Vol. 277, Issue 1, H279-H289, July 1999
pH regulation of K+ efflux
from myocytes in isolated rat hearts:
87Rb,
7Li, and
31P NMR studies
V. V.
Kupriyanov,
B.
Xiang,
B.
Kuzio, and
R.
Deslauriers
Institute for Biodiagnostics, National Research Council, Winnipeg,
Manitoba, Canada R3B 1Y6
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ABSTRACT |
This study investigates the effects of
intracellular (pHi) and
extracellular pH (pHe) on the
efflux of Rb+ and
Li+ in isolated rat hearts.
87Rb and
7Li NMR were used to measure
Rb+ and
Li+ content, respectively, of
hearts, and 31P NMR was used to
monitor pHi,
pHe, and phosphate levels. After 30-min equilibration with Rb+ or
Li+, effluxes were initiated by
switching perfusion to a Rb+- or
Li+-free,
high-K+ (20.7 mM) Krebs-Henseleit
buffer with 15 µM bumetanide. Monensin (2 µM) increased
pHi from 7.10 ± 0.05 to 7.32 ± 0.07 and resulted in activation of
Rb+ efflux; the first-order rate
constant (k × 103, in
min
1) increased from 42 ± 2 to 116 ± 16. Glibenclamide (4 µM) did not inhibit
monensin-activated Rb+ efflux
(k = 110 ± 17), whereas quinine
(0.2 mM) slightly inhibited it by 19 ± 9%. Infusion of 15 mM
NH4Cl during
Rb+ washout increased
k for
Rb+ efflux by 93% (81 ± 8),
which was glibenclamide and quinine insensitive, and caused a transient
increase in pHi to 7.25 ± 0.08. Intracellular Li+ inhibited
NH4Cl-stimulated
Rb+ efflux by 55%. Monensin and
NH4Cl stimulated
Li+ efflux by 40%, increasing
k from 29 ± 3 to 43 ± 7 and 41 ± 3, respectively. The stimulation was not sensitive to 10 µM
dimethylamiloride. Intracellular acidosis that resulted from the
washout of NH4Cl (pH 6.86 ± 0.2) slightly inhibited Rb+ efflux
(k = 36 ± 5), whereas
NH4Cl itself in the absence of
pHi changes did not markedly
affect Rb+ efflux. A moderate
increase in pHi (7.17 ± 0.06)
produced by washout of 15 mM 2,2-dimethylpropionate (DMP)-Tris from
hearts preequilibrated with DMP did not markedly affect
Rb+ efflux. Neither global
alkalosis (pHi 7.4, pHe 7.55) nor acidosis (pHi 
pHe 6.8) produced by 3 mM Tris
base or 5 mM MES, respectively, affected
Rb+ efflux. We suggest that
intracellular alkalosis stimulates
Rb+
(K+) and
Li+ effluxes by activating a
nonselective sarcolemmal K+
(Li+)/cation exchanger or a
K+
(Li+)-anion symporter.
rubidium (potassium) ion efflux; lithium ion efflux; intracellular
pH; cation/cation exchanger
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INTRODUCTION |
INTRACELLULAR AND EXTRACELLULAR pH
(pHi and
pHe, respectively) are important
regulators of cation transport in myocytes. In early studies it was
noted that pH affects the distribution of K+ in skeletal muscle such that
the ratio of intracellular to extracellular K+ concentration
([K+]i/[K+]e)
correlates with the ratio of intracellular to extracellular H+ concentration
([H+]i/[H+]e)
(41). Intracellular alkalosis results in a net efflux of K+ from the intracellular space
(17, 28, 29). Acidification of the extracellular medium by increasing
the ratio of CO2 to HCO3 concentration
([CO2]/[HCO3]) produces a similar effect (1). In cardiac muscle similar results were
obtained for rabbit atria (35) and isolated rat heart (14) preparations. In contrast, in vivo experiments in dog hearts
demonstrated a net uptake of K+ in
response to respiratory acidosis
(PCO2 increase) (38). Radiotracer
studies (42K) on perfused rat and
rabbit septa showed that this effect was caused by the inhibition of
unidirectional K+ efflux by
respiratory acidosis (33).
Under ischemic conditions, acidosis is more profound than respiratory
acidosis and may affect the observed net efflux of
K+ (18, 44, 45) mediated by
ATP-sensitive K+
(KATP) channels (44, 45) and
K+-anion cotransporters (43).
Patch-clamp experiments have shown that acidosis can increase (7, 11),
decrease (5, 11), or not change (12, 27) the activity of
KATP channels when the ATP
concentration exceeds 0.1 mM. In addition to pH modulation of the
above-mentioned K+ efflux
pathways, H+ may exchange with
K+ and other monovalent cations
through the nonselective cation/H+
exchanger. This exchanger has been characterized by Periyasamy et al.
(32) in isolated sarcolemmal vesicles from bovine hearts.
Thus K+ efflux from myocytes in
cardiac muscle is affected by pH changes under both normal and
pathological conditions; however, the data are controversial.
Measurements of net fluxes are somewhat difficult to interpret because
they depend on both unidirectional influx and efflux rates. The
evaluation of unidirectional fluxes using tracers, either radioisotope
or magnetic resonance, may provide information on unidirectional influx
and efflux rates. Rb+ has been
used successfully as an NMR-sensitive tracer
(87Rb, 28% natural abundance) for
K+ in studies of unidirectional
K+ uptake and efflux rates in
isolated heart preparations (2, 6, 20, 21).
In this study we used 87Rb and
7Li NMR spectroscopy to
investigate the effects of changes in
pHi and
pHe on the unidirectional rates of
Rb+ and
Li+ efflux from myocytes in
isolated rat hearts. It was found that intracellular alkalosis produced
by infusion of NH4Cl or monensin results in a two- to threefold activation of
Rb+ efflux, which is not
glibenclamide sensitive, and 40% activation of dimethylamiloride
(DMA)-insensitive Li+ efflux. We
suggest that this effect could be caused by activation of either the
cation/cation exchanger or the cation-anion cotransporter.
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METHODS |
The investigation was performed in accordance with the
Guide to the Care and Use of Experimental
Animals published by the Canadian Council on Animal
Care (2nd ed., Ottawa, ON, Canada, 1993).
Heart perfusion.
