 3-second depolarizing
and hyperpolarizing pulses to potentials between +60 mV and -120 mV.
The I-V curves obtained from these experiments are shown in
Figure 5. Under control conditions, the
inward proton current reversed at a potential of -6 to -8 mV and
showed slight outward rectifying properties at a potential greater than
0 mV (Figure 5A). The average slope conductances at potentials
between 0 to +60 mV and between -20 to -120 mV were 19.1±1.8 nS
(n=6) and 10.6±1.1 nS (n=6), respectively. On exposure to 30
µmol/L AA for 5 minutes (Figure 5A), both the outward and
inward slope conductances of the ventricular cells were
significantly reduced to 13.1±0.2 nS (n=6, P<0.05) and
7.0±0.6 nS (n=6, P<0.05), respectively. The addition of
1 mmol/L Zn2+ to AA-treated cells completely
abolished the inward current (Figures 4C and 5A) and
reduced the outward current conductance to 3.2±0.6 nS (n =6;
P<0.05). In another set of control experiments, 1
mmol/L Zn2+ alone was found to cause similar
inhibition of the membrane current (Figure 5B) to that seen in
the presence of AA (Figure 5A). These results suggest that, in
the resting state, the cardiac cell possesses a
Zn2+-sensitive H+
conductance that is inhibited, rather than stimulated, by AA.
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After ruling out possible involvement of an AA-activated
H+ current, another possibility was simple
diffusion of AA, resulting in acidosis. Albumin has been shown
to extract FAs from the lipid bilayer and to increase the rate of
Hi efflux.20 21 22 If this were to
occur in our system, a pHi increase should be
seen on addition of FA-free BSA in the presence of AA. When 0.3% BSA
was added, such a phenomenon was indeed seen (95±4% recovery, n=8,
Figure 6A). Furthermore, as shown in
Figure 6B, the initial rate of pHi
recovery (R1=0.023±0.005 pH unit/min, n=5) seen
when the external AA was removed was significantly less than that seen
after the subsequent addition of BSA
(R2=0.234±0.025 pH unit/min, n=5;
P<0.05), suggesting that BSA can extract AA from the plasma
membrane. Figures 6C and 6D show that the effect of BSA on the
pHi recovery was specific to AA-induced acidosis,
because it had no effect on recovery using the other two acid loading
methods (Na-free medium in Figure 6C and propionate/EIPA in
Figure 6D). Interestingly, when the NHE was blocked by
Nao removal (Figure 6E), BSA in the
continued presence of AA had only a partial effect (50±7% recovery
compared with its own control, n=5), and complete
pHi recovery was only seen when
Nao was added back (Figure 6E), resulting
from the reactivation of the NHE. Thus, there was an acid extrusion
(see Discussion), via the NHE, throughout the AA-induced intracellular
acidosis. The pHi recovery seen during BSA
treatment of cardiac cells, as opposed to artificial membranes,
therefore involves an acid extrusion mechanism, which is
activated at the same time as BSA extracts AA from the plasma
membrane.
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We also tested the effect of two other nonesterified FAs, oleic acid
(10 µmol/L; Figure 7A) and
linoleic acid (10 µmol/L; Figure 7B), which are released
in large amounts during ischemia.3 4 Both caused a
significant pHi decrease (-0.14±0.02 pH units
for oleic acid, n=6; -0.36±0.04 pH units for linoleic acid, n=6),
which was also reversed by 0.3% BSA. In contrast, addition of AA
methyl ester (10 µmol/L, Figure 7C, n=4) for 10 minutes
had no effect on the pHi. Tetradecylamine, in its
neutral form, has been shown to be transported by a simple diffusion
mechanism and then attract intracellular protons, causing an
intracellular alkalosis.20 21 22 After perfusion with
10 µmol/L tetradecylamine, a pHi increase
of 0.19±0.02 pH units was seen (Figure 7D, n=4), which was
again reversed, to a large extent, by addition of 0.3% BSA. Finally, a
possibility that the three nonesterified FAs, including 10
µmol/L AA, linoleic acid, and oleic acid, contribute to the
intracellular acidosis seen during cardiac ischemia is
investigated. Owing to technical reasons, conditions that only partly
mimic cardiac ischemia were used. At pHo
6.4 (containing 30 mmol/L lactate and 14.5 mmol/L
KCl),50 51 the pHi significantly
decreased by 0.9 pH unit (from pHi 7.2 to
6.3), as also seen in a previous study.50 Subsequent
addition of these nonesterified FAs (Figure 8) induced a further substantial
intracellular acidosis (0.30±0.02 pH unit, n=4).
