Role of JNK in hypertonic activation of
Cl
-dependent Na+/H+ exchange
in Xenopus oocytes
Greg G.
Goss1,
Lianwei
Jiang2,3,
David H.
Vandorpe2,3,
Dawn
Kieller1,
Marina N.
Chernova2,3,
Marilyn
Robertson1, and
Seth L.
Alper2,3,4
1 Department of Biological Science, University of Alberta,
Edmonton, Alberta, Canada T6G 2E9, 2 Molecular Medicine and
Renal Units, Beth Israel Deaconess Medical Center, Boston; and
Departments of 3 Medicine and 4 Cell Biology, Harvard
Medical School, Boston, Massachusetts 02215
 |
ABSTRACT |
In the course of studying the hypertonicity-activated ion
transporters in Xenopus oocytes, we found that activation of
endogenous oocyte Na+/H+ exchange activity
(xoNHE) by hypertonic shrinkage required Cl
, with an
EC50 for bath [Cl
] of ~3 mM. This
requirement for chloride was not supported by several nonhalide anions
and was not shared by xoNHE activated by acid loading.
Hypertonicity-activated xoNHE exhibited an unusual rank order of
inhibitory potency among amiloride derivatives and was blocked by
Cl
transport inhibitors. Chelation of intracellular
Ca2+ by injection of EGTA blocked hypertonic activation of
xoNHE, although many inhibitors of Ca2+-related signaling
pathways were without inhibitory effect. Hypertonicity activated oocyte
extracellular signal-regulated kinase 1/2 (ERK1/2), but inhibitors of
neither ERK1/2 nor p38 prevented hypertonic activation of xoNHE.
However, hypertonicity also stimulated a Cl
-dependent
increase in c-Jun NH2-terminal kinase (JNK) activity. Inhibition of JNK activity prevented hypertonic activation of xoNHE but
not activation by acid loading. We conclude that hypertonic activation
of Na+/H+ exchange in Xenopus
oocytes requires Cl
and is mediated by activation of JNK.
Na+/H+ exchange; c-Jun
NH2-terminal kinase; extracellular signal-regulated kinase; p38; transport; SP600125; U-0126
 |
INTRODUCTION |
CELL VOLUME
REGULATION in response to anisosmotic perturbation of the
surrounding medium is a fundamental property of most cell types. The
cell swelling that follows exposure to hypotonic medium leads to
cellular release of intracellular K+, Cl
, and
osmotically obliged water in a process termed regulatory volume
decrease (RVD). Conversely, cell shrinkage elicited by a hyperosmotic
solution promotes subsequent influx of ions and water in a process
termed regulatory volume increase (RVI). RVI is mediated either by
Na+-K+-Cl
cotransport (NKCC) or
by the coupled activities of Na+/H+ exchange
(NHE) and Cl
/HCO
exchange (anion
exchange, or AE) (30, 36, 42).
There are at least seven known mammalian isoforms of NHE
(42). Each cloned mammalian isoform displays a
characteristic set of pharmacological sensitivities to amiloride and
its derivatives, specific cellular and tissue localization, and
signaling mechanisms including characteristic responses to hyperosmotic
shock (19, 36). NHEs also have been cloned from tissues of
many nonmammalian species, including Saccharomyces
cerevisiae (40), Amphiuma
(38), and Xenopus laevis oocyte (XL-NHE)
(9). XL-NHE shares 69% amino acid identity with rat NHE1,
showing highest divergence in the amino- and carboxy-terminal
sequences. Xenopus oocytes and eggs are widely used for
studies of early development. Progesterone-activated postmeiotic
maturation of Xenopus oocytes is accompanied by a c-Mos-mediated NHE activation leading to increased intracellular pH
(pHi) (49). Although Xenopus
oocytes also are widely used as heterologous expression systems
for channels and transporters, including on occasion mammalian NHEs
(10), regulation of endogenous Xenopus oocyte
NHE activity remains less extensively studied (12, 57).
Xenopus oocytes respond to hyperosmotic shock with an extracellular
Na+-dependent, amiloride-sensitive elevation in
pHi, mediated by Na+/H+ exchange
(xoNHE) (30). Hypertonic activation of the AE2 anion exchanger expressed in Xenopus oocytes appears to require
intracellular alkalinization by xoNHE (29). Although
hypertonic activation of xoNHE does not suffice for RVI, oocytes
expressing heterologous AE2 (but not AE1) exhibit a secondary RVI that
requires functional coupling of AE2 with the endogenous xoNHE to
mediate uptake of extracellular Na+ and Cl
(30). In the course of studying the coupled activation of
AE and NHE activities required to mediate RVI in Xenopus
oocytes, we observed that hypertonic activation of xoNHE itself
requires bath Cl
. Cl
-dependent NHE activity
was first noted in high-Na+ dog red blood cells
(44) but has been observed subsequently in
high-K+ trout red blood cells (23), barnacle
muscle fibers (18), apical membrane vesicles prepared from
rat colonic crypts (45-47), and cultured mesangial
cells (39). Sensitivity of Cl
-dependent NHE
to inhibitors of Cl
transport also has been reported in
several of these cell types.
A Cl
-requirement for NHE provides a plausible mechanism
contributing toward functional coupling of AE and NHE activity.
Nonetheless, the mechanisms of functional coupling between AE and NHE
activities remain poorly understood, as are the signaling pathway(s)
initiated in oocytes by hypertonic shrinkage. Hypertonic shrinkage of
diverse cell types has been shown to regulate numerous signaling
pathways in cell type-specific manners (25). However, the
pathways leading to and from those activation events remain
incompletely defined.
In this study, we have characterized endogenous
Cl
-dependent xoNHE activity by measurement of the rate of
change in pHi and 22Na+ influx and
efflux. We report that bath Cl
was required for
activation of xoNHE by hypertonicity but not for xoNHE-mediated
recovery from intracellular acid load. Cl
-dependent,
hypertonicity-activated xoNHE exhibited a distinct profile of
inhibition by amiloride derivatives. Hypertonic activation of xoNHE,
but not activation by acid load, was blocked by pharmacological inhibition of the Cl
-dependent stimulation of
c-Jun-NH2-terminal kinase (JNK).
 |
METHODS |
Materials.