Male Sprague-Dawley rats (300-400 g,
n = 107) were obtained from
the animal facility of the Institute for Biodiagnostics. Isolated
hearts were perfused in the Langendorff mode with Krebs-Henseleit (KH)
buffer aerated with 95% O2-5%
CO2 at a constant flow (~10 ml · min1 · g
wet wt1) maintained by a
peristaltic pump. KH buffer contained (in mM) 118 NaCl, 25 NaHCO3, 4.7 KCl, 1.75 CaCl2, 1.2 MgSO4, 11 glucose, and 0.5 EDTA.
Temperature of the perfusate was maintained at 36.0-36.5°C by
using water-jacketed reservoirs and perfusion line. Functional parameters such as left ventricular systolic pressure (LVSP), end-diastolic pressure (LVEDP), heart rate (HR), perfusion pressure (PP), and coronary flow were monitored continuously before, during, and
after the acquisition of NMR spectra by a Gould four-channel physiological recorder. A water-filled balloon was inserted into the LV
and connected to a Statham P23 Db pressure transducer to measure LVSP,
LVEDP, and HR, which were 129 ± 17 mmHg, 2.9 ± 3.0 mmHg, and
277 ± 33 beats/min, respectively, after the equilibration period.
PP was measured by the separate pressure transducer connected to the
aortic line and was 68 ± 8 mmHg after the equilibration period.
Cardiac performance was evaluated using the rate-pressure product (RPP)
as HR × (LVSP 
LVEDP), which was 34,900 ± 4,800 beats · mmHg · min1.
Experimental protocols.
For measurements of Rb+ and
Li+ effluxes, perfusion was
switched to a RbCl- or LiCl-containing solution
([Rb+] = 2.14 mM,
[K+] = 3.76 mM, or
[Li+] = 15 mM,
[K+] = 4.7 mM) for 30 min. Rb+ or
Li+ washout was then initiated by
switching the perfusion to a Rb+-
and Li+-free,
high-K+ (20.7 mM) solution
containing 15 µM bumetanide, a
Na+-K+-2Cl
cotransport inhibitor (10). A
high-K+ medium was used for the
following reasons: 1) it causes
cardiac arrest and eliminates the dependence of
Rb+ and
Li+ efflux on HR, which is
important because some interventions may significantly affect HR;
2) the conductance of
K+ channels increases at high
[K+]e,
which makes the assay more sensitive;
3) high
[K+]e
significantly dilutes the Rb+
released from cardiac cells, preventing its reuptake by
Na+-K+-ATPase;
4) high
K+ dramatically decreases energy
consumption [~15% of that in beating hearts (19)] that
prevents potential deleterious effects of drugs increasing
[Ca2+]i
(monensin, veratridine) by maintaining sufficient energy reserve. Two
data points obtained during the first 4 min after
Rb+ or
Li+ efflux was initiated (0-2
and 2-4 min), which corresponded to Rb+ or
Li+ washout from the extracellular
space, were excluded from the analyses. The remaining data were
presented as semilogarithmic plots and analyzed using linear regression
(see Figs. 2 and 4). The slopes of the straight lines yielded
pseudo-first-order rate constants
(k) that are presented for
convenience as k × 103 (in
min1).
Protocol A, which describes effects of
monensin and related drugs on Rb+
efflux, is shown in Fig.
1A.
Pharmacological agents, such as monensin (2 µM), nigericin (1-2
µM), or veratridine (16 µM), were added at the beginning of
Rb+ washout to ensure that
anticipated changes in
[Na+]i
(monensin, veratridine),
[K+]i
(nigericin), and
[H+]i
(monensin, nigericin) had developed. In some experiments
monensin was added after 6 min of
Rb+ washout (Fig.
1A; see Fig.
2B) to reveal better kinetics of
Rb+-efflux activation.
Glibenclamide (4 µM) and quinine (0.2 mM) were added at the beginning
of Rb+ washout where indicated
(Fig. 1A; see Fig.
2B and Table 2).
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Fig. 1.
Experimental protocols. A:
protocol A describes experiments with
monensin (Mon) and controls for these experiments.
B: protocol
B describes experiments with
NH4Cl and controls for these
experiments. C:
protocol C describes experiments with
2,2-dimethylpropionate (DMP). D:
protocol D describes
Li+ efflux experiments. Bum,
bumetanide; Nig, nigericin; Ver, veratridine; Glib, glibenclamide; Qn,
quinine; 4-AP, 4-aminopyridine; DNDS,
4,4'-dinitrostilbene-2,2'-sulfonate; CNHCA,
 -cyano-4-hydroxycinnamic acid; DMA, dimethylamiloride. Shaded or
hatched bars show time intervals when
Rb+,
Li+, KCl + Bum,
NH4Cl, and DMP were present in
perfusate; solid bars show time intervals when drugs were infused
separately or in combination. For details see
METHODS and
RESULTS.
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Protocol B (Fig.
1B) describes effects of changes in
pHi and
pHe on
Rb+ efflux produced by other
agents, such as NH4Cl, Tris base,
and MES. Intracellular alkalosis was induced by infusion of 15 mM NH4Cl at 6 min of
Rb+ washout. Intracellular
acidosis was induced by washout of
NH4Cl at 6 min after
Rb+ efflux was initiated from
hearts equilibrated with NH4Cl for 16 min. In control experiments
NH4Cl was present before (10 min) and after initiation of Rb+
efflux. The NH4Cl prepulse and
washout method (8) is based on the following principles. Ammonia, which
is in equilibrium with NH+4, penetrates into
the cytoplasm faster than NH+4, where it
binds H+, resulting in transient
alkalinization of the cytoplasm. After NH4Cl reaches equilibrium between
compartments, pHi returns to normal. Washout of NH4Cl results
in faster removal of NH3 from the
cytoplasm and shifts the equilibrium (NH+4 
NH3 + H+) toward
NH+4 dissociation and release of
H+, which results in transient
intracellular acidosis.
Global alkalosis was induced by infusion of Tris base (3 mM). Global
acidosis (pHi and
pHe) was induced by infusion of
5 mM MES at 6 min of Rb+ washout.
Pharmacological agents (see Table 1) such as glibenclamide (4 µM),
4-aminopyridine (4-AP; 1.5 mM),
4,4'-dinitrostilbene-2,2'-sulfonate (DNDS; 0.1 mM), and
quinine-HCl (0.1-0.2 mM) were added at the beginning of
Rb+ washout. In
Li+-inhibition experiments, hearts
were loaded with 15 mM LiCl for 30 min simultaneously with
Rb+ loading.
Li+ washout was started
simultaneously with Rb+ washout
such that at the moment of NH4Cl
infusion (after 6-min washout) Li+
was present only in the intracellular space (22).
Protocol C (Fig.