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Discussion |
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The accumulation of large amounts of AA during ischemia has been demonstrated to correlate closely with cardiac arrhythmia52 and loss of cell viability.53 The present study shows that AA can also induce a marked intracellular acidosis of 0.3 to 0.4 pH units and that this acidification results from simple diffusion, rather than from activation of NADPH oxidase or proton conductance, as is suggested to occur in macrophages and neutrophils.24 25 26
A simple diffusion model of AA-induced acidosis, as proposed by Kamp and Hamilton for protein-free vesicles,17 18 19 20 21 22 seems to explain our results in cardiac cells:
(1) According to Fick’s first law for the simple diffusion of a
substance, the rate of flux (J) is proportional to the concentration
gradient across the membrane (C), when other factors are constant
(Fick’s first law states that J=-DA(C/X), where D is the
diffusion coefficient, A is the area of the membrane, X is the
thickness of the membrane, and X is the change in the thickness of
the membrane; D, A, and X are regarded as constant). A plot of the
initial rate of acid flux (JH) versus
[AA]o (=C at time zero) is linear (Figure 1B), and no saturation (Vmax) can be seen,
suggesting that a simple diffusion model54 may be
involved. However, membrane carrier protein–mediated AA transport
cannot be completely ruled out when the AA concentration is above
50 µmol/L, because high concentrations of AA (>50
µmol/L) have a detergent effect on myocytes (data not shown). The
reason for calculating the initial rate of acid flux
(JH) was to avoid effects due to activation of
the NHE during AA-induced marked acidosis.
(2) Nonesterified FAs with a -COOH group (eg,
AA, oleic acid, and linoleic acid) induced intracellular acidification,
whereas an esterified FA with a
-COOCH3 group (AA methyl
ester) had little effect on the pHi (Figure 7).
(3) Inward movement of an FA amine (tetradecylamine) was accompanied by
a pHi increase (Figure 7D).
(4) Recovery from AA-induced acidosis was seen in the presence of large
amounts of BSA, suggesting that the FAs are removed by the BSA (Figures 6 and 7).
Observations (numbers 2 through 4 above) also suggest that a membrane carrier protein may not be involved in AA-evoked acidosis. A cytosolic FA binding protein has been identified and characterized in neonatal or adult rat cardiac cells,55 56 and AA and FAs accumulated in the inner leaflet are rapidly transported by this protein and metabolized.
Using the Kamp and Hamilton model,20 21 22 it is possible to predict that a significant intracellular acidosis will occur on addition of a concentration of AA as low as 10 µmol/L. For convenience, we used the result produced on addition of 1 mL of 10 µmol/L AA to the cell bath (ie, a total of 10 nmol of AA), which resulted in a 0.29±0.04 pH unit decrease in adult rat cardiac myocytes (see Results) for the following estimation.
At a resting pHi of 7.29, the
ßi of the rat cardiac myocyte is 6.6
mmol/L.29 Because the acid injection equals
ßix0.29 pH units, the concentration of
H+ ions needed to induce a 0.29 pH unit decrease
is 1.91 mmol/L (1.91x10-3 mol/L). If a
ventricular myocyte is regarded as a cylinder, given the
averaged diameter (2r) and cell length of a myocyte are 14.4±1.0
µm and 86.7±3.8 µm, respectively (n=30 cells), the calculated
single cell volume is 1.41x10-11 L (cell
volume=cell lengthx
r2=14 112
µm3, 1 L=1015
µm3. A single cardiac cell therefore requires
2.69x10-14 mole of protons
(=1.91x10-3
mol/Lx1.41x10-11 L) for a 0.29 pH unit
decrease.
If a total of 10 nmol of AA in 1 mL of solution is applied to a single cell lipid bilayer, according to the Henderson-Hasselbalch equation and the Kamp and Hamilton model,20 21 22 6.1 nmol of nonionized AA (AA-H) and 3.9 nmol of ionized AA- are formed in the outer leaflet at pHo 7.4 (the apparent pK of FAs in the biomembrane of both leaflets is also 7.6.22 ). Because AA-H diffuses more rapidly than AA-, almost all of the 6.1 nmol of AA-H diffuses to the inner membrane. At pHi 7.29 and pK 7.6, 2.0 nmol of the 6.1 nmol of AA-H dissociates into 2.0 nmol of AA-+2.0 nmol of H+ ions, which can be released into the cytosol. Given that a single cell requires only 2.69x10-14 mole of H+ ions for a 0.29 pH unit decrease, 2.0 nmol of H+ ions could therefore supply -7.4x104 cells. In the present study, 1 mL of medium contained 1.6x104±1.3x103 cells (5 hemocytometer measurements). However, in the presence of NHE activity, it is probable that the observed change of 0.29 pH unit is an underestimation. Thus, the amount of acid injection required for a single cardiac myocyte (2.69x10-14 mole of protons) may be underestimated, resulting in an overestimate of the number of cells (7.4x104) that could be affected.
One interesting question is therefore raised, namely, why at least
20 mmol/L sodium propionate is required to evoke a similar
magnitude of acidosis (-0.29 pH unit; data not shown). One possible
explanation is as follows. Because propionic acid does not accumulate
in the lipid bilayer, intracellular protonation depends on the amount
of propionic acid (HA) that dissolves in, and fluxes from, the external
solution, resulting in the release of H+ in the
cytosol. Because the pK for propionate in both the external and
internal solutions is known to be low (4.8,57 ), the
concentration of sodium propionate required would therefore have to be
high enough (mmol/L range) to produce sufficient nonionized HA,
resulting in H+ release.