22Na+ (as NaCl) was obtained from ICN (Irvine,
CA). Collagenase A was from Roche (Indianapolis, IN). The amiloride
analogs ethylisopropyl amiloride (EIPA), benzamil, and phenamil
were purchased from BioMol (Plymouth Meeting, PA). HOE-694 was a kind
gift from Dr. H. J. Lang (Hoechst, Frankfurt, Germany).
2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM, fura 2-AM, DIDS, and nystatin were from Molecular Probes
(Eugene, OR). U-0126
[1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto) butadiene]
was purchased from Promega, and SB-203580
[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1 H-imidazole]
was from Sigma (St. Louis, MO). SP600125
{anthra[1,9-c,d]pyrazol-6(2h)-one} was provided by the Signal
Research Division of Celgene (San Diego, CA). All other chemicals were
obtained from Sigma.
Preparation of oocytes.
Mature female Xenopus (Nasco, Fort Atkinson, WI or Xenopus
1, Dexter, MI) were maintained in running charcoal-filtered water and
fed frog brittle twice weekly. Frogs were anesthetized (1.7% MS-222,
4°C), and ovarian fragments obtained by partial ovariectomy as
described previously (30) were dissociated by 30 min of
gentle agitation in the presence of 2 mg/ml collagenase A, followed by manual defolliculation and incubation for 1-4 days at 19°C in isosmotic (210 mosM) ND-96 containing (in mM) 96 NaCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES, 2.5 Na-pyruvate, and
100 U/ml gentamycin, pH 7.40. All subsequent measurements were carried
out in the absence of pyruvate and gentamycin. Solutions were made
hypertonic (300 mosM for 22Na+ flux, 280 mosM
for pHi measurements, unless otherwise noted) by the
addition to ND-96 of additional NaCl or (for voltage-clamp experiments)
90 mM mannitol. Addition of either NaCl or mannitol as hypertonic
stimulus produced indistinguishable effects on
22Na+ efflux and pHi responses.
N-methyl-D-glucamine was used to substitute for
extracellular Na+. Gluconate substituted for
Cl
except when indicated [nitrate, bromide, isethionate,
sulfamate, or 2-N-morpholinoethanesulfonate (MES)].
Gluconate salts of Ca2+, Mg2+, and
K+ were used when other anions substituted for
Cl
. In gluconate substitution experiments,
Ca2+-D-gluconate was increased to 11 mM to
counter the Ca2+-chelating effect of gluconate
salts.1 All solutions
contained 20 µM bumetanide. All
22Na+-containing flux solutions also contained
100 µM ouabain. Measurements of membrane potential by
two-microelectrode voltage clamp indicated no significant rundown of
membrane potential during 1.5 h of ouabain treatment.
pHi measurements.
Oocytes were monitored for changes in pHi during
hyperosmotic shock as described previouosly (30). Briefly,
oocytes were placed in ND-96 containing 2.5 µM BCECF-AM for 45 min. A
single oocyte was then washed and mounted in a closed perfusion chamber mounted on a microscope (Olympus IMT-2) with the vegetal pole facing
the excitation beam and with the focal plane of the ×10 objective
positioned at the oocyte equator. Oocyte pHi was monitored at 1-min intervals during continuous superfusion by ratiometric imaging
of serial 510-nm emission images elicited by alternating excitation at
495 and 440 nm (Universal Imaging, Westchester, PA). Chamber fluid
exchange was >95% complete within 1 min after solution change.
Calibration of pHi was performed as described previously
(30).
Unidirectional 22Na+
influx studies.
Measurement of unidirectional 22Na+
influx was performed by modification of the method of Towle et al.
(57). Briefly, groups of 10-12 oocytes were placed in
wells of a 24-well plate and preincubated for 1 h at 18°C in
isosmotic (210 mosM) flux solution containing (in mM) 10 NaCl, 86 N-methyl-D-glucamine Cl, 1.8 CaCl2,
1 MgCl2, and 10 HEPES, pH 7.0. Oocytes were then
transferred to another well containing 1 ml of the above solution. At
specific intervals, the isosmotic flux solution was removed and
replaced with 150 µl of either isotonic or hypertonic flux solution
containing 1 µCi 22Na+, 20 µM bumetanide,
100 µM ouabain, and other drugs as indicated. Solutions were made
hyperosmotic by addition of 90 mM mannitol to give a final osmolarity
of 300 mosM. After 45 min, the oocytes were removed, rapidly washed
four times in large volumes of ice-cold wash buffer (100 mM NaCl, and
10 mM HEPES, pH 7.4, 200 mosM), and counted in individual tubes with a
gamma counter (Packard Cobra or Packard Canberra). Duplicate 10-µl
samples of the flux solution were counted for calculation of specific
activity. Influx (Jin) was calculated according
to the following equation:
|
(1)
|
where cpm is counts per minute.
Unidirectional 22Na+
efflux studies.
22Na+ efflux was measured by a method modified
from Humphreys et al. (27). Oocytes were preincubated in
ND-96 without pyruvate and gentamycin for 1 h and then
microinjected with 50 nl (~10% of oocyte water space) of a solution
containing 0.05 µCi 22Na+ and (in mM) 90 KCl
and 10 NaCl, pH 7.4. After a 10-min recovery period, each oocyte was
placed individually in 1 ml of test solution. At 5- or 10-min
intervals, a 950-µl volume was removed for gamma counting and
immediately replaced by an equal volume of the same solution. To test a
different condition, two rapid 4-ml washes were followed by addition of
1 ml of the new test solution. Nominal total injected cpms were
computed as the sum of all cpms from each flux period plus the cpm
remaining in the oocyte at the end of the experiment. Efflux rate
constants were calculated for each oocyte from the slope of plots of
ln (%22Na+ remaining in the oocyte) vs. time
for each experimental condition. Changes in efflux rate constants were
in some cases normalized relative to the initial condition. Relative
efflux rate constants measured in media containing different anions
were normalized to that measured in NaCl within the same lot of oocytes
in the same experiment.
Oocyte electrophysiology.