1C) describes effects of
pHi changes on
Rb+ efflux produced by the weak,
nonmetabolizable carboxylic acid 2,2-dimethylpropionate (DMP)-Tris salt
(pH 7.4). Its principle of action is similar to that of
NH4Cl. Protonated DMP (DMPH)
penetrates faster than its anion into the cytoplasm, where DMPH
releases H+, resulting in
transitory acidification of the cytoplasm (16). Subsequent washout of
DMP results in rapid diffusion of its protonated form
(pKa ~5.8) from
the cytoplasm to the interstitium and shifts the cytoplasmic
equilibrium toward proton binding to the DMP anion, thereby producing
an increase in pHi. Intracellular
alkalosis was induced by washout of DMP at 6 min after
Rb+ efflux was initiated from
hearts preequilibrated with 15 mM DMP-Tris for 16 min. Intracellular
acidosis was produced by infusion of 15 mM DMP at 6 min of
Rb+ washout. In control
experiments DMP was present before (10 min) and after
Rb+ efflux was initiated.
Protocol D (Fig.
1D) describes effects of monensin
and NH4Cl on
Li+ efflux. Hearts were loaded
with Li+ by 30-min perfusion with
KH buffer containing 15 mM LiCl at an unchanged
Na+ concentration (143 mM) as
described previously (22). Li+
washout was initiated by switching perfusion to the buffer used for
Rb+ washout (20.7 mM KCl, 15 µM
bumetanide). DMA (10 µM), an inhibitor of
Na+/H+
exchanger (25), was infused for 20 min at the beginning of Li+ washout.
Similar protocols were used in the absence of
Rb+ for the measurements of
phosphates and pHi. Because the
Pi level is very low in
KCl-arrested hearts, it is very difficult to measure
pHi using the
Pi peak. Therefore, before KCl
arrest and pHi manipulations, the
hearts were treated with 10 mM
2-deoxy-D-glucose (DG) for 8 min, which was then washed out for 6 min. This resulted in the appearance of a small but clearly detectable peak of 2-deoxyglucose 6-phosphate (DG-6-P), which was used as a
pHi probe. A short period of
treatment with DG resulted in only slight decreases in the phosphocreatine (PCr) and ATP levels to 83 ± 14 and 89 ± 2% of their pretreatment levels, respectively. 2-Aminoethylphosphonic acid
(AEPA)-Na salt (3 mM, pH 7.4) was used as a
pHe probe in the experiments with
monensin (protocol A) and Tris and
MES (protocol B) and was added 6 min
before the above agents were added.
NMR spectroscopy.
All NMR spectra were obtained using a Bruker AM-360 WB spectrometer
with a 20-mm Morris Instruments broad-band probe placed in a wide-bore,
vertical 8.4-T magnet. The 23Na
signal (95.2 MHz) from the heart and surrounding bath was used for
adjusting the homogeneity (shimming) of the magnetic field. The NMR
tube was continuously heated from outside with a flow of air, the
temperature of which was maintained at 36°C by the temperature
control unit of the NMR spectrometer. The temperature of the heart was
maintained mainly by a high flow (14-17 ml/min) of thermostated
perfusate. The temperature was checked in some experiments by placing
the thermocouple in the right ventricle and was in the range of
36.0-36.5°C.
87Rb NMR spectra were acquired at
117.8 MHz with a 2-min time resolution. The pulse duration was 55 µs
(90° flip angle), recycle time was 10 ms, sweep width was 18 kHz,
and memory size was 512 data points. A capillary containing 1 M RbCl
and 5 M KI was used as a concentration reference (2) in which
I serves as a shift reagent. The reference signal was
stable during experiments carried out on any given day (
4 hearts)
such that the standard deviation did not exceed 5%.
87Rb NMR spectra were obtained in
a "dry" perfusion mode by aspirating the perfusate from the
bottom of the 20-mm NMR tube (21). Postprocessing by exponential
filtering with a line-broadening factor of 150 Hz was applied. After
baseline correction (spline), peak areas were measured using an
integration subroutine (Bruker). Because only the kinetics of
Rb+ efflux were pursued (rate
constant) in this study, no attempt was made to measure absolute
concentrations of Rb+.
7Li NMR spectra were acquired as described previously (22)
on a Bruker AM-360 spectrometer using a broad-band probe (Morris Instruments) operating at 139.95 MHz.
7Li NMR spectra were collected
every 2 min (60° pulse, 2.2-s repetition time). A capillary
containing 0.5 M LiCl plus 50 mM
(Tris)3DyTTHA-3Tris · HCl
(7 µmol of Li; Dy, dysprosium; TTHA, triethylenetetraminehexaacetate) was used as a chemical shift and concentration reference. Before Fourier transformation, free induction decays were exponentially multiplied using a line-broadening factor of 20 Hz. Peak heights were
used for spectral quantification using a peak-picking subroutine provided by Bruker.
31P NMR spectra were acquired at
145.8 MHz with a 2-min time resolution using a broad-band Bruker probe.
The pulse duration was 35 µs (60° flip angle), recycle time was 2 s, sweep width was 10 kHz, memory size was 4 K data points, and the
line-broadening factor was 20 Hz. After baseline
correction (spline), the heights and chemical shifts of the PCr,
Pi, and 
-phosphate of ATP peaks were measured using a peak-picking subroutine (Bruker) and referred to
their initial values. Cytoplasmic pH was assessed from the chemical shift of the DG-6-P and
Pi peaks using calibration curves, as described previously (20, 21).
pHe was measured using dependence of the chemical shift of AEPA on pH as a calibration curve. The curve
was obtained at 36°C using a medium identical to KH buffer except
for the absence of NaHCO3, which
was replaced with 25 mM HEPES/HEPES-Na (pH 6.0-8.0).
Rate constants.
First-order rate constants for Rb+
and Li+ efflux
(k × 103
min1) were calculated as
follows. Natural logarithms of Rb+
peak intensity (ln Rb) were plotted as a function of time (see Figs. 2
and 5). In all experiments, the first two data points (0-2 and
2-4 min) were ignored because they were affected by changes in the
extracellular Rb+ or
Li+ concentration (20-22).
The rest of the data were subjected to the linear regression analysis,
and k was calculated as the slope of a
straight line (see Figs. 2 and 5). In the experiments in which
pHi was changed at 6 min of
Rb+ or
Li+ washout, the data points for
the 6- to 20-min time interval were analyzed (see Figs. 2 and 4). In
these experiments, only linear parts of the experimental curves were
used for calculations of k (5 data
points between 9 and 17 min) because activation of
Rb+ efflux developed in time
[see Figs. 2, A and
B (monensin), and 5 (NH4Cl)].
