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-dependent Na+/H+ exchange
in Xenopus oocytes
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
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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
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).
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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:
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(1) |
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.
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).
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.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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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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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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 × 108 M
for amiloride, 6 × 107 M for EIPA, 7 × 105 M for HOE-694, and 6 × 105 M for
benzamil. At the 10-fold higher [Na+] of the
22Na+ efflux assay, ID50 values
were ~3 × 107 M for amiloride and
~3-5 × 106 M for EIPA, HOE-694, and
benzamil.
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(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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-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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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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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) (GTPS) 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).
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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. 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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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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.
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 GTPS, aluminum fluoride, or cholera
toxin (26). G13 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 GTPS or GDPS inhibited
Li+ uptake in isotonic conditions. However, these studies
contrast with the lack of effect of GTPS 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).
-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 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.
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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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Aharonovitz, O,
Demaurex N,
Woodside M,
and
Grinstein S.
ATP dependence is not an intrinsic property of Na+/H+ exchanger NHE1: requirement for an ancillary factor.
Am J Physiol Cell Physiol
276:
C1303-C1311,
1999
2.
Aharonovitz, O,
and
Granot Y.
Stimulation of mitogen-activated protein kinase and Na+/H+ exchanger in human platelets. Differential effect of phorbol ester and vasopressin.
J Biol Chem
271:
16494-16499,
1996
3.
Aharonovitz, O,
Kapus A,
Szaszi K,
Coady-Osberg N,
Jancelewicz T,
Orlowski J,
and
Grinstein S.
Modulation of Na+/H+ exchange activity by Cl.
Am J Physiol Cell Physiol
281:
C133-C141,
2001
4.
Aharonovitz, O,
Zaun HC,
Balla T,
York JD,
Orlowski J,
and
Grinstein S.
Intracellular pH regulation by Na+/H+ exchange requires phosphatidylinositol 4,5-bisphosphate.
J Cell Biol
150:
213-224,
2000
5.
Bagowski, CP,
Xiong W,
and
Ferrell JE.
c-Jun N-terminal kinase activation in Xenopus laevis eggs and embryos. A possible non-genomic role for the JNK signaling pathway.
J Biol Chem
276:
1459-1465,
2001
6.
Bennett BL, Sasaki D, Murray B, O'Leary E, Satoh Y, Xu W, Motiwala A,
Pierce S, Leisten J, Bhagwat S, Manning A, and Anderson D. SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase.
Proc Natl Acad Sci USA. In press.
7.
Bianchini, L,
L'Allemain G,
and
Pouyssegur J.
The p42/p44 mitogen-activated protein kinase cascade is determinant in mediating activation of the Na+/H+ exchanger (NHE1 isoform) in response to growth factors.
J Biol Chem
272:
271-279,
1997
8.
Bouaboula, M,
Bianchini L,
McKenzie FR,
Pouyssegur J,
and
Casellas P.
Cannabinoid receptor CB1 activates the Na+/H+ exchanger NHE-1 isoform via Gi-mediated mitogen activated protein kinase signaling transduction pathways.
FEBS Lett
449:
61-65,
1999[Web of Science][Medline].
9.
Busch, S.
Cloning and sequencing of the cDNA encoding for a Na+/H+ exchanger from Xenopus laevis oocytes (Xl-NHE).
Biochim Biophys Acta
1325:
13-16,
1997[Medline].
10.
Busch, S,
Burckhardt BC,
and
Siffert W.
Expression of the human sodium/proton exchanger NHE-1 in Xenopus laevis oocytes enhances sodium/proton exchange activity and establishes sodium/lithium countertransport.
Pflügers Arch
429:
859-869,
1995[Web of Science][Medline].
11.
Busch, S,
Rosskopf D,
Lang HJ,
Weichert A,
and
Siffert W.
Expression, functional characterization and tissue distribution of a Na+/H+ exchanger cloned from Xenopus laevis oocytes (XL-NHE).