Microelectrodes of borosilicate glass were filled with 3 M KCl-agar and
had resistances between 2 and 5 M
. Oocytes were impaled, and the
resting membrane voltage was measured. Oocytes were chosen in which the
initial resting membrane potential was more negative than
40 mV. To
ensure that either seal breakage or rundown of the membrane potential
did not occur because of the presence of ouabain, we monitored resting
membrane potential during the entire protocol. All oocytes had resting
potentials greater than
30 mV at the end of the experiment.
Whole oocyte currents were recorded in ND-96 under two-electrode
voltage-clamp conditions with a Geneclamp 500 amplifier (Axon Instruments, Foster City, CA) as previously described
(58). Before and 2, 5, 10, 20, and 30 min after
application of a hypertonic shock (addition to ND-96 of 70 mosM
mannitol, final osmolarity = 280 mosM), oocytes were stepped from
100 to +100 in 20-mV steps. The resulting data were filtered at 5 kHz
(8-pole Bessel filter; Frequency Devices) and sampled at 1 kHz. Data
were acquired and analyzed using pCLAMP version 6.0.
Measurement of kinase activity.
JNK activity was measured by a substrate phosphorylation assay. Groups
of 30 oocytes were placed in 1 ml of lysis buffer (1% Triton X-100/PBS
containing 100 µg/ml aprotinin, 100 µg/ml leupeptin, 1 mM
pepstatin, 1 mM phenylmethylsulfonyl fluoride, 100 µM
Na3VO4, 1 mM benzamidine, and 50 µM NaF,
4°C) and rapidly lysed by hand pestle in a small microcentrifuge
tube. The oocyte lysate was then incubated for at least 20 min on ice,
followed by centrifugation for 5 min at 10,000 g to pellet
cellular debris. The cleared lysate was incubated (3 h overnight at
4°C) with 20 µl (~2 µg) glutathione-S-transferase (GST)-c-Jun(5-89) conjugated to
glutathione beads (kind gift of Dr. James Woodgett, Princess Margaret
Hospital and University of Toronto). The beads were then washed at
least five more times in lysis buffer, sedimented, resuspended in 20 µl of kinase buffer containing 2 µCi [
-32P]ATP and
(in mM) 50 Tris · HCl, 1 EGTA, 10 MgCl2, and 4 K+-ATP, pH 7.5, and incubated for 30 min at 30°C. The
reaction was stopped by addition of 20 µl of 2× Laemmli sample
buffer. Samples were fractionated by SDS-PAGE (10%), stained with
Coomassie blue, destained, and dried. 32P incorporation
into GST-c-Jun was quantified directly by PhosphorImager (Molecular Dynamics, Sunnyvale, CA) or by analysis of scanned autoradiograph images (Scion Image software, Frederick, MD).
Extracellular signal-regulated kinase 1/2 (ERK1/2) activity was
measured by detection of the phosphorylated form of the kinase. Groups
of 30 oocytes were washed briefly in ice-cold buffer, homogenized in
lysis buffer (3.6 ml/oocyte), and centrifuged (18,000 g for 5 min at 4°C) to separate cytosol from cellular debris and yolk proteins. Cleared lysate was withdrawn with a Hamilton syringe, added
to an equivalent volume of 2× Laemmli sample buffer, fractionated on a
10% SDS-polyacrylamide gel, and transferred to nitrocellulose. Blots
were developed with phosphospecific ERK antibody (New England Biolabs,
Beverley, MA) and horseradish peroxidase-coupled secondary antibody,
exposed to preflashed X-ray film for enchanced chemiluminescence detection, and quantitated as described above. Kinase inhibitors were
injected into oocytes or added to the bath solution as indicated.
Statistical analysis.
22Na+ efflux or pHi measurements
within the same oocytes were analyzed by two-tailed t-tests
or ANOVA followed by Dunnet's test as appropriate. P < 0.05 was used as the limit of significance.
 |
RESULTS |
Hypertonic activation of xoNHE is Cl
dependent.
Figure 1A shows the time
course of pHi change in three individual oocytes subjected
to hypertonic stress. Whereas the oocyte exposed to additional NaCl
underwent substantial alkalinization following a lag period of about
7-9 min, the oocytes in Cl
-free baths as well as in
Na+-free baths exhibited severely attenuated rates
(dpHi/dt) and magnitudes (
pHi) of
alkalinization. Figure 1, B and C, and Table 1 demonstrate that exposure of oocytes to
a Cl
-free hypertonic solution resulted in 60-75%
inhibition of
pHi and dpHi/dt, comparable to
those in either the absence of Na+ or the presence of 100 µM amiloride. Depletion of intracellular Cl
by
overnight incubation of oocytes in Cl
-free medium
(28) only minimally enhanced the observed inhibition of
hypertonic xoNHE activation in the absence of bath Cl
(Table 1).

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Fig. 1.
Hypertonic activation of amiloride-sensitive
intracellular alkalinization requires bath Cl as well as
Na+. A: time-dependent changes in intracellular
pH (pHi) in individual oocytes superfused with hypertonic
medium containing NaCl, Na-gluconate (Cl -free), or
N-methyl-D-glucamine-Cl (Na+-free).
B: magnitude of pHi increase
( pHi) after 20-25 min of exposure to the indicated
hypertonic media. C: maximal rate of pHi
increase (dpHi/dt) following shift to hypertonic
media. Values are means ± SE. *P < 0.001 compared with control.
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Figure 2 shows amiloride-sensitive
22Na+ efflux as an index of xoNHE activity,
with each trace representing an individual oocyte. The efflux rate
constant was 14-fold higher in hypertonic than in isotonic conditions.
This 22Na+ efflux represented largely
Na+/Na+ exchange through the xoNHE, since
removal of extracellular Na+ inhibited
22Na+ efflux as effectively as did addition of
amiloride (Fig. 2B). As was true for hypertonically
activated xoNHE, Cl
removal inhibited
Na+/Na+ exchange by ~65% (Fig.