Reagents.
Monensin, nigericin, veratridine, bumetanide, glibenclamide, 4-AP,
RbCl, LiCl, quinine-HCl, Tris base, MES,
NH4Cl, DG,
-cyano-4-hydroxycinnamic acid (CNHCA), tetramethylammonium chloride
(TMA-Cl), and DMP (pivalic acid) were purchased from Sigma Chemical
(St. Louis, MO). DNDS was supplied by Molecular Probes (Eugene, OR),
and DMA was supplied by Research Biochemicals International (Nattick,
MA). Glucose and salts from BDH (Canada) were reagent and analytic
grades. Monensin and nigericin were prepared as stock solutions in 95% EtOH and were diluted with water (6% EtOH vol/vol) to prepare solutions for infusion (120 µM each). The concentration of EtOH in
the perfusion solution did not exceed 0.1%. Glibenclamide and veratridine were dissolved in DMSO and then diluted with water to yield
240 µM and 1 mM solutions, respectively, in 6-10% (vol/vol) DMSO. To prevent glibenclamide from precipitating from the DMSO-water solution, it was made alkaline by the addition of 1 mM NaOH. Bumetanide was dissolved in DMSO and added to the solution used for
Rb+ washout to bring its
concentration to 15 µM and DMSO to 0.05%. The final concentration of
DMSO in the perfusion solution did not exceed 0.15%. 4-AP (120 mM),
NH4Cl (900 mM), quinine-HCl (12 mM), CNHCA (60 mM), and DNDS (6 mM) were used as aqueous solutions; pH
was adjusted to 7.4 using HCl and NaOH. DMP (900 mM) was neutralized with Tris base to bring the pH to 7.4. Tris base (180 mM), MES (300 mM), TMA-Cl (900 mM), and DMA (0.6 mM) were dissolved in water.
Statistics.
Data are presented as means ± SD. Comparisons were made using the
StatView computer program for Macintosh. A two-tailed paired Student's
t-test was used for comparisons within
groups (pH changes), and a one-factor ANOVA was used for comparisons
between groups at a significance level of 95%.
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RESULTS |
Effects of monensin.
The rate constant for Rb+ efflux
increased by 180% in response to the infusion of monensin (2 µM), a
Na+-specific ionophore (Fig.
2, A and
B, and Tables
1 and 2). The activation
developed in time and reached maximum after 8 min of monensin
application. This time dependence of the activating effect is clearly
seen in Fig. 2B, where the experiments
with different times of monensin addition (0 and 6 min after initiation
of Rb washout) are compared. Monensin also caused alkalinization of the
intracellular medium such that pH increased from 7.1 to 7.4 (maximum)
over an 8-min period and reached a steady-state level of ~7.35, which
was maintained for another 8 min (Fig. 3).
The "instantaneous" rate constant calculated as [ln
Rb(t) ln
Rb(t + 2)]/2 increased over 8 min and reached a maximal level of 120-140 (Fig.
4). Time courses of
k and
pHi increases were qualitatively similar, although dependence for k was
shifted to the right (Fig. 4). This shift probably reflects a nonlinear
dependence of k on pH. The average
increase in pH caused by monensin over the last 8 min of its action on
Rb+ efflux was 0.21 ± 0.06 units (1.6-fold decrease in
[H+]i)
(Table 2). The pHe (Fig. 3 and
Table 2) and PCr (102 ± 17%, averaged over 14 min) did not change
in response to the addition of monensin, whereas ATP decreased slightly
to 89 ± 12% of its initial value. Glibenclamide (4 µM), an
inhibitor of KATP channels (Table
1), did not markedly suppress the activating effect of monensin on
Rb+ efflux (Fig. 2 and Table 2).
Quinine (0.2 mM), an inhibitor of the mitochondrial and sarcolemmal
K+/H+
exchanger (9, 32), moderately inhibited
Rb+ efflux in the presence of
monensin (28%) and did not affect basal Rb+ efflux (not shown). This
corresponds to 43% inhibition of the monensin-stimulated fraction of
Rb+ efflux. However, treatment
with quinine plus monensin depressed the recovery of contractile
function (RPP; by 32 ± 17%), measured after 20-min washout of the
drugs and high KCl (K+) plus bumetanide. It was caused by
some decrease in HR (19 ± 16%) and an increase in LVEDP
by 21.0 ± 20.0 mmHg at unchanged LVSP. After treatment with
monensin alone, cardiac function (RPP) recovered completely to 97 ± 16% of its pretreatment level. These data, combined with the lack of
any significant changes in PCr and ATP, show that monensin did not
cause any detectable damage to cardiac cells under our experimental
conditions. Thus activation of Rb+
efflux by monensin was not associated with any increase in sarcolemmal permeability associated with energy deprivation and cell damage. This
conclusion is consistent with the lack of inhibition of
monensin-activated Rb+ efflux by
glibenclamide, because it was shown previously that this inhibitor
completely blocked activation of
Rb+ efflux caused by energy
deprivation with cyanide (21) and 2,4-dinitrophenol (24).
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Fig. 2.
Stimulation of Rb+ washout from
isolated rat hearts by monensin.
Rb+ efflux was initiated by
switching perfusion to a Rb+-free,
high-K+ (20.7 mM) solution
containing 15 µM bumetanide. Monensin was added either simultaneously
with Rb+ washout
(A and
B) or 6 min after beginning of
Rb+ washout
(B). Glibenclamide (4 µM) and
quinine (0.2 mM) were added at beginning of
Rb+ washout (time
0). Rb/Rb0 is
ratio of intracellular 87Rb NMR
peak intensity to its initial value before initiation of
Rb+ washout.
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Fig. 3.
Effect of monensin on intracellular
(pHi) and extracellular pH
(pHe) in isolated rat hearts.
pHi was measured from pH
dependence of chemical shift of intracellular
Pi.
pHe was measured using pH
dependence of chemical shift of 2-aminoethylphosphonic acid (AEPA).
Hearts were arrested with a
high-K+ (20.7 mM) buffer in
presence of 15 µM bumetanide. For details see
METHODS.
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Fig. 4.
Time course of changes in pHi and
Rb+ efflux rate constant
(k, expressed as
k × 103 in
min1) induced by
monensin. pH data are from Fig. 3. "Instantaneous" rate constant
was calculated from data presented in Fig. 2 using relationship
k(t + 1) = [ln Rb(t) ln
Rb(t + 2)]/2.
