Gastrointestinal Division, Departments of Medicine and Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2195
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ABSTRACT |
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Intestinal neutral NaCl absorption, which is made up of
brush-border (BB)
Na+/H+
exchange linked to BB
Cl/HCO
3
exchange, is up- and downregulated as part of digestion and diarrheal
diseases. Glucocorticoids stimulate ileal NaCl absorption and BB
Na+/H+
exchange. Intestinal BB contains two
Na+/H+
exchanger isoforms, NHE2 and NHE3, but their relative roles in rabbit
ileal BB
Na+/H+
exchange has not been determined. A technique to separate the contribution of NHE2 and NHE3 to ileal BB
Na+/H+
exchange activity was standardized by using an amiloride-related compound, HOE-694. Under basal conditions, both NHE2 and NHE3 contribute ~50% to ileal
Na+/H+
exchange. Glucocorticoids (methylprednisolone) increase BB
Na+/H+
exchange (2.5 times) but increase only ileal NHE3 activity (4.1 times),
without an effect on NHE2 activity. Thus ileal BB
Na+/H+
exchange in animals treated with glucocorticoids is 69% via NHE3. A
quantitative Western analysis for NHE3 was developed, using as an
internal standard a fusion protein of the COOH-terminal 85 amino acids
of NHE3 and maltose binding protein. Glucocorticoid treatment increased
the amount of BB NHE3. The quantitative Western analysis showed that
NHE3 makes up 0.018% of ileal BB protein in control rabbits and
0.042% (2.3 times as much) in methylprednisolone-treated rabbits.
Methylprednisolone treatment did not alter the amount of ileal BB NHE2
protein. NHE3 turnover number was estimated to be 458 cycles/s under
basal conditions and 708 cycles/s in glucocorticoid-treated ileum. Thus
methylprednisolone stimulates ileal BB
Na+/H+
exchange activity only by an effect on NHE3 and not on NHE2; it does so
primarily by increasing the amount of BB NHE3, although it also
increases the NHE3 turnover number.
HOE-694; plasma membrane vesicle; quantitative Western analysis; turnover number
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INTRODUCTION |
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SODIUM-HYDROGEN EXCHANGERS are plasma membrane transport proteins. They exchange extracellular Na+ for intracellular H+ with a stoichiometry of 1:1. In eukaryotic cells, the plasma membrane Na+/H+ exchangers have multiple functions, including pH homeostatis, volume regulation, cell proliferation, and transcellular Na+ absorption. Up to now, four mammalian Na+/H+ exchanger isoforms have been fully cloned and expressed: NHE1, NHE2, NHE3, and NHE4 (2, 21, 25-30). One additional isoform has recently been reported (15).
Functionally, there are two identified classes of mammalian Na+/H+ exchangers, the housekeeper isoforms, including NHE1, and the epithelial isoforms, NHE2 and NHE3. Kinetically, there is a major difference in the short-term regulation by protein kinases and growth factors of NHE1 vs. NHE2 and NHE3. Nearly all short-term regulation of NHE1 by protein kinases is by a change in affinity for intracellular H+, whereas both short-term regulation and more prolonged transcriptional regulation of NHE2 and NHE3 include changes in the maximum velocity (Vmax). The meaning of these changes is not yet understood, nor is it known whether the Vmax regulation of NHE2 and NHE3 represents changes in the number of active plasma membrane exchangers, in the turnover number of each exchanger, or in both.
Glucocorticoids stimulate intestinal water and NaCl absorption and do so by affecting NHE3 (3, 32). Yun et al. (32) showed that methylprednisolone increased the ileal brush-border (BB) Na+/H+ exchange rate two- to threefold within 24 h of injection and stimulated the NHE3 mRNA by four- to sixfold at 24 h but did not affect the mRNA levels of NHE1 and NHE2. This was one of the main reasons to initially suggest that NHE3 probably was the Na+/H+ exchanger involved in ileal Na+ absorption. Cho et al. (4) also showed that the increased NHE3 expression in dexamethasone-treated rats was tissue and region specific. They found that NHE3 mRNA was increased 3.5-fold in rat ileum and 2.0-fold in the proximal colon and that there was no increase in the jejunum, distal colon, or kidney, whereas expression of NHE1 and NHE2 mRNA was not affected by dexamethasone administration. How methylprednisolone quantitatively affects the amount of ileal BB NHE3 and NHE2 protein has not yet been determined. Also, although NHE2 and NHE3 are BB Na+/H+ exchangers and are present in at least most small intestinal villus epithelial cells (12), their relative roles in Na+ absorption have not been determined.