2C). Replacement of extracellular Na+ with
Li+ also supported 22Na+ efflux at
~70% of Na+/Na+ exchange rates (not shown),
consistent with earlier reports that measured amiloride-sensitive
Li+ uptake (10). However, amiloride-sensitive
xoNHE-mediated recovery from acid load was not Cl
dependent (Table 1), whether the acid load was imposed either at
constant extracellular pH (pHo) by NH4Cl
exposure (27) or by preincubation at pHo 5.0 (65). Figure 3A
shows the extracellular Cl
concentration dependence of
hypertonically activated 22Na+ efflux.
Half-maximal activation of efflux required ~3 mM Cl
.
Activation was maximal at 20 mM and was sustained at 140 mM extracellular Cl
. Figure 3B shows the
selectivity of the anion requirement for hypertonic activation of
22Na+ efflux. Nitrate and iodide completely,
and bromide partially, substituted for Cl
in supporting
xoNHE activity, but anion substitution with isethionate or gluconate
prevented xoNHE activation by hypertonicity.

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Fig. 2.
Hypertonic activation of 22Na+ efflux
requires bath Cl as well as Na+.
A: amiloride (100 µM) inhibited hypertonic activation of
22Na+ efflux. Left: each trace
represents an individual oocyte. Right: relative rate
constants (kNa) are normalized to the isotonic
condition. B: hypertonic activation of
22Na+ efflux required bath Na+.
C: a substantial component of hypertonicity-activated
22Na+ efflux required bath Cl .
Amil, amiloride; Iso, isotonic conditions; Hyper, hypertonic
conditions. Values are means ± SE. *P < 0.001 compared with control (Iso).
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Fig. 3.
Anion concentration dependence and selectivity of
hypertonicity-activated 22Na+ efflux.
A: extracellular Cl concentration
([Cl ]o) dependence of
hypertonicity-activated 22Na+ efflux. The
apparent half-maximal activation of efflux
(k1/2) for extracellular Cl
derived from the hyperbolic fit to the data is 2.8 mM. B:
extracellular anion selectivity of hypertonicity-activated
22Na+ efflux. Values are means ± SE;
n = 14 experiments for Cl group
(normalized to 1.0) and n = 6-8 for other
groups.
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Inhibitor pharmacology of hypertonically stimulated xoNHE.
The rate of hypertonicity-stimulated 22Na+
influx from medium containing 10 mM Na+ (Fig.
4A) and of
hypertonicity-stimulated 22Na+ efflux into
medium containing 100 mM Na+ (Fig. 4B) was
measured in the presence of increasing doses of various NHE inhibitors.
Amiloride was the most potent inhibitor of xoNHE as measured by both
efflux and influx. EIPA inhibited xoNHE more potently in the influx
studies conducted in 10 mM bath [Na+] than in the efflux
experiments conducted in higher bath [Na+], in which
EIPA, HOE-694, and benzamil were of equivalent potency. Phenamil was
ineffective at concentrations up to 1 mM. ID50 values for
22Na+ influx were 1 × 10
8 M
for amiloride, 6 × 10
7 M for EIPA, 7 × 10
5 M for HOE-694, and 6 × 10
5 M for
benzamil. At the 10-fold higher [Na+] of the
22Na+ efflux assay, ID50 values
were ~3 × 10
7 M for amiloride and
~3-5 × 10
6 M for EIPA, HOE-694, and
benzamil.

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Fig. 4.
Inhibitory profile of Xenopus oocyte
hypertonicity-activated 22Na+ flux by amiloride
and its analogs. A: inhibition of hypertonicity-activated
22Na+ efflux. B: inhibition of
hypertonicity-activated 22Na+ influx. EIPA,
ethylisopropyl amiloride.
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Activation of endogenous nonspecific cation conductances has been
observed in Xenopus oocytes under numerous conditions
(33, 64). Chan and Nelson (13) observed in
airway epithelial cells a nonspecific cation conductance activated by
hypertonicity and requiring extracellular Cl
(13). We used the two-microelectrode voltage-clamp
technique to assess the contribution of conductive pathways to
hypertonicity-activated 22Na+ flux. Figure
5A shows that 30-min exposure
to hypertonicity produced inward current of 10 ± 5 nA at a
holding potential of
50 mV, in contrast to the outward
Na+ current of 34 nA that was predicted if all measured
22Na+ efflux were conductive. The
hypertonicity-induced currents were amiloride insensitive, and
hypertonicity altered neither the reversal potential nor (not shown)
the resting potential. Depolarization in isotonic high-K+
medium (Na+ = 10 mM, K+ = 88 mM) did
not reproduce the increase in 22Na+ efflux
induced by hypertonicity (Fig. 5B). Moreover, the oocyte stretch-activated cation channel inhibitor Gd3+ (10 µM)
did not inhibit hypertonicity-activated 22Na+
efflux. These data suggest that previously described cation
conductances of oocytes (18, 20, 64) do not mediate the
observed hypertonicity-activated 22Na+ efflux.

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Fig. 5.
Hypertonicity does not activate cation currents of
magnitude adequate to account for the 22Na+
fluxes. A: current-voltage profile of oocytes treated for 30 min in isotonic medium ( , n = 5) or
hypertonic medium in the absence ( , n = 5) or presence of 100 µM amiloride ( ,
n = 3). B: high K+
depolarization did not activate 22Na+ efflux as
did hypertonicity.
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The Cl
-dependent NHE of the rat colonic crypt apical
membrane is distinguished by its sensitivity to inhibition by
Cl
channel blockers (46, 47). As is evident
in Table 2, 500 µM DIDS, 500 µM
5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), and 50 µM
niflumic acid also inhibited hypertonic activation of xoNHE as measured
as dpHi/dt and
pHi. Similarly, 50 µM niflumic acid inhibited hypertonicity-activated 22Na
efflux by ~40%, while even the reduced concentration of 50 µM NPPB
marginally inhibited hypertonicity-activated 22Na efflux by
~25% (P = 0.056). In contrast, DIDS activated
22Na efflux by 80% in both isotonic and hypertonic
conditions (Table 3), consistent with the
DIDS-activated cation and Na+ conductances observed by
Diakov et al. (20).
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Table 3.