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A series of control experiments was performed to test whether the
activation of Rb+ efflux by
monensin was caused by 1) the direct
transfer of Rb+ across the
sarcolemma as a complex with monensin (because of its limited
Na+/Rb+
selectivity), 2) an increase in
[Na+]i
and an associated increase in
[Ca2+]i,
or 3) an increase in
pHi. Nigericin (1-2 µM), a
K+-specific ionophore (see Table
1) structurally related to monensin, produced a modest stimulation of
K+ efflux and did not
significantly change pHi (Table
2). Veratridine (16 µM), an activator of
Na+ channels (26) (see Table 1)
that raises
[Na+]i,
caused an increase in Rb+ efflux
of ~50%, which is significantly less than the effect of monensin
(180%). In two experiments, ouabain, an inhibitor of Na+-K+- ATPase (see Table 1) that reduces
Na+ efflux and increases both
[Na+]i
and
[Ca2+]i,
did not affect the Rb+ efflux rate
(k = 46 ± 2 and 36 ± 2, respectively). These data support the hypothesis that intracellular
alkalosis is the main factor activating
Rb+ efflux from rat heart myocytes
during monensin treatment. None of the above agents significantly
affected the recovery of contractile function after their washout
simultaneously with the removal of KCl plus bumetanide.
Effects of NH4Cl infusion and washout.
To eliminate the effects of increases in
[Na+]i
and
[Ca2+]i
on Rb+ efflux, a
NH4Cl pulse technique (8) was used
to increase pHi. The rate of
Rb+ efflux and its rate constant
increased by 93% in response to the infusion of 15 mM
NH4Cl (Fig.
5 and Table 2), which increased pHi from 7.1 to 7.3 during the
first 4 min of infusion and increased the osmolarity of the perfusate
by 30 mM. pHi returned to its prepulse level 6-8 min after reaching its peak (Fig.
6). The average increase in
pHi over the 8-min period was 0.15 ± 0.07 units. Rb+ efflux
kinetics followed pH changes: the level of activation increased in
time, reached a maximum, and then decreased (see Fig. 5). High-energy
phosphates and Pi remained
unchanged (not shown). The rate constant for
Rb+ efflux decreased slightly
(k = 36 ± 5, n = 3) relative to the control when
pHi decreased (6.86 ± 0.2, n = 3, P < 0.05) in response to
the washout of NH4Cl (Fig. 6).
NH4Cl itself did not markedly affect either Rb+ efflux
(k = 49 ± 4, n = 3, P = 0.005) or
pHi (7.10 ± 0.07, n = 3), when it was infused 16 min
before the rate of Rb+ efflux was
measured. TMA-Cl (15 mM) was infused instead of
NH4Cl to test whether the
activating effect of NH4Cl is
caused by increased osmolarity of the intracellular medium and
associated cell volume change. TMA-Cl did not significantly affect
Rb+ efflux
(k = 44 ± 3, n = 3). To probe the nature of the
transporter(s) responsible for the observed alkalosis-stimulated
Rb+ efflux, a variety of
inhibitors (see Table 1) was tested:
1) glibenclamide (4 µM), an
inhibitor of KATP channels (Table
2); 2) 4-AP (1.5 mM,
n = 4, k = 83 ± 25), an inhibitor of some
voltage-gated K+ channels;
3) DNDS (0.1 mM,
n = 4, k = 72 ± 10), an inhibitor of
anion exchange and cotransport (3); and
4) quinine (0.1-0.2 mM) (Fig. 5
and Table 2). None of these substances significantly affected
alkalosis-stimulated Rb+ efflux.
Quinine in combination with NH4Cl
depressed the recovery of cardiac function (by 33 ± 5%) measured after 20-min washout of
NH4Cl, quinine, and high
K+ plus bumetanide. It was mainly
caused by a decrease in HR (by 36 ± 5%) and some increase in LVEDP
(by 6.0 ± 6.3 mmHg) at unchanged LVSP. Quinacrine, which is a more
potent inhibitor of
K+/H+
exchanger [inhibition constant
(Ki) = 10 µM] than quinine
(Ki = 60 µM)
(32), did not affect
NH4Cl-stimulated
Rb+ efflux at low concentrations
of 20 and 40 µM and abolished the efflux at 100 µM. However,
treatment with this dose produced toxic effects, such as contracture
and cardiac arrest, which persisted even after 20 min of drug washout.
NH4Cl alone or in combination with
drugs other than quinacrine did not produce any changes in cardiac
performance measured after their washout.
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Fig. 5.
Stimulation of Rb+ washout from
isolated rat hearts by NH4Cl.
Rb+ efflux was initiated by
switching perfusion to a Rb+-free,
high-K+ (20.7 mM) solution
containing 15 µM bumetanide.
NH4Cl was added 6 min after
initiation of Rb+ washout.
Glibenclamide (4 µM) and quinine (0.2 mM) were added at beginning of
Rb+ washout (time
0). LiCl was added to perfusate simultaneously with
Rb+ and removed at
time 0.
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Fig. 6.
Effects of NH4Cl infusion and
washout on pHi of isolated rat
hearts. pHi was measured using pH
dependence of chemical shift of 2-deoxyglucose 6-phosphate peak that
was preformed by short-term perfusion (8 min) with buffer containing 10 mM 2-deoxyglucose. Hearts were subsequently arrested with
K+ (20.7 mM) buffer containing 15 µM bumetanide. For details see
METHODS.
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NH4Cl infusion stimulated
Rb+ efflux not only under
conditions of KCl arrest but also in beating hearts in the presence of bumetanide. However, the effect was smaller, with the rate constant increasing from 47 ± 4 to 63 ± 2, a 39 ± 14% increase
(n = 5, P < 0.05). The drugs were not tested
on beating hearts because activation was relatively small and some
drugs affected heart rate. Quinine caused cardiac arrest, perhaps
blocking Na+ channels in a manner
similar to that of its stereoisomer, quinidine (37).
Effects of DMP infusion and washout.
Washout of DMP from preloaded hearts (Fig.
7; see Fig.