The purpose of this investigation was to 1) develop a technique to quantitate the contributions of NHE2 and NHE3 to ileal BB Na+/H+ exchange activity, 2) use that technique to determine whether methylprednisolone-stimulated BB Na+/H+ exchange activity is due to NHE2 and/or NHE3, 3) develop a quantitative Western analysis to measure amount of ileal BB NHE3, and 4) use that technique to determine the magnitude of change in NHE3 protein caused by methylprednisolone treatment. In so doing, we developed a technique allowing determination of the number of molecules of NHE3 present in the ileal BB.
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MATERIALS AND METHODS |
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Cell Studies
Stable transfection of cloned rabbit NHE2 and NHE3 in PS120 cells. A Chinese hamster lung fibroblast cell line selected to lack all endogenous Na+/H+ exchangers was stably transfected, using Lipofectin, with rabbit NHE2 or rabbit NHE3, as previously described (20, 25, 28). To obtain PS120 cells stably transfected with NHE3 cDNA tagged with a previously described vesicular stomatitis virus G protein (VSVG) epitope tag (NHE3V construct) and also transfected with NHE2 (PS120/NHE2-NHE3V cells) (12), PS120/NHE2 cells were further transfected with a cDNA for NHE3V and selected on the basis of ability to survive three to four consecutive acid killing treatments and recovery in 2 mM Na+ plus 100 µM 3-methylsulfonyl-4-piperidino benzoyl guanidine methanesulfonate (HOE-694) (25, 28). The surviving colonies were subcloned and screened for NHE3V expression by Western blot. Four clones were selected that differed in relative amounts of NHE2 vs. NHE3V.
All transfected cells were maintained in DMEM supplemented with 25 mM NaHCO3, 10 mM HEPES, pH 7.4, 50 IU/ml penicillin, 50 µg/ml streptomycin, 10% fetal bovine serum, and 800 µg/ml G418 in a 5% CO2-95% O2 incubator at 37°C. The cells were acid loaded weekly, as described above, and recovered in 2 mM Na+ with (PS120/NHE2-NHE3V) or without (PS120/NHE2 and PS120/NHE3) 100 µM HOE-694.Immunoprecipitation. PS120 cells expressing NHE2, NHE3V, and NHE2-NHE3V were grown to confluency in 10-cm dishes, rinsed with ice-cold PBS (in mM: 136.9 NaCl, 2.7 KCl, 4.3 Na2HPO4, and 1.5 KH2PO4, pH 7.3), and scraped into buffer A, consisting of (in mM) 150 NaCl, 60 HEPES, pH 7.4, 3 KCl, 5 EDTA, 3 Na+, 5 EGTA, 1 sodium orthovanadate, 25 sodium pyrophosphate, and 50 NaF, plus protease inhibitors [phenylmethylsulfonyl fluoride (PMSF), iodoacetamide, aprotinin, and 1,10-phenanthroline]. The cells were pelleted and homogenized in buffer A plus 1% Triton X-100 (buffer B) using a sonicator (30 s) at 4°C, followed by shaking at 4°C for 30 min. The homogenate was centrifuged at 14,000 g for 30 min at 4°C to remove insoluble material, and the supernatant was collected and incubated overnight with 5 µl of either polyclonal anti-NHE2 antibody 597 (Ab 597) or polyclonal anti-VSVG Ab 1648, both of which have been previously described by us (see Anti-VSVG antibody). The antibody complexes were precipitated after exposure to Pansorbin (Calbiochem) for 1 h at 4°C with rocking, washed three times with buffer B, and processed for Western analysis with 9% SDS-PAGE, using Laemmli sample buffer. Each sample was split into two aliquots and run in parallel in SDS-PAGE. The samples were transferred to nitrocellulose membrane and analyzed with Ab 597 or Ab 1648 (1:1,000) followed by peroxidase-conjugated donkey anti-rabbit IgG (Santa Cruz Biotechnology).