Rate constants for 22Na+
efflux from Xenopus oocytes measured first in isotonic and then in
hypertonic medium
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Role of intracellular Ca2+ in
hypertonic activation of xoNHE.
Calmodulin binds to defined regions of the carboxy-terminal cytoplasmic
domain of mammalian NHE (60). Ca2+/calmodulin
binding to NHE1 appears to promote activation by relief of tonic
inhibition by the unliganded calmodulin-binding regions. Chelation of
intracellular Ca2+ by injection of EGTA (1 mM final
intracellular concentration) did not change resting pHi or
resting xoNHE activity as measured by 22Na+
efflux but significantly attenuated both the hypertonicity-activated increases in pHi and 22Na+ efflux
by ~50% (Fig. 6, A-C).
In contrast, removal of extracellular Ca2+ did not inhibit
hypertonic stimulation of xoNHE and did not enhance the inhibitory
effect of intracellular Ca2+ chelation (Table
4). Similarly, oocytes preincubated in
the intracellular Ca2+ chelating agent,
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid- AM (50 µM), exhibited a 50% reduction in hypertonic
activation of xoNHE (Table 4). Blockade of endogenous Ca2+
permeability pathways using the metals Ni2+ and
Cd2+ also resulted in an ~50% reduction in
hypertonicity-activated 22Na+ efflux (Fig.
6D). Despite the inhibition of xoNHE by intracellular Ca2+ chelation, many injected modulators of
Ca2+ signaling were without effect on
hypertonicity-activated 22Na+ efflux (not
shown). These included calmodulin, small molecule calmodulin
inhibitors, calmodulin kinase II inhibitory peptide, calcineurin, and
calcineurin inhibitory protein.

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Fig. 6.
Role of intracellular Ca2+ concentration
([Ca2+]i) in hypertonicity-activated
Na+/H+ exchange. A: injected EGTA (5 mM final concentration) blunted hypertonicity-activated increase in
pHi. Each trace represents a single oocyte. B:
injected EGTA blunted hypertonicity-activated increase in
22Na+ efflux. Values are means + SE;
n = 8. *P < 0.01. C: rate
constants for 22Na+ efflux from uninjected and
EGTA-injected oocytes in isotonic or hypertonic medium in absence or
presence of 100 µM amiloride. Values are means + SE;
n = 9. *P < 0.01 compared with Iso;
+P < 0.05 compared with untreated oocytes.
D: extracellular Cd2+ (1 mM) and
Ni2+ (1 mM) each partially inhibited
hypertonicity-activated 22Na+ efflux. Values
are means + SE; n = 4-5. *P < 0.01 compared with Iso; +P < 0.05 compared with
untreated oocytes.
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Role of selected other signaling pathways.
G proteins regulate Cl
-dependent NHE activity in barnacle
muscle (26). The GTPase activity of G proteins exhibits
Cl
-dependence, and Ca2+ signaling can be
regulated by G proteins. However, injection of neither guanosine
5'-O-(3-thiotriphosphate) (GTP
S) nor guanosine 5'-O-(2-thiodiphosphate) (GDP
S) (100 µM final
intracellular concentration) altered hypertonic activation of
22Na+ efflux. Lipid agonists can regulate
Ca2+ signaling, but injection of neither sphingosine (50 µM final concentration) nor 17-octadecynoic acid (25 µM final
concentration) altered hypertonic activation of
22Na+ efflux. Cytoskeletal active drugs modify
Ca2+ signaling, but injected cytochalasin D (2 µg/ml
final concentration) was without effect on hypertonic stimulation of
22Na+ efflux. The kinase antagonists H-7 (100 µM), ML-7 (10 µM), calphostin C (1 µM), and staurosporine (100 µM) also were without effect, as were the phosphatase inhibitors
deltamethrin D (10 nM), okadaic acid (500 nM), and calyculin (5 nM) and
the adenylate cyclase activator forskolin (100 µm).
As previously reported (12), the protein kinase C
activator phorbol 12-myristate 13-acetate (1 µM) increased isotonic
22Na+ efflux sixfold and was blocked by the
inhibitor H-89 (1 µM). Similarly, the nonspecific tyrosine kinase
inhibitors genistein D (40 µM) and herbimycin A (2 µg/ml) also
increased 22Na+ efflux in isotonic conditions
(not shown).
Effects of mitogen-activated protein kinase inhibitors.
The p38 family is activated by hypertonicity in many cell types
(41). In our study, inhibition of p38 with the inhibitor SB-203580 (5 µM) activated basal isotonic
22Na+ efflux approximately twofold, whereas
hypertonic activation of 22Na+ efflux was
unaffected (n = 20; not shown). Hypertonicity activated ERK1/2 activity, as shown in Fig. 7,
A and B, by the time-dependent approximately
sixfold increase in phospho-ERK, peaking 60 min after initiation of the
hypertonic stimulus. To determine whether hypertonicity-activated ERK
was involved in the regulation of xoNHE, we monitored xoNHE activity as
a function of exposure time to hypertonicity. Injection of the ERK
inhibitor U-0126 (50 µM), recently demonstrated to block completely
oocyte ERK activation (6), had no effect on hypertonic
activation of 22Na+ efflux (Fig.
7C), suggesting that this pathway is not required for xoNHE
activation.

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Fig. 7.
Hypertonic activation of extracellular signal-regulated
kinase 1/2 (ERK1/2) is not essential for activation of
22Na+ efflux. A: hypertonicity
activated endogenous Xenopus oocyte ERK1/2, as shown by
immunoblot with anti-phospho-ERK (p-ERK1/2). B: time course
of phospho-ERK activation. Values are means + SE;
n = 4. *P < 0.05. C: rate
constant for 22Na+ efflux from oocytes exposed
to hypertonicity was not reduced by bath exposure to the ERK inhibitor
U-0126 (50 µM final concentration).
|
|
Figure 8A shows activation of
JNK in lysates prepared from Xenopus oocytes exposed to
hypertonicity in the presence of Cl
. JNK activity
increased threefold within 60 min (Fig. 8B). Activation of
JNK by hypertonicity in intact oocyte required the presence of
extracellular Cl
. Activation was prevented by
extracellular Cl
substitution with gluconate, sulfamate,
or MES (Fig. 8B). In contrast, gluconate replacement of
Cl
in the in vitro kinase assay solution did
not effect JNK activity in lysates prepared from oocytes previously
exposed to hypertonic NaCl medium (not shown).