Pflügers Arch
436:
828-833,
1998[Web of Science][Medline].
12.
Busch, S,
Wieland T,
Esche H,
Jakobs KH,
and
Siffert W.
G protein regulation of the Na+/H+ antiporter in Xenopus laevis oocytes. Involvement of protein kinases A and C.
J Biol Chem
270:
17898-17901,
1995
13.
Chan, HC,
and
Nelson DJ.
Chloride-dependent cation conductance activated during cellular shrinkage.
Science
257:
669-671,
1992
14.
Chang, L,
and
Karin M.
Mammalian MAP kinase signaling cascades.
Nature
410:
37-40,
2001[Medline].
15.
Cheng, HF,
Wang JL,
Zhang MZ,
McKanna JA,
and
Harris RC.
Role of p38 in the regulation of renal cortical cyclooxygenase-2 expression by extracellular chloride.
J Clin Invest
106:
681-688,
2000[Web of Science][Medline].
16.
Chernova, MN,
Jiang L,
Crest M,
Hand M,
Vandorpe DH,
Strange K,
and
Alper SL.
Electrogenic sulfate/chloride exchange in Xenopus oocytes mediated by murine AE1 E699Q.
J Gen Physiol
109:
345-360,
1997
17.
Cooper, GJ,
and
Hunter M.
Na+/H+ exchange in frog early distal tubule: effect of aldosterone on the set-point.
J Physiol (Lond)
479:
423-432,
1994
18.
Davis, BA,
Hogan EM,
and
Boron WF.
Shrinkage-induced activation of Na+/H+ exchange in barnacle muscle fibers.
Am J Physiol Cell Physiol
266:
C1744-C1753,
1994
19.
Demaurex, N,
and
Grinstein S.
Na+/H+ antiport: modulation by ATP and role in cell volume regulation.
J Exp Biol
196:
389-404,
1994
20.
Diakov, A,
Koch JP,
Ducoudret O,
Muller-Berger S,
and
Fromter E.
The disulfonic stilbene DIDS and the marine poison maitotoxin activate the same two types of endogenous cation conductance in the cell membrane of Xenopus laevis oocytes.
Pflügers Arch
442:
700-708,
2001[Web of Science][Medline].
21.
Gekle, M,
Freudinger R,
Mildenberger S,
Schenk K,
Marschitz I,
and
Schramel H.
Rapid activation of Na+/H+ exchange in MDCK cells by aldosterone involves MAP-kinases ERK1/2.
Pflügers Arch
441:
781-786,
2001[Web of Science][Medline].
22.
Gillis, D,
Shrode LD,
Krump E,
Howard CM,
Rubie EA,
Tibbles LA,
Woodgett J,
and
Grinstein S.
Osmotic stimulation of the Na+/H+ exchanger NHE1: relationship to the activation of three MAPK pathways.
J Membr Biol
181:
205-214,
2001[Web of Science][Medline].
23.
Guizouarn, H,
and
Motais R.
Swelling activation of transport pathways in erythrocytes: effects of Cl, ionic strength, and volume changes.
Am J Physiol Cell Physiol
276:
C210-C220,
1999
24.
Han, H,
Boyle D,
Chang L,
Bennett B,
Karin M,
Yang L,
Manning AM,
and
Firestein GS.
c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis.
J Clin Invest
108:
73-81,
2001[Web of Science][Medline].
25.
Hoffmann, EK,
and
Pedersen SF.
Sensors and signal transduction in the activation of cell volume regulatory ion transport systems.
Contrib Nephrol
123:
50-78,
1998[Web of Science][Medline].
26.
Hogan, EM,
Davis BA,
and
Boron WF.
Intracellular Cl dependence of Na-H exchange in barnacle muscle fibres under normotonic and hypertonic conditions.
J Gen Physiol
110:
629-639,
1997
27.
Humphreys, BD,
Chernova MN,
Jiang L,
Zhang Y,
and
Alper SL.