22Na+ uptake. Stably transfected PS120/NHE2, PS120/NHE3V, and PS120/NHE2-NHE3V were seeded on 24-well plates and examined for Na+/H+ exchanger activity when they reached confluency. The cells were washed three times with PBS and acidified by incubation in NH4Cl buffer for 20 min, after which the cells were washed three times and allowed to stand for 1 min in tetramethylammonium (TMA) buffer. Then the TMA solution was aspirated and replaced with either transport buffer alone, transport buffer plus 1 mM amiloride, or transport buffer with different concentrations of HOE-694 (0.01 µM to 1 mM) (6). The cells were allowed to stand in transport buffer for exactly 4 min (during which Na+ uptake has been shown to be linear), after which the buffer was aspirated and the plates were rinsed in ice-cold PBS. The cells were lysed in 0.5 M NaOH for 30 min, and the lysates were assayed for gamma-radiation using a Beckman gamma counter. All transport experiments were performed in triplicate. The composition of the NH4Cl buffer was (in mM) 40 NH4Cl, 90 TMA chloride, 5 KCl, 2 CaCl2, 1 MgSO4, 20 HEPES, pH 7.4, 1 H3PO4, and 8.58 TMA hydroxide. The TMA buffer was obtained by replacing the NH4Cl completely with TMA. The transport buffer was composed of (in mM) 1 NaCl, 129 TMA chloride, 5 KCl, 2 CaCl2, 1 MgSO4, 20 HEPES, pH 7.4, 8 NaOH, 0.83 Na2HPO4, 7 H2O, and 1 ouabain, plus 1 µCi/ml of 22NaCl.
Animal Studies
Male New Zealand White rabbits weighing ~2.5 kg were injected subcutaneously once with 40 mg methylprednisolone or an equal volume (1 ml) of 0.9% NaCl and killed 24 h later by intravenous nembutal. This model has been characterized by us (32).Ileal BB membrane preparation. Immediately after the rabbits were killed, ~90 cm of distal ileum was removed. After the intestine was sequentially washed with saline (0.9%) and a protease inhibitor solution (containing 0.05 mM PMSF, 6 µM phosphorhamidone, and 0.3 trypsin inhibitory units/ml aprotinin), the ileal mucosa was lightly scraped with glass slides on ice. BB membrane vesicles were prepared as previously described (5, 8). All steps were at 4°C. Cells were homogenized with a Brinkmann Polytron homogenizer using 10 bursts of 10 s each, with 20-s intervals, in a solution containing (in mM) 60 mannitol, 2.4 Tris · HCl, pH 7.1, and 1 EGTA, plus the protease inhibitors PMSF (0.32 mM) and phosphorhamidone (0.006 mM) and the trypsin inhibitor aprotinin (0.3 units/ml). Homogenate was treated with 10 mM MgCl2 for 15 min and centrifuged (3,000 g, 15 min). After this pellet was discarded, the supernatant was centrifuged (27,000 g, 30 min) and the pellet was resuspended in (in mM) 60 mannitol, 5 EGTA, and 10 Tris, pH 7.1. The 10 mM MgCl2 steps were repeated, and the pellet was suspended in (in mM) 300 mannitol, 5 magnesium gluconate, and 20 HEPES-Tris, pH 7.1, and centrifuged (27,000 g, 40 min). The final pellet was resuspended in the same buffer with a 25-gauge needle. Membranes were stored in liquid nitrogen and generally used <1 wk after preparation.
The protein concentration was measured by using Bio-Rad reagent, with ovalbumin as a standard. The enrichment of the ileal BB compared with homogenate was determined by sucrase-isomaltase assay using glucose as a standard, as described (5).BB Na+/H+ exchange. On the day of study, BB membranes were homogenized in 6 ml of membrane buffer consisting of (in mM) 200 mannitol, 40 3-(N-morpholino)-2-hydroxypropanesulfonic acid-11.4 Tris, pH 6.5, and 5 magnesium gluconate, and were then centrifuged (110,000 g, 60 min). The pellet was needle homogenized in the same membrane buffer and allowed to equilibrate 30 min on ice and then for 30 min at room temperature before study.