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Fig. 8.
Hypertonicity-associated activation of
c-Jun-NH2-terminal kinase (JNK) activity in Xenopus
oocytes requires bath Cl . A: lysate from
oocytes exposed to hypertonic NaCl medium for the indicated times
increased 32P incorporation into
glutathione-S-transferase-c-June (GST-c-Jun) fusion protein.
Lysate from oocytes exposed to hypertonic Na-gluconate medium had
minimal effect. B: time dependence of hypertonic activation
of JNK activity in Na+ media containing either
Cl ( ),
2-N-morpholinoethanesulfonate (MES; ),
sulfamate ( ), or gluconate ( ). Values
are means ± SE; n = 4. *P < 0.05, Cl vs. other anions.
|
|
Injection of the specific JNK inhibitor SP600125 (25 µM final
estimated intracellular concentration) 1 h (Fig.
9, A and B) or
18 h (not shown) before to hypertonic shock also abolished hypertonicity-induced JNK activation in oocytes. Overnight bath application of the drug at 25 or 50 µM or drug injection (25 or 50 µM final concentration) immediately before assay were without effect
(not shown). The acidification of resting pHi associated with injection of SP600125 (Fig. 9C) should serve only as an
additional stimulus for activation of xoNHE. However, SP600125
inhibited the hypertonicity-induced increase in pHi but did
not inhibit pHi recovery from NH4Cl-induced
acid load (Fig. 9C and Table
5). Moreover, the ~6 mM per pH unit
increase in intrinsic buffer capacity (52) predicted to
accompany the basal acidification produced by SP600125 accounts for
only a small fraction of the decrease in hypertonicity-induced
alkalinization.

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Fig. 9.
Hypertonic activation of Xenopus oocyte
Na+/H+ exchange is blocked by pharmacological
inhibition of JNK. A: injected SP600125 (25 µM final
concentration) inhibited activation by hypertonicity of Xenopus
oocyte JNK. B: inhibition of JNK in oocyte lysate by
injection of intact oocytes with SP600125 for the times indicated
before lysis. Values are means ± SE; n = 3.*P < 0.05. C: injection of SP600125
inhibited hypertonic increase in oocyte pHi. Each trace
represents an individual oocyte. D: injection of SP600125
inhibited hypertonic activation of 22Na+ efflux
from Xenopus oocytes. Values are means ± SE;
n = 13. *P < 0.01 compared with Iso;
+P < 0.05 compared with untreated oocytes.
|
|
Figure 9D and Table 5 show that 25 µM injected
SP600125 also inhibited hypertonicity-activated
22Na+ influx. Similarly, injected selenite (1 mM final concentration), a less specific inhibitor of JNK
(43), also inhibited hypertonic activation of
22Na+ efflux (not shown). SP600125 at 25 µM
selectively inhibits JNK activation in vitro and in cultured mammalian
cells, without concomitant inhibition of p38 or ERK1/2 activities
(6, 24).
 |
DISCUSSION |
NHE activity in Xenopus oocytes.
In unstimulated Xenopus oocytes, xoNHE mediates
pHi recovery from intracellular acid load and
pHi increase in response to hypertonic shrinkage. In the
presence of heterologous AE2 anion exchanger, xoNHE activation by
shrinkage results in NaCl influx that can confer secondary RVI in
Xenopus oocytes (30). The only cloned X. laevis NHE, XL-NHE, is 68% identical with human NHE1 (hNHE1)
(11), but Xenopus oocyte lysate contained no
polypeptide recognized by the anti-rat NHE1 monoclonal antibody 4E9
that recognized Amphiuma erythrocyte NHE (79% identical to
hNHE1) (38) or by other anti-NHE1 antibodies tested (Alper
and Goss, unpublished observation).
Although XL-NHE has been tacitly assumed to mediate xoNHE activity
elicited by all stimuli, the present data show distinct properties of
xoNHE activity elicited by different stimuli. Amiloride-sensitive pHi recovery from two types of acid load was independent of
extracellular Cl
. In contrast, hypertonic activation of
xoNHE, measured as intracellular alkalinization and as
amiloride-sensitive Na+/Na+ exchange, was
inhibited up to 75% by removal of extracellular Cl
. In
addition, the high-potency amiloride inhibition (ID50
~0.3 µM) of hypertonicity-stimulated xoNHE differs greatly from the 130 µM ID50 of amiloride for
Na+/Li+ exchange (10). The
pharmacology of hypertonicity-activated xoNHE also contrasts with the
rank order of inhibitor potency of cloned XL-NHE expressed in PS-120
cells (EIPA > HOE-642 > amiloride) (11),
whereas the relative HOE-694 insensitivity of hypertonic xoNHE activity
resembles that noted previously for endogenous oocyte
Na+/Li+ exchange measured in isotonic
conditions (10). These combined differences in inhibitor
pharmacology and Cl
dependence suggest the presence in
oocytes of two or more differentially regulated NHE isoforms. However,
as might explain the modified EIPA sensitivity of NHE in murine
sarcoma virus-transformed Madin-Darby canine kidney (MDCK) cells
(35), a regulatory modification by Cl
removal of xoNHE inhibitor potency cannot be ruled out.
Cl
-dependent NHE.
Modulation of NHE activity by Cl
could be physiologically
useful as a means of regulating independently the relative impacts of
NHE activity on cell volume and cell pHi. Despite the
apparent universal importance of this discrimination,
Cl
-dependent NHE activities vary in detail among the cell
types in which they have been observed.
Our experiments suggest that extracellular Cl
is
important in the hypertonic regulation of xoNHE but do not rule out the
possibility that all effects of extracellular Cl
might be
mediated by changes in Cl
. Global intraoocyte
[Cl
]i measured in isotonic
Cl
-free medium changes very little over times of up to 90 min as judged by assays of 36Cl
efflux,
Cl
content, or Cl
-sensitive microelectrodes
(16) (Goss and Alper, unpublished results). However, these
measurements have not been reported in Cl
-free hypertonic
medium. In addition, local [Cl
]i near the
oocyte plasma membrane has not yet been measured during extracellular
Cl
removal.