1C) resulted in a moderate and
transient alkalinization of intracellular medium. In the first 2 min,
pHi reached a maximum of 7.20 ± 0.03 and returned to normal. The average pH for the 6-min period
that followed DMP removal was 7.17 ± 0.06 [Fig. 7,
DMP(+/)], and the change in pH (
pH) was 0.07. This
modest and transient alkalosis caused no activation of
Rb+ efflux
(k = 42 ± 3, n = 4) relative to the control in the
absence of DMP. Transient acidosis (pH 6.92 ± 0.06) caused by the
infusion of DMP resulted in moderate inhibition of
Rb+ efflux
[k = 35 ± 2, n = 3, DMP(/+)]. Hearts
preequilibrated with DMP for 16 min showed a slight decrease in
pHi (7.01 ± 0.05) and
Rb+ efflux
[k = 33 ± 4, n = 3, DMP(+/+)]. Thus there is
little difference (27%) in k
value between hearts with DMP-induced alkalosis and DMP control. CNHCA
(1 mM) did not affect the response of
Rb+ efflux to DMP, indicating a
lack of Rb+-DMP cotransport
through the monocarboxylate carrier. DMP did not affect levels of
high-energy phosphates and recovery of contractile function after
washout of DMP and simultaneous removal of KCl and bumetanide.
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Fig. 7.
Dependence of rate constant of Rb+
efflux on pHi.
NH4Cl(/+) (intracellular
alkalosis) was produced by infusion of 15 mM
NH4Cl at 6 min after initiation of
Rb+ washout.
NH4Cl(+/) (intracellular
acidosis) was produced by infusion of 15 mM
NH4Cl at 10 min before initiation
of Rb+ washout and then stopping
infusion 6 min after initiation of
Rb+ washout.
NH4Cl(+/+):
NH4Cl was added 10 min before
initiation of Rb+ washout and was
present during entire 20-min washout period. DMP(+/)
(intracellular alkalosis) was produced by infusion of 15 mM DMP at 10 min before initiation of Rb+
washout and then stopping infusion at 6 min after initiation of
Rb+ efflux. DMP(/+)
(intracellular acidosis) was produced by infusion of 15 mM DMP at 6 min
after initiation of Rb+ washout.
DMP(+/+): DMP was added 10 min before initiation of
Rb+ washout and was present during
entire 20-min washout period. Dashed line represents regression line
fitted to 5 data points in pH range 7.1-7.35. Slope of line is
2.10 ± 0.42 (R = 0.94, P = 0.016).
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Effects of global alkalosis and acidosis.
To test whether Rb+ efflux was
affected by changes in both intra- and extracellular pH, the effects on
Rb+ efflux of a weak base, Tris,
and a weak acid, MES, were investigated. The efflux was not affected by
global alkalosis or global acidosis produced by the infusion of 3 mM
Tris base or 5 mM MES acid, respectively (Table 2). Tris caused a rapid
increase in pHe and
pHi such that they reached new
steady-state levels of 7.55 and 7.4, respectively (Table 2) within 2 min (time course not shown). MES infusion resulted in a rapid decrease
in pHe and
pHi to 6.8 and 6.7, respectively (Table 2). Fast changes in pHi
imply that any pH gradients imposed by a
pHe jump do not persist any longer
than 2 min. Tris and MES did not affect levels of high-energy
phosphates and subsequent recovery of contractile function.
Effect of intracellular alkalosis on
Li+ efflux.
To test the ionic specificity of alkalosis-activated
Rb+
(K+) transporter(s), hearts were
loaded with Li+ as described
previously (22) (Fig. 1D). The rate
constant of Li+ efflux increased
by ~40% from 29 ± 3 (n = 4) to
43 ± 7 (n = 5, P = 0.008) and 41 ± 3 (n = 5, P = 0.007) in response to
NH4Cl and monensin infusions,
respectively. An inhibitor of
Na+/H+
exchanger, DMA (10 µM), inhibited neither
NH4Cl-stimulated
(k = 45 ± 8, n = 3, P = 0.01) nor monensin-stimulated
(k= 49 ± 3, n = 3, P = 0.01)
Rb+ efflux. These data indicate
that the stimulated fraction of
Li+ efflux is not caused by the
operation of amiloride-sensitive Na+/H+
exchanger and can be mediated by the pH-dependent carrier(s) involved
in Rb+ efflux. To test this
hypothesis, the effect of intracellular Li+ on
Rb+ efflux was investigated.
Hearts loaded with Li+
([Li+]i = 21.5 ± 3.4 mM) (22) showed a reduced response to
NH4Cl stimulation (Fig. 5); the
rate constant for Rb+ efflux
decreased from 81 ± 11 to 60 ± 9 (n = 4, P = 0.004). Thus NH4Cl-stimulated fraction of
Rb+ efflux decreased to ~45%
[(60 42)/ (81 42)] of maximal
NH4Cl effect. This inhibitory
effect reflects competition between intracellular ions
(Rb+ and
K+ with
Li+) because
Li+ was present only in the
intracellular space. This inhibition was not caused by decreased
[Rb+]i,
because Li+ loading only slightly
decreases
[Rb+]i
plus
[K+]i
(12%) (22) and Rb+ efflux rate
constant does not depend on
[Rb+]i
in a wide range of its concentrations (24). A lack of deleterious effects of 15 mM LiCl on cardiac energetics and contractile function was demonstrated (22). The addition of monensin,
NH4Cl, and DMA during
Li+ washout did not change
subsequent recovery of contractile function.
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DISCUSSION |
Mechanisms of activation of
Rb+ efflux by
monensin, NH4Cl, and DMP.
Monensin functions as an ionophore, carrying
Na+ into the cell (down its
concentration gradient) and extruding
H+ into the interstitium (31, 34,
36). Under similar experimental conditions (hyperkalemia and cardiac
arrest by carbachol) we have shown that 2 µM monensin increases
[Na+]i
by ~30% in 10 min relative to its level in arrested hearts (23).
However, cardiac arrest resulted in a decrease in
[Na+]i
relative to that in beating hearts (20, 23) such that monensin in
effect nearly restored the level of
Na+ to that observed in beating
hearts. Nonetheless, an increase in
[Na+]i
(if any) could activate
Na+-dependent
K+ channels (30) and stimulate
Rb+ efflux. However, the moderate
stimulatory effect of veratridine on
Rb+ efflux (~50%), which is
known to increase
[Na+] and
[Ca2+] (26), indicates
that these ions could contribute only partially to the stimulatory
effect of monensin, which was 3.6 times larger (~180%). Furthermore,
the stimulatory effect of veratridine could be associated in part with
Rb+ efflux through the
Na+ channels, which becomes less
selective to Na+ in the presence
of veratridine (26).
A fraction of the monensin-stimulated
Rb+ efflux could be ascribed to
limited selectivity of monensin for
Na+ relative to
K+
(Rb+), which could transfer
Rb+ across the sarcolemma in the
form of a Rb+-monensin complex.