Transport buffer pH 8.4 (final pH of 8.0 when mixed with membrane buffer at a ratio of 2:1, vol/vol) contained (in mM) 207 mannitol, 28 Tris-9.6 MES, 5 magnesium gluconate, and 1.0 sodium gluconate (~40 µCi/ml 22Na+). Methylpropyl amiloride (MPA) was used at a final concentration of 200 µM, and HOE-694 was used at concentrations of 1-1,000 µM. In conditions containing MPA or HOE-694, additions were only to the transport buffer. HOE-694 (stock solutions made in water) was used within 1.5 h of being dissolved. 22Na+ initial rates of uptake were measured by mixing 15 µl of membrane with 30 µl of transport buffer at 22°C, as described (5, 9), with the reaction electronically stopped after 8 s by injection of 1 ml of ice-cold stop solution. Reaction mixture was then vacuum filtered through nitrocellulose filters (0.45 µM pore size; Millipore, Bedford, MA) and rinsed with 6 ml of stop solution. Stop solution contained (in mM) 40 mannitol, 90 potassium gluconate, 15.6 MES, and 20 Tris, pH 8.0. The filters were dissolved in 4 ml of scintillant, and radioactivity was determined in a liquid scintillation spectrometer (Beckman L5 7500). Na+/H+ exchange was defined as the 22Na+ uptake that was inhibited by 200 µM MPA. Initial uptake rates were expressed in picomoles per milligram protein per 8 s.Antibodies and Fusion Proteins
Glutathione S-transferase/E3-C85 fusion
protein.
A fusion protein of glutathione
S-transferase (GST) and the
COOH-terminal 85 amino acids of NHE3 was constructed by PCR using the
269 bp that encode the last 85 amino acids of NHE3, amplified from cDNA
by paired primers (5'-ACC TCC GAC TCC CCTG-3' and
5'-CGGAGCTCGAG TCA CAT GTG TGTGG-3'). PCR conditions used
were 30 cycles of denaturation at 95°C for 30 s, primer annealing
at 55°C for 30 s, and extension by
Taq polymerase at 70°C for 45 s.
The 269-bp PCR fragment was subcloned into pCRII (InVitrogen),
sequenced, and subcloned into the EcoR
I site of pGEX-2T to produce the construct pGEX/E3-C85. Fusion protein
was expressed in Escherichia
coli (NM 522) by induction with 0.3 mM
isopropyl
-D-thiogalactopyranoside
(IPTG) and was affinity purified from bacterial lysates by batch
absorption with glutathione Sepharose 4B, as previously described (12).
Maltose binding protein/E3-C85 fusion protein. pMal-c2 (New England Biolab) was linearized by digestion with restriction enzyme Hind III and blunt ended with Klenow, followed by digestion with BamH I. To construct maltose binding protein/E3-C85 (MBP/E3-C85), pGEX/E3-C85 was linearized with Xho I, blunt ended with Klenow, and then cut with BamH I. This cut cDNA fragment was cloned into pMal-c2 by unidirectional cloning into BamH I and blunt-ended sites. The construct was confirmed by sequencing. The pMal-c2/E3-C85 construct was transformed into E. coli (NM 522) for expression of the fusion protein, according to the manufacturer's instructions. As a control, pMal-c2 without an insert was similarly transformed.
The fusion protein was affinity purified. Briefly, 1 liter of transformed bacteria was propagated in 2 XYT medium at 1:100 dilution from an overnight culture until the optical density at 600 nm was 0.5 and was induced with 0.3 mM IPTG for 2 h. The cells were collected by centrifuging for 20 min at 4,000 g. Cells were resuspended in 50 ml of column buffer (30 mM NaCl, 10 mM HEPES, brought to pH 7.2 with Tris base) plus protease inhibitors (100 µM PMSF and 1 mM iodoacetamide) plus 0.25% Tween 20. Then the cells were sonicated to clarity on ice using a Banson Sonifier 450 probe sonicator. The cell suspension was centrifuged for 30 min at 9,000 g, and then the supernatant was applied to an amylose-agarose column and washed extensively with column buffer with protease inhibitors. The bound fusion protein was eluted with column buffer and 10 mM maltose and then dialyzed against PBS.Anti-NHE3 and anti-NHE2 antibodies. Anti-NHE3 Ab 1380 was produced in rabbits after injection of affinity-purified GST/E3-C85 fusion protein, as previously described (12). Ab 1380 has been shown to specifically recognize NHE3 and not NHE1 or NHE2 expressed in PS120 cells by Western analysis and immunocytochemistry (12).
Anti-NHE2 Ab 597 and Ab 1797 were produced in rabbits after injection with affinity-purified GST/E2-C87, as previously described (27). Both antibodies have been shown to specifically recognize NHE2 and not NHE1 or NHE3 expressed in PS120 cells by Western analysis and immunocytochemistry.Anti-VSVG antibody. Polyclonal Ab 1648 was made in rabbits to the 11 amino acid epitope of the VSVG and has been described previously (12).