NH4Cl activates AE2 anion exchanger in Xenopus oocytes at acidic pHi.
Am J Physiol Cell Physiol
272:
C1232-C1240,
1997
28.
Humphreys, BD,
Jiang L,
Chernova MN,
and
Alper SL.
Functional characterization and regulation by pH of murine AE2 anion exchanger expressed in Xenopus oocytes.
Am J Physiol Cell Physiol
267:
C1295-C1307,
1994
29.
Humphreys, BD,
Jiang L,
Chernova MN,
and
Alper SL.
Hypertonic activation of AE2 anion exchanger in Xenopus oocytes via NHE-mediated intracellular alkalinization.
Am J Physiol Cell Physiol
268:
C201-C209,
1995
30.
Jiang, L,
Chernova MN,
and
Alper SL.
Secondary regulatory volume increase conferred on Xenopus oocytes by expression of AE2 anion exchanger.
Am J Physiol Cell Physiol
272:
C191-C202,
1997
31.
Kang, MG,
Kulisz A,
and
Wasserman WJ.
Raf-1 kinase, a potential regulator of intracellular pH in Xenopus oocytes.
Biol Cell
90:
477-485,
1998[Web of Science][Medline].
32.
Klein, JD,
Lamitina ST,
and
Neill WC.
JNK is a volume-sensitive kinase that phosphorylates the Na-K-2Cl cotransporter in vitro.
Am J Physiol Cell Physiol
277:
C425-C431,
1999
33.
Kuruma, A,
Hirayama Y,
and
Hartzell HC.
A hyperpolarization- and acid-activated nonselective cation current in Xenopus oocytes.
Am J Physiol Cell Physiol
279:
C1401-C1413,
2000
34.
Kusuhara, M,
Takahashi E,
Peterson TE,
Abe J,
Ishida M,
Han J,
Ulevitch R,
and
Berk BC.
p38 Kinase is a negative regulator of angiotensin II signal transduction in vascular smooth muscle cells: effects on Na+/H+ exchange and ERK1/2.
Circ Res
83:
824-831,
1998
35.
Lagana, A,
Vadnais J,
Le PU,
Nguyen TN,
Laprade R,
Nabi IR,
and
Noel J.
Regulation of the formation of tumor cell pseudopodia by the Na+/H+ exchanger NHE1.
J Cell Sci
113:
3649-3662,
2000[Abstract].
36.
Lang, F,
Busch GL,
Ritter M,
Volkl H,
Waldegger S,
Gulbins E,
and
Haussinger D.
Functional significance of cell volume regulatory mechanisms.
Physiol Rev
78:
247-306,
1998
37.
Marunaka, Y,
and
Niisato N.
The essential role of cytosolic Cl in Ca2+ regulation of an amiloride-sensitive channel in fetal rat pneumocyte.
J Membr Biol
180:
91-99,
2001[Web of Science][Medline].
38.
McLean, LA,
Zia S,
Gorin FA,
and
Cala PM.
Cloning and expression of the Na+/H+ exchanger from Amphiuma RBCs: resemblance to mammalian NHE1.
Am J Physiol Cell Physiol
276:
C1025-C1037,
1999
39.
Miyata, Y,
Muto S,
Yanagiba S,
and
Asano Y.
Extracellular Cl modulates shrinkage-induced activation of Na+/H+ exchanger in rat mesangial cells.
Am J Physiol Cell Physiol
278:
C1218-C1229,
2000
40.
Nass, R,
Cunningham KW,
and
Rao R.
Intracellular sequestration of sodium by a novel Na+/H+ exchanger in yeast is enhanced by mutations in the plasma membrane H+-ATPase. Insights into mechanisms of sodium tolerance.
J Biol Chem
272:
26145-26152,
1997
41.
Ono, K,
and
Han J.
The p38 signal transduction pathway: activation and function.
Cell Signal
12:
1-13,
2000[Web of Science][Medline].
42.