This fraction cannot be large because the stability of monensin
complexes in water decreases in the following order: Na+,
Li+,
K+,
Rb+ (526, 286, 77, <10
M1, respectively) (15).
Furthermore, nigericin, a
K+-specific ionophore (K:Na
selectivity ratio of 24), did not produce any significant stimulation
of Rb+ efflux. These selectivity
profiles indicate that the fraction of
K+/Rb+
transferred as a monensin complex cannot exceed that of nigericin. Indeed, nigericin might produce an even greater stimulatory effect than
monensin if it were the major mechanism for monensin stimulation. This
is to be anticipated because of the higher selectivity of nigericin for
K+
(Rb+), comparable pK values for
both ionophores, and the assumption of similar permeabilities for both
ionophore-K+
(Rb+) complexes. Theoretically,
nigericin should produce intracellular acidosis (25, 34) by exchanging
intracellular K+ for extracellular
H+. This was not observed in our
experiments because of the small activation of
K+ efflux and the presence of a
functional
Na+/H+
exchanger, which can reduce intracellular acidosis.
We postulate that the largest contribution to the monensin effect
resulted from intracellular alkalosis. Indeed, alkalosis produced by
the infusion of NH4Cl resulted in
a high degree of stimulation of
Rb+ efflux (~100% increase). In
this case,
[Na+]i
and
[Ca2+]i
are not expected to increase. The infusion of
NH4Cl and DMP also increased the
osmolarity of the extracellular medium by 30 mM and possibly caused
transient cell shrinkage. However,
NH4Cl increased and DMP decreased
Rb+ efflux. Furthermore, the
addition of 15 mM TMA-Cl instead of NH4Cl did not affect the
Rb+ efflux. In contrast, monensin,
which increases
[Na+]i,
should result in transient cell swelling, i.e., an effect opposite to
shrinkage in the presence of
NH4Cl. However, activation of
Rb+ efflux occurred in both cases.
In other words, there was no correlation between direction of volume
changes and activation of Rb+
efflux. Note that the
Na+-K+-2Cl
cotransporter, which is activated by volume changes (10), was blocked
by 15 µM bumetanide.
It is also possible that extracellular NH+4
was exchanged for intracellular
Rb+ through the
K+/cation exchanger, thereby
increasing Rb+ efflux. However,
stimulation of the hypothetical
Rb+/NH+4
exchange did not occur when the intra- and extracellular compartments
were equilibrated with NH+4, which was
present on both sides of the sarcolemma. Modest alkalosis induced by
washout of the anion of the weak carboxylic acid DMP did not result in
stimulation of Rb+ efflux.
Contrariwise, pHi decrease
produced by DMP infusion or NH4Cl
washout slightly inhibited Rb+
efflux (~20%). Interestingly, transport of monocarboxylates into guinea pig myocytes inhibited KATP
current, whereas their washout activated the current, possibly caused
by intracellular acidosis and alkalosis, respectively (5). Thus the
stimulation of Rb+ efflux by
monensin and NH4Cl indicates that
the major factor activating Rb+
efflux is intracellular alkalosis. The logarithm of the
Rb+ efflux rate constant depended
linearly on pHi in the narrow pH range between 7.1 and 7.35 (Fig. 7), revealing a strong
activation of Rb+ efflux, and was
pH independent in the range between 6.85 and 7.05. The slope of the
straight line is ~2, which indicates participation of more than one
proton or OH in the
transport process. For instance, one
H+ or
OH could be transported as
a substrate, whereas another H+
could play a regulatory role. Alternatively, transport of 2 H+ or 2 OH could be coupled to the
efflux of 2 Rb+. The pK of
ionizable group(s) involved in the activation of
Rb+ efflux is ~7.0-7.1.
Mechanisms responsible for alkalosis-activated
Rb+ efflux.
The large increase in Rb+ efflux
with increasing pHi could be
associated with a positive pH dependence
(dk/dpH > 0) of various K+ transporters, such as
voltage-gated and ATP-, Na+-, and
Ca2+-sensitive
K+ channels as well as
K+-anion cotransporters. However,
increasing pH on both sides of the sarcolemma did not produce any such
activation. This implies that if the above transporters are involved in
intracellular alkalosis-stimulated Rb+ efflux, extracellular
alkalosis abolishes this effect. The lack of sensitivity to
glibenclamide, 4-AP, and DNDS argues against involvement of
KATP, 4-AP-sensitive channels, and
HCO3/Cl-dependent
cation transporters, respectively. Similarly, the
Na+-K+-2Cl
cotransporter can be excluded because all measurements were performed in the presence of 15 µM bumetanide. Patch-clamp experiments have shown that neither global acidosis nor alkalosis affects the activity of KATP channels, whereas
intracellular alkalosis inhibits these channels (12). The increase in
Rb+ efflux could be caused by the
activation of a sarcolemmal
K+/H+
exchanger (32), which is similar to the mitochondrial exchanger (9,
13), by intracellular alkalosis. However, the inhibitor of the
sarcolemmal and mitochondrial
K+/H+
exchanger quinine has some effect on monensin-activated and no effect
on NH4Cl-activated
Rb+ efflux at moderate
concentrations (0.1-0.2 mM), which exceeded the
Ki for the
sarcolemmal exchanger (60 µM) (32). Note, however, that this
Ki value was
obtained using sarcolemmal vesicles isolated from bovine heart at a low
[Rb+] or
[K+] of 1 mM (32). In
our experiments
[Rb+]i + [K+]i
is ~100 mM and
[K+]e
is 20.7 mM, which could dramatically reduce the efficiency of potential
competitive quinine inhibition. In addition, rat heart exchanger can be
less sensitive to quinine than bovine heart exchanger. Furthermore, the
drug may inhibit other transport systems, such as
Na+ and
K+ channels, similar to its
stereoisomer, quinidine (37). At the least, cardiac arrest produced by
0.2 mM quinine, which was observed in our experiments with beating
hearts, is consistent with this interpretation. In the experiments on
KCl-arrested hearts, quinine depressed recovery of contractile function
assessed after washout of quinine and high
K+. For these reasons the
experiments with quinine on the intact hearts are not conclusive.