Anti-actin antibody.
As an internal control for separation and efficiency of transfer in
Western analysis, use was made of a general anti-actin monoclonal
antibody (Sigma A-4700). This antibody is known to recognize the
-isoform of actin, which is present in the intestine.
SDS-PAGE and Western Analysis
The ileum and MBP/E3-C85 samples were resolved on 8-10% SDS-PAGE gels by using the mini protean II electrophoresis cell (Bio-Rad). The samples were run at 150 V for 1.25 h (running buffer was 14.4 g/l glycine, 3 g/l Tris, and 1 g/l SDS). The gel was electrotransferred onto nitrocellulose at 100 V for 1 h (transfer buffer was 200 ml/l methanol, 14.4 g/l glycine, and 3 g/l Tris). Then the blot was blocked using 5% nonfat dry milk in Tris-buffered saline (TBS; 150 mM NaCl and 13 mM Tris, pH 8.0) for 1 h at room temperature. After blocking, the blot was incubated with Ab 1380 in 1:1,000 dilution (in blocking solution) overnight at 4°C. For NHE2, we used Ab 597 or Ab 1797 in 1:1,000 dilution. After five 5-min washes with TBS plus 0.02% Triton X-100, the blot was incubated with secondary antibody (donkey anti-rabbit IgG horseradish peroxidase) in 1:8,000 dilution for 1 h at room temperature. Then the blot was again washed (five 5-min washes with washing buffer). The bound secondary antibody was detected by enhanced chemiluminescence (ECL; Renaissance, NEN). Simultaneously studied molecular mass markers (Bio-Rad prestained SDS-standard high range) were used on each gel. The nitrocellulose membranes were exposed to preflashed X-ray film.The developed X-ray films were scanned and densitometry was performed using ImageQuant software. To determine the absolute amount of NHE3 per milligram of ileal BB membrane, a dilution series of MBP/E3-C85 was loaded in the same gel as the ileal BB membrane samples of the control and methylprednisolone-treated animals. We have assumed that the affinities of Ab 1380 for NHE3 and for MBP/E3-C85 fusion protein are the same, since they have the identical epitope, i.e., the last 85 amino acids of NHE3. A standard curve of amount of fusion protein loaded vs. density of the bands was determined empirically. Concentrations of BB NHE3 were empirically determined to be within the linear range of the standard curve.
Materials
HOE-694 was a kind gift of Dr. H. J. Lang (Hoechst, Frankfurt, Germany). MPA was from Research Biochemicals (Natick, MA), 22Na+ was from DuPont NEN, and all other chemicals were from Sigma unless stated otherwise. ![]() |
RESULTS |
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Cell Model of PS120 Cells Coexpressing NHE2 and NHE3V
As a result of the double transfection (stable transfection of NHE3V into PS120/NHE2 cells), four clones were obtained that expressed both NHE2 and NHE3V, as shown by Western analysis (Fig. 1). As expected, all clones displayed a similar expression level for NHE2 (Fig. 1B) but differed widely in NHE3V expression (Fig. 1A). Clones A3 and B3, clone B4, and clone B1, displaying high, low, and intermediate NHE3 expression levels, respectively, were selected to perform subsequent transport experiments.
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As shown in Fig. 2, both NHE3V and NHE2 could be immunoprecipitated by their corresponding antibodies from crude membranes of PS120/NHE3V and PS120/NHE2 cells, respectively (lanes 2 and 3 and lanes 6 and 8, respectively, numbering from left). However, no NHE3 was detected in the NHE2 immunoprecipitate and vice versa (Fig. 2, lane 4 and lane 7, respectively). This suggested that NHE2 and NHE3V do not heterodimerize in vivo when coexpressed in the same cell.
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22Na+ Uptake Studies in PS120 Cells Demonstrating HOE-694 Sensitivity of NHE2, NHE3V, and NHE2 Plus NHE3V
Figure 3 shows the HOE-694 sensitivity curves for NHE2 and NHE3V expressed alone in PS120 cell lines. As expected, NHE2 is more sensitive to the inhibitor than NHE3V, showing an IC50 of 3 µM, similar to what was reported previously (5 µM) (16, 20). On the other hand, NHE3V was unaffected at HOE-694 concentrations up to 70-100 µM and was inhibited by ~50% at the solubility limit of HOE-694, i.e., ~1 mM. This is also in agreement with the reported NHE3 IC50 of 650 µM (16, 20). NHE3V was found to have an HOE-694 sensitivity curve identical to that of untagged NHE3 (not shown).