Orlowski, J,
and
Grinstein S.
Na+/H+ exchangers of mammalian cells.
J Biol Chem
272:
22373-22376,
1997
43.
Park, HS,
Park E,
Kim MS,
Ahn K,
Kim IY,
and
Choi EJ.
Selenite inhibits the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) through a thiol redox mechanism.
J Biol Chem
275:
2527-2531,
2000
44.
Parker, JC.
Na-proton exchange in dog red blood cells.
In: Na-H Exchange, Intracellular pH, and Cell Function, edited by Aronson PM,
and Boron WF.. Orlando, FL: Academic, 1997, vol. 26, p. 101-114.
45.
Rajendran, VM,
Geibel J,
and
Binder HJ.
Chloride-dependent Na-H exchange. A novel mechanism of sodium transport in colonic crypts.
J Biol Chem
270:
11051-11054,
1995
46.
Rajendran, VM,
Geibel J,
and
Binder HJ.
Role of Cl channels in Cl-dependent Na/H exchange.
Am J Physiol Gastrointest Liver Physiol
276:
G73-G78,
1999
47.
Rajendran, VM,
Geibel J,
and
Binder HJ.
Characterization of apical membrane Cl-dependent Na/H exchange in crypt cells of rat distal colon.
Am J Physiol Gastrointest Liver Physiol
280:
G400-G405,
2001
48.
Reifarth, FW,
Amasheh S,
Clauss W,
and
Weber W.
The Ca2+-inactivated Cl channel at work: selectivity, blocker kinetics and transport visualization.
J Membr Biol
155:
95-104,
1997[Web of Science][Medline].
49.
Rezai, K,
Kulisz A,
and
Wasserman WJ.
Protooncogene product, c-mos kinase, is involved in upregulating Na+/H+ antiporter in Xenopus oocytes.
Am J Physiol Cell Physiol
267:
C1717-C1722,
1994
50.
Robertson, MA,
and
Foskett JK.
Na+ transport pathways in secretory acinar cells: membrane cross talk mediated by [Cl]i.
Am J Physiol Cell Physiol
267:
C146-C156,
1994
51.
Roger, F,
Martin PY,
Rousselot M,
Favre H,
and
Feraille E.
Cell shrinkage triggers the activation of mitogen-activated protein kinases by hypertonicity in the rat kidney medullary thick ascending limb of the Henle's loop. Requirement of p38 kinase for the regulatory volume increase response.
J Biol Chem
274:
34103-34110,
1999
52.
Romero, MF,
Henry D,
Nelson S,
Harte PJ,
Dillon AK,
and
Sciortino CM.
Cloning and characterization of a Na+-driven anion exchanger (NDAE1). A new bicarbonate transporter.
J Biol Chem
275:
24552-24559,
2000
53.
Sabri, A,
Byron KL,
Samarel AM,
Bell J,
and
Lucchesi PA.
Hydrogen peroxide activates mitogen-activated protein kinases and Na+-H+ exchange in neonatal rat cardiac myocytes.
Circ Res
82:
1053-1062,
1998
54.
Schoenmakers, TJ,
Visser GJ,
Flik G,
and
Theuvenet AP.
CHELATOR: an improved method for computing metal ion concentrations in physiological solutions.
Biotechniques
12:
870-879,
1992[Web of Science][Medline].
55.
Shrode, LD,
Rubie EA,
Woodgett JR,
and
Grinstein S.
Cystolic alkalinization increases stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) activity and p38 mitogen-activated protein kinase activity by a calcium-independent mechanism.
J Biol Chem
272:
13653-13659,
1997
56.
Snabaitis, AK,
Yokoyama H,
and
Avkiran M.
Roles of mitogen-activated protein kinases and protein kinase C in 1A-adrenoceptor-mediated stimulation of the sarcolemmal Na+/H+ exchanger.
Circ Res
86:
214-220,
2000
57.
Towle, DW,
Baksinski A,
Richard NE,
and
Kordylewski M.