We also ruled out involvement of the following possible mechanisms of
AA-induced acidosis. The acidosis was not due to (1) changes in basal
[Ca2+]i or
[Na+]i or inhibition or
stimulation of the pHi regulators (Figure 2); (2) PKC activation or the generation of AA metabolites or
free radicals (Table 2); or (3) NADPH oxidase activation or
H+ conductance (see below). The reason for little
change in [Na+]i seen
during AA-evoked acidosis (Figure 2B) is not clear, but at least
two explanations are possible. After complete inhibition of the NHE,
the AA-evoked acidosis ("Na-/Ca-free," -0.56 pH unit in Table 2) was only 0.14 pH unit greater than that seen under control
conditions (-0.42 pH unit, Table 2). We therefore suggest that
(1) the NHE is partially blocked by AA or (2) the NHE activity might be
low so that a slow influx of Na+ would be
balanced by efflux of Na+ via the Na/K ATPase
with very little rise in
[Na+]i.
In macrophages and leukocytes, it has been demonstrated that AA
activates NADPH oxidase, resulting in depolarization of the
membrane potential and outward H+ conductance (a
pHi increase).24 25 26 27 We observed
that addition of 100 mmol/L KCl/valinomycin, which depolarizes the
membrane potential, reversed the AA-induced acidosis
(pHi increase, Figure 3A). Moreover, this
pHi increase was not seen when using two other
internal acid loading methods (Figures 3B and 3C), suggesting
the possible existence of AA-activated H+
channels in myocytes. However, other results in the present study
do not support this hypothesis. First, little effect was seen in the
presence of DPI/BCNU (Table 2), ruling out the involvement of
NADPH oxidase. Second, using the whole-cell patch-clamp technique, we
found that AA significantly inhibited both the inward and outward slope
conductances (Figures 4 and 5), suggesting that
AA-induced acidosis does not result from the activation of an inward
H+ current at a negative resting membrane
potential. The reason for the high K-induced recovery in the presence
of AA is not clear, but it is possible that AA activates a
K-dependent acid extrusion mechanism (eg, K-H exchanger). Other
K-/AA-dependent acid extrusion mechanism(s) should also be
considered.
Possible Physiological or Pathophysiological Roles of AA-Induced Intracellular Acidosis. In the present study, the addition of a concentration of AA lower than 2 µmol/L had little effect on the pHi. Since the concentration of unbound AA normally present in the extracellular fluid is less than 0.5 µmol/L,1 this suggests that AA-induced acidosis does not occur under physiological conditions and therefore plays a minor role in pHi-dependent cellular activities. However, during the first 1 to 3 hours after a sudden reduction in blood flow (ie, the acute phase of myocardial ischemia), marked accumulation of AA and other nonesterified FAs, including oleic and linoleic acids, occurs in the myocardium.3 4
The present study shows that intracellular acidosis was caused by
the addition of AA (-0.42 pH unit), linoleic acid (-0.36 pH unit), or
oleic acid (-0.14 pH unit). Furthermore, even at very acid conditions
(pHi 6.3), as seen during cardiac
ischemia,50 51 a substantial FA-induced acidosis
(0.3 pH unit, Figure 8) was still seen. In theory,
intracellular protonation of FAs should be less when at a low
pHi (Henderson-Hasselbalch equation, see above
discussion). One possible explanation for the 0.3 pH unit change
under acid conditions is that the three added FAs provide
H+ ions (Figure 7). During cardiac
ischemia, however, the magnitude of the FA-induced acidosis may
be even larger, because these FAs may also be released from the inner
leaflet of the plasma membrane. For example, at ischemic
pHi 6.3, much more [HA] is formed in the inner
leaflet (given that
6.3=7.5+log[A-]/[HA], see above) than at
a normal pHi of 7.29. Therefore, the possibility
that released AA and other nonesterified FAs contribute to the late
phase of the ischemic-induced acidosis should be considered,
because a marked acidosis (1.0 pH units) is known to
occur.50 51 At this stage, the left
ventricular developed pressure (LVDP) is almost
zero.58 59 Because the contractility of
cardiac muscle is known to be very sensitive to intracellular
acidosis,11 12 13 14 15 16 FA-induced acidosis may therefore
contribute, at least in part, to the ischemic-induced LVDP
decrease.
In summary, the present study is the first to show that, in cardiac cells, AA, oleic acid, and linoleic acid, which may be released in large amounts in ischemic cardiac tissue, evoke a marked intracellular acidosis. In contrast to the results from studies on macrophages and lymphocytes, this acidosis is probably due to simple diffusion of AA. Because a marked intracellular acidosis may be involved in many pathophysiological abnormalities during ischemia, the present findings may provide at least a partial explanation of the ischemic-induced negative inotropic effect.
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Acknowledgments |
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This study was supported by the National Taiwan University Hospital (NTUH89S1003). We gratefully acknowledge the technical help of Dr Meng Li Tsai and Wei-Luen Chang.
Received January 3, 2000; accepted January 14, 2000.
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References |
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