The conditions used to elicit Cl
-dependent
Na+/H+ exchange activities have differed in
different systems. In the present oocyte studies, hypertonic solutions
were applied at constant neutral bath pH without acid loading.
Cl
removal inhibited hypertonic alkalinization but not
isotonic recovery from acid load. Studies of apical membrane vesicles
from rat colonic crypt (45, 47) and studies of dog red
blood cells (44) varied both extracellular and
[Cl
]i while maintaining their near
equivalence. The studies of colonic crypt apical vesicles measured
Cl
-dependent 22Na+ influx in
isotonic media under pH-gradient or Na+-gradient conditions
(47). The studies in rat mesangial cells (39)
varied [Cl
]o under conditions in which
[Cl
]i also changed. These studies showed
that Cl
removal abolished both hypertonicity-induced
alkalinization and pHi recovery from acid load in
hypertonic conditions but not pHi recovery from acid load
in isotonic conditions.
In contrast, Na+/H+ exchange activity in
dialyzed barnacle muscle fiber clearly exhibited dependence on
[Cl
]i and not on
[Cl
]o (26). In these studies,
Cl
dependence was measured as recovery from acid load,
was evident in both isotonic and hypertonic conditions, and was
absolute, extrapolating to zero activity at zero
[Cl
]i. Moreover, this
[Cl
]i dependence was bypassed by the
heterotrimeric G
activators GTP
S, aluminum fluoride, or cholera
toxin (26). G
13 similarly stimulated
hNHE1-mediated recovery from acid load in HEK-293 cells as well as NHE1
activation by ligation of the D2 dopamine receptor (59). Busch et al. (10) reported that
injection into oocytes of either GTP
S or GDP
S inhibited
Li+ uptake in isotonic conditions. However, these studies
contrast with the lack of effect of GTP
S on hypertonic activation of
xoNHE, suggesting that the oocyte employs different regulatory
mechanisms. A recent paper (3) demonstrated
[Cl
]i dependence of acid-stimulated
activation of mammalian NHE-1, NHE-2, and NHE-3 expressed in
antiport-deficient fibroblasts. After [Cl
]i
depletion by removal of [Cl
]o and
equilibration, a rapid return to normal
[Cl
]o did not restore NHE activity in each
of the isoforms. This suggests that [Cl
]i
is responsible for mediating the noted acid-activated Cl
dependence in heterologously transfected fibroblasts.
Mechanism of xoNHE modulation by Cl
.
As in rat colonic crypt apical vesicles (in which the ability of
Cl
channel blockers to inhibit Cl
-dependent
Na+/H+ exchange was first reported) and rat
mesangial cells (39), Cl
channel blockers
inhibited hypertonicity-activated xoNHE activity. The ability of
Cl
channel blockers to inhibit hypertonicity-activated
xoNHE supports possible involvement of Cl
channels in
this activity, but the identities of these candidate regulatory
channels remain unknown. Moreover, the lack of pharmacological specificity of these agents has been most recently shown in
Xenopus oocytes by the ability of DIDS to activate two types
of cation channels also activated by maitotoxin (20). This
effect likely explains why, in contrast to its inhibition of
hypertonicity-induced increase in pHi, DIDS activated
22Na+ efflux from oocytes in both isotonic and
hypertonic media (Table 2).
The anion selectivities of Cl
-dependent anion exchange
differ in different cell types. Oocytes resembled mesangial cells in that I
substituted for Cl
. In contrast, in
apical membrane vesicles from rat distal colonic crypt, I
was as ineffective in replacing Cl
as was gluconate. In
antiport-deficient fibroblasts transfected with either NHE-1 or NHE-2,
there was no Cl
dependence when hypertonicity was used to
activate the exchanger (3). However, the substituting
anion in this study was NO
, an anion that supported
xoNHE activity in this study. It is possible that establishing
hypertonicity with other substituent anions may have demonstrated
Cl
dependence in the antiport-deficient fibroblasts.
The mechanism(s) by which intracellular and/or extracellular
Cl
modulate xoNHE activity also are obscure. One possible
explanation may be linked to the inhibition of hypertonicity-activated
xoNHE by intracellular chelation of Ca2+. It is thus
possible that Cl
removal decreases Ca2+
sensitivity of xoNHE activation by hypertonicity, either directly or
through element(s) of the hypertonicity signaling pathway. However,
although Ca2+-liganded calmodulin binds to and activates
mammalian NHE1 (60), many modulators of Ca2+
signaling failed to alter hypertonic activation of
22Na+ efflux.
Interestingly, reduction of [Cl
]i in rat
parotid acinar cells activated rather than inhibited NHE activity. The
elevation of [Ca2+]i that accompanied
reduction in [Cl
]i further potentiated NHE
activity (50). Similarly, in basolaterally permeabilized
Rana distal tubule, reduction in basolateral bath [Cl
] increased NHE activity, likely secondary to
release of Ca2+ from intracellular stores
(17). Marunaka and Niisato (37) described a
terbutaline-activated Ca2+-stimulated nonspecific cation
channel in fetal rat pneumocytes for which agonist-stimulated reduction
in [Cl
]i enhanced channel sensitivity to
activation by [Ca2+]i.
In rat mesangial cells, the pHi set point for activation
was acid shifted by hypertonicity, suggesting that Cl
might regulate the set point value. For NHE1, the set point value has
been associated with an ATP requirement (1), likely
reflecting turnover of and signaling by phosphatidylinositol
bisphosphate (4). Hypertonicity also has been reported to
retard endocytic processes (see references in Ref. 27),
perhaps elevating the number of transporters at the oocyte surface.
These regulatory pathways have yet to be tested in oocytes.
Role of Cl
in oocyte mitogen-activated protein kinase
family response to hypertonicity.