Characterization of an endogenous Na+/H+ antiporter in Xenopus laevis oocytes.
J Exp Biol
159:
359-369,
1991
58.
Vandorpe, DH,
Shmukler BE,
Jiang L,
Lim B,
Maylie J,
Adelman JP,
de Franceschi F,
Cappellini MD,
Brugnara C,
and
Alper SL.
cDNA cloning and functional characterization of the mouse Ca2+-gated K+ channel, mIK1. Roles in regulatory volume decrease and erythroid differentiation.
J Biol Chem
273:
21542-21553,
1998
59.
Voyno-Yasenetskaya, T,
Conklin BR,
Gilbert RL,
Hooley R,
Bourne HR,
and
Barber DL.
G13 stimulates Na-H exchange.
J Biol Chem
269:
4721-4724,
1994
60.
Wakabayashi, S,
Bertrand B,
Ikeda T,
Pouyssegur J,
and
Shigekawa M.
Mutation of calmodulin-binding site renders the Na+/H+ exchanger (NHE1) highly H+-sensitive and Ca2+ regulation-defective.
J Biol Chem
269:
13710-13715,
1994
61.
Watts, BA,
Di M,
Davis RJ,
and
Good DW.
Hypertonicity activates MAP kinases and inhibits HCO
62.
Weber, WM,
Liebold KM,
and
Clauss W.
Amiloride-sensitive Na+ conductance in native Xenopus oocytes.
Biochim Biophys Acta
1239:
201-206,
1995[Medline].
63.
Yang, T,
Park JM,
Arend L,
Huang Y,
Topaloglu R,
Pasumarthy A,
Praetorius H,
Spring K,
Briggs JP,
and
Schnermann J.
Low chloride stimulation of prostaglandin E2 release and cyclooxygenase-2 expression in a mouse macula densa cell line.
J Biol Chem
275:
37922-37929,
2000
64.
Yang, XC,
and
Sachs F.
Block of stretch-activated ion channels in Xenopus oocytes by gadolinium and calcium ions.
Science
243:
1068-1071,
1989
65.
Zhang, Y,
Chernova MN,
Stuart T,
Jiang L,
and
Alper SL.
The cytoplasmic and transmembrane domains of AE2 both contribute to regulation of anion exchange by pH.
J Biol Chem
271:
5741-5749,
1996
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 S. F. Pedersen, S. A. King, R. R. Rigor, Z. Zhuang, J. M. Warren, and P. M. Cala Molecular cloning of NHE1 from winter flounder RBCs: activation by osmotic shrinkage, cAMP, and calyculin A Am J Physiol Cell Physiol, June 1, 2003; 284(6): C1561 - C1576. [Abstract] [Full Text] [PDF]
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 M. N Chernova, L. Jiang, B. E Shmukler, C. W Schweinfest, P. Blanco, S. D Freedman, A. K Stewart, and S. L Alper Acute regulation of the SLC26A3 congenital chloride diarrhoea anion exchanger (DRA) expressed in Xenopus oocytes J. Physiol., May 15, 2003; 549(1): 3 - 19. [Abstract] [Full Text] [PDF]
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M. N. Chernova, A. K. Stewart, L. Jiang, D. J. Friedman, Y. Z. Kunes, and S. L. Alper Structure-function relationships of AE2 regulation by Ca2+i-sensitive stimulators NH+4 and hypertonicity Am J Physiol Cell Physiol, May 1, 2003; 284(5): C1235 - C1246. [Abstract] [Full Text] [PDF]
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J. Aleu, M. Martin-Satue, P. Navarro, I. P. de Lara, L. Bahima, J. Marsal, and C. Solsona Release of ATP induced by hypertonic solutions in Xenopus oocytes J. Physiol., February 15, 2003; 547(1): 209 - 219. [Abstract] [Full Text] [PDF]
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, 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.
),
2-N-morpholinoethanesulfonate (MES; 
), or gluconate (