Other transport systems responsible for activation of
Rb+ efflux by alkalosis could be
1) a cation-anion cotransporter
capable of symporting K+ and
OH or
Cl and
2) a hypothetical
H+-sensitive
K+ channel with pH regulatory
sites on both sides of the sarcolemma. Interestingly, there is some
resemblance to nonselective ion channels of gap junctions, which can be
activated by alkaline pHi (39). However, Findlay (12) did not find any significant effect of NH4Cl-evoked intracellular
alkalosis on the background K+
current in guinea pig myocytes over a wide range of membrane potentials
(from 100 to 45 mV). Moreover, intracellular alkalosis inhibited
KATP channels (by ~35%)
activated pharmacologically (12). Therefore, the channel hypothesis
seems to be less likely.
If Rb+ efflux is coupled to the
movement of acid equivalents
(K+/H+
exchanger or
K+-OH
cotransporter), the Rb+ efflux is
not driven by pH. Under the conditions used in the majority of our
experiments, the K+ gradient was
~7 (log
[K+]i/[K+]e ~ 0.85) and the H+ gradient
changed from negative (outwardly directed, pH = 0.30) to slightly
negative (pH = 0.10-0.20) and was much less than the
K+ gradient. A similar situation
occurs for the
K+/H+
exchanger in Amphiuma red blood cells,
where K+ efflux coupled to
H+ uptake is activated by osmotic
swelling and is involved in regulatory volume decrease (4). In our
experiments the kinetics of
K+(Rb+)
efflux are controlled by relatively small changes in transmembrane pH, which decreased from 0.35 to 0.10-0.20 during intracellular alkalosis. Furthermore, global alkalosis and acidosis also decreased pH to 0.10-0.15 (see Table 2) without any effect on
Rb+ efflux. In other words, it is
hardly possible that a small increase in driving force due to a
decrease in pH is responsible for very significant activation of
Rb+ efflux. Rather, it is a
reflection of kinetic regulation of the efflux by
[H+]i.
Interestingly, it was demonstrated (32) that in sarcolemmal vesicles
Rb+/H+
exchange was not active under conditions of global acidosis
(pHi = pHe ~6.0) or alkalosis
(pHi = pHe ~8.0).
According to Periyasamy et al. (32) the
K+/H+
exchanger can also catalyze partial
K+/K+,
K+/Li+,
Na+/Na+,
K+/Na+,
and
Li+/Na+
exchanges. In our Rb+ efflux
experiments the
K+/H+
exchanger could exchange intracellular
Rb+ for extracellular
K+ such that
Rb+ efflux would be coupled to
H+ and
K+ entry. If so,
[K+]e
may affect Rb+ efflux, depending
on the transporter kinetics model. In this study we have found that
under normokalemic conditions
([K+]e = 4.7 mM, beating hearts) activation of
Rb+ efflux resulting from
NH4Cl-induced alkalosis decreased
(k = 63 ± 2 vs. 81 ± 8 in
KCl-arrested hearts). This can be explained by a higher maximal rate
for
Rb+/K+
exchange relative to
Rb+/H+
exchange. Alternatively, the inhibition of alkalosis-activated Rb+ efflux by a decrease in
[K+]e
may result from the more negative plasma membrane potential, implying
positive net charge movement during
Rb+ efflux from the cytoplasm. In
this case a decrease in
[K+]e
renders the diastolic membrane potential more negative and decreases
the driving force that moves Rb+
from the cell.
Inhibition of NH4Cl-activated
Rb+ efflux by intracellular
Li+ and activation of
Li+ efflux by intracellular
alkalosis imply that Rb+ and
Li+ interact with similar
pH-sensitive transporter(s). The quantitative difference in the levels
of activation of Rb+ efflux (100 and 180%) and Li+ efflux (40%)
can be explained by competition between higher
[Rb+]i + [K+]i
(~100 mM) and lower
[Li+]i
(~22 mM) (22) as well as by lower maximal rates for
Li+ efflux. Note that the
differences between absolute values of basal and stimulated
Rb+ and
Li+ fluxes
(J = k[ion]i)
are even greater. In fact, Rb+ + K+ flux increased from 4.2 to
8.1-11.6 mM/min (J = 3.9-7.4 mM/min), whereas Li+
flux increased from 0.62 to 0.92 mM/min
(J = 0.3 mM/min). These data show
that the carrier(s) responsible for alkalosis-stimulated Rb+ efflux is not absolutely
selective to
Rb+(K+),
corresponding to properties of sarcolemmal monovalent cation/cation antiporter described by Periyasamy (32).
The above mechanisms imply the direct participation of
H+ or
OH in
Rb+ transport or their direct
effects on ion transporters. We can exclude any indirect effects of
intracellular alkalosis, such as effects on signaling pathways or
affinity of Ca2+-binding proteins
to Ca2+, because global alkalosis
produced by Tris base did not activate Rb+ efflux.
Physiological relevance.
Intracellular alkalosis is a relatively rare occurrence in
cardiomyocytes relative to acidosis, which is characteristic of ischemia. Transient alkalosis can occur early in
ischemia due to the breakdown of PCr at unchanged ATP and
cessation of aerobic CO2
production. If this occurs, the K+
release that takes place early in ischemia (0-3 min) (18,
45) can be, in part, an alkalosis-activated process. In addition, it
has been shown in rat myocytes that stimulation of
Na+/H+
exchanger by pharmacological activation of protein kinase C results in
intracellular alkalosis (42). A parallel decrease in extracellular HCO3 and
CO2 also results in intracellular
alkalosis (14). In this study, an increase in extracellular
K+ was observed, probably caused
by K+ efflux from the cells
(17).
In conclusion, intracellular alkalosis stimulates unidirectional
effluxes of Rb+ and
Li+ from myocytes in isolated rat
hearts. We suggest that this phenomenon reflects the activation of a
sarcolemmal
K+(Li+)/cation
exchanger that extrudes K+,
Rb+, and
Li+ and takes up cations such as
H+ and
K+. We do not yet have any direct
evidence for transport of H+, such
as stoichiometric Rb+ efflux and
H+ influx, because no selective
inhibitor of alkalosis-activated Rb+ efflux has yet been found. If
such an inhibitor is identified, it should increase the alkalosis
produced by either monensin or NH4Cl and allow calculation of the
K+/H+ stoichiometry.
| |
ACKNOWLEDGEMENTS |
This work was supported in part by a grant from the Heart and
Stroke Foundation of Manitoba.
| |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: V. V. Kupriyanov, Inst. for Biodiagnostics, National Research Council of
Canada, 435 Ellice Ave., Winnipeg, MB, Canada R3B 1Y6 (E-mail:
kupriyanov{at}ibd.nrc.ca).
Received 7 August 1998; accepted in final form 2 March 1999.
| |
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