Hypertonicity is among the stressor stimuli that activate
mitogen-activated protein (MAP) kinases in a wide range of cell types
(14). ERK1/2 and JNK activities in oocytes were indeed activated by mild hypertonic stress. However, whereas hypertonic activation of xoNHE was unaffected by inhibition of ERK1/2, it was
severely attenuated by inhibition of JNK. We could not easily measure
p38 activity in oocytes, but the p38 inhibitors SB-200125 and SB-203580
each stimulated 22Na+ efflux in isotonic
conditions. These data suggest that whereas JNK mediates hypertonic
activation of xoNHE, p38 suppresses xoNHE activity in isotonic conditions.
This pattern of NHE regulation by the MAP kinases in Xenopus
oocytes differs from those in other cell types. MAP kinase
inhibition alters NHE activity differently in different cell types.
Thus ERK1/2 inhibitors inhibited NHE1-like activity stimulated by
angiotensin II in vascular smooth muscle cells (34),
aldosterone in MDCK cells (21), thrombin in platelets
(2),
1A-adrenoceptor stimulation (56), hydrogen peroxide treatment in rat cardiomyocytes
(53), and cannabinoid agonist in CB1-transfected Chinese
hamster ovary cells (8).
Hypertonicity activated ERK1/2 and p38 but not JNK in rat medullary
thick ascending limb segments (61), and those activations were due to shrinkage per se (51). Inhibition of these MAP
kinases did not alter hypertonicity-induced inhibition of apical
NHE3-mediated bicarbonate absorption (61), but inhibition
of p38 activation did block RVI (51), likely mediated by
basolateral NHE activity. Hypertonic activation of hNHE1 overexpressed
in lung fibroblast cells was insensitive to inhibition of any MAP
kinase, whereas ERK inhibition blocked the stimulatory effects of
growth factors on NHE1 (7). A recent paper describing
either mouse fibroblasts deficient in stress-activated protein/ERK
kinase 1/MAP kinase kinase 4 (SEK1/MKK4), an upstream component of the
JNK signaling pathway, or transfection of COS-7 cells with dominant
negative SEK1/MKK4 A-L failed to prevent hyperosmotic stimulation of
NHE1 (22), suggesting that JNK does not play a role in
activation of NHE1 in these cells. Hypertonic or isotonic shrinkage of
bovine aortic endothelial cells activated JNK, which could in vitro
phosphorylate NKCC1 fusion proteins (32).
The Xenopus oocyte appears to be the first example of a
requirement for JNK in the hypertonic activation of NHE, leading to intracellular alkalinization, as well as the first example of a
Cl
requirement for hypertonic activation of JNK. This
Cl
requirement appears to be upstream of JNK, since in
vitro kinase activity is itself Cl
independent. In U-937
cells, intracellular alkalinization by any means sufficed to activate
JNK and p38, but hypertonic activation of these MAP kinases did not
require NHE activity (55). In the progesterone-stimulated
oocyte undergoing meiotic maturation, JNK is activated by Mos, Raf, and
constitutively activated ERK. However, whereas the inhibitor U-0126
blocked progesterone activation of p42 (ERK1/2), it was without effect
on JNK activation (5). Thus upstream MEK-independent
regulatory steps may reveal the locus of Cl
sensitivity
for hypertonic activation of JNK. c-Raf may be one such
candidate (31). Interestingly, a role for extracellular or
intracellular Cl
has not been reported in the
fertilization-associated activation of xoNHE in Xenopus
eggs.
Reduction in extracellular NaCl elicited distinct effects on MAP
kinases in cells derived from the diluting segment of the nephron. Low
NaCl activated both p38 and ERK1/2 kinases in mouse macula densa cells,
leading to prostaglandin E2 release and, ultimately, increased cyclooxygenase-2 expression (63). In rabbit
cortical thick ascending limb cells, low NaCl activated both p38 and
JNK but not ERK1/2 (15).
The MAP kinases exhibit diverse patterns of regulation among the many
cell types in which they are intricately and tightly regulated. We have
shown here that in Xenopus oocytes, JNK activation by
hypertonicity is Cl
dependent and is required for
Cl
-dependent activation of xoNHE by hypertonicity. The
Xenopus oocyte, a workhorse for expression of heterologous
proteins, should continue to prove valuable for the study of its
intrinsic ion transport proteins and their modes of regulation.
Knowledge of these regulatory pathways is critical for optimal use of
the oocyte as a vehicle for heterologous gene expression and for
studies of regulation of those gene products.
 |
ACKNOWLEDGEMENTS |
The JNK inhibitor SP600125 was provided by the Signal Research
Division of Celgene (San Diego, CA).
 |
FOOTNOTES |
G. G. Goss was supported by grants from the Canadian Heart and
Stroke Foundation and the Alberta Heritage Foundation for Medical Research. S. L. Alper was supported by grants from the American Heart Association and by National Institutes of Health Grants DK-43459,
DK-34854 (Harvard Digestive Diseases Center), and HL-15157 (Boston
Sickle Cell Center).
Address for reprint requests and other correspondence: G. Goss,
Rm Z-512 Biological Sciences Bldg., Dept. of Biological Sciences, Univ.
of Alberta, Edmonton, Alberta, Canada T6G 2E9 (E-mail:
greg.goss{at}ualberta.ca) or S. L. Alper, RW763 East Campus, Beth
Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215 (E-mail: salper{at}caregroup.harvard.edu).
1
In gluconate-substituted solutions, a total
[Ca2+] of 11 mM is calculated to yield a free
[Ca2+] similar to that of 1.8 mM total
[Ca2+] in a Cl
solution (Chelator; Ref.
54). Activation of intracellular alkalinization in
hypertonic gluconate medium was indistinguishable in 1.8 and 11 mM Ca
(not shown). In the presence of hypertonic Cl
-free
gluconate medium containing a total [Ca2+] of 1.8 mM,
many but not all oocytes developed a high-magnitude, time-dependent,
100 µM amiloride-insensitive 22Na+ efflux
unrelated to xoNHE (not shown). This low bath
[Ca2+]-associated 22Na+ efflux
may represent Ca2+-inactivated cation channels
(62), with possible contribution from cation permeation
via the relatively nonselective Ca2+-inactivated Cl-
channels (48).
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.
Received 11 June 2001; accepted in final form 16 August 2001.
 |
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