Role of JNK in hypertonic activation of Clminus -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
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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).


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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:
J<SUB>in</SUB><IT>=</IT>cpm<IT>∗</IT>oocyte<SUP><IT>−</IT>1</SUP><IT>∗</IT>min<SUP><IT>−</IT>1</SUP><IT>∗</IT><FENCE><FR><NU>pmol Na<SUP>+</SUP><IT>/</IT>10<IT> &mgr;</IT>l solution</NU><DE>cpm<IT>/</IT>10<IT> &mgr;</IT>l solution</DE></FR></FENCE> (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 MOmega . 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 [gamma -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.


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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 (Delta 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 Delta 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 (Delta 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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Table 1.   Chloride dependence of Na+/H+ exchange

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.

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.

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 (open circle , n = 5) or hypertonic medium in the absence (triangle , n = 5) or presence of 100 µM amiloride (, n = 3). B: high K+ depolarization did not activate 22Na+ efflux as did hypertonicity.

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 Delta 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 2.   Cl- dependence of Na+/H+ exchange activated by hypertonicity


                              
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Table 3.   Rate constants for 22Na+ efflux from Xenopus oocytes measured first in isotonic and then in hypertonic medium

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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Table 4.   Ca2+ dependence of Na+/H+ exchange activated by hypertonicity

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) (GTPgamma S) nor guanosine 5'-O-(2-thiodiphosphate) (GDPbeta 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; open circle ), sulfamate (), or gluconate (triangle ). 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.


                              
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Table 5.   Inhibition of hypertonic activation of Na+/H+ exchange by SP600125

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Galpha activators GTPgamma S, aluminum fluoride, or cholera toxin (26). Galpha 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 GTPgamma S or GDPbeta S inhibited Li+ uptake in isotonic conditions. However, these studies contrast with the lack of effect of GTPgamma 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<UP><SUB>3</SUB><SUP>−</SUP></UP>, 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), alpha 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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