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Of the three PS120/NHE2-NHE3V clones assayed, only clone B4 showed an intermediate HOE-694 sensitivity between that of NHE2 and NHE3V (Fig. 3). More than 90% of Na+/H+ exchanger activity in clones A3, B1, and B3 was resistant to 100 µM HOE-694, resembling the resistance of wild-type NHE3. This presumably is due to NHE3 overexpression in these clones and probably resulted from the method of selection used (the presence of 100 µM HOE-694). A plateau of inhibition is apparent for clone B4 between 30 and 100 µM HOE-694. Because of the limits imposed by the solubility of the HOE-694, it was not possible to obtain a full biphasic sigmoidal curve, as would be expected from the hypothesis that NHE2 and NHE3 have distinct, nonoverlapping sensitivities to HOE-694. Nevertheless, the existence of a plateau in the concentration range in which NHE2 is completely inhibited while NHE3V is unaffected when expressed separately suggests that NHE2 and NHE3V expressed in the same cells have the same sensitivities to HOE-694 as when NHE2 and NHE3V are expressed individually in PS120 cells. Furthermore, if the percentage of Na+/H+ exchanger activity that remains at 30 µM HOE-694 is considered to be accounted for solely by NHE3, this amount of Na+/H+ exchange can be subtracted from all data points that include NHE2 to yield the contribution of NHE2 to total Na+/H+ exchange in PS120/NHE2-NHE3V cells. Figure 4 shows this amount of NHE2 activity in the absence of HOE-694 set to 100% and the NHE2 HOE-694 response plotted from 0.01 to 30 µM HOE-694. In Fig. 4, it is shown that HOE-694 sensitivity in PS120 cells containing only NHE2 is very similar (calculated Ki, 3 µM) to the NHE2 sensitivity to HOE-694 in cells containing NHE2 and NHE3 (calculated Ki, 5 µM). Although results with NHE3 are less complete due to the solubility limits of HOE-694, the concentration of HOE-694 that first inhibits NHE3 and the shape of the HOE-694 sensitivity curves are also similar when NHE3 is expressed alone in PS120 cells and when NHE2 and NHE3 are present together (Fig. 3). Thus NHE2 and NHE3 have similar sensitivities to HOE-694 whether NHE2 and NHE3 are both present in the same cell or each is present alone.
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NHE2 and NHE3 Contribute to Ileal BB Na+/H+ Exchange Under Basal Conditions
The contribution of NHE2 and NHE3 to ileal BB Na+/H+ exchange under basal and methylprednisolone-treated conditions was determined by use of HOE-694. BB Na+/H+ exchange was determined, as previously reported (8), as the difference in Na+ uptake into ileal BB vesicles in the presence of an acid intravesicular driving force (pH in 6.5, pH out 8.0) minus the uptake in the presence in the transport buffer of 200 µM MPA (MPA Ki for NHE2, 0.08 µM; for NHE3, 2.4 µM). Uptake was performed at 8 s, which is within the linear range of Na+ uptake in these vesicles (5, 8). The percent inhibition of Na+/H+ exchange caused by addition of varying concentrations of HOE-694 to the transport buffer was determined. As shown in Fig. 5, total basal ileal Na+/H+ exchange was inhibited in a concentration-dependent manner by HOE-694, and the curve had two components, closely resembling the HOE-694 concentration curve for PS120 cells containing NHE2 plus NHE3V shown in Fig. 3. One component started at ~1 µM HOE-694 and reached a maximum inhibition at 10-50 µM; 50% inhibition of this component occurred at ~3 µM HOE-694 (based on calculations with Enzfitter). This is interpreted as representing the contribution of NHE2. The second component began at concentrations >50 µM and reduced Na+/H+ exchange to only 10% of total at 1 mM HOE-694, which is interpreted as partial but not complete inhibition of NHE3. We did not use a higher concentration of HOE-694 due to inability to consistently dissolve higher concentrations.
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Total BB Na+/H+ exchange and that contributed by NHE2 and NHE3 are shown in Table 1. NHE2 was calculated as Na+/H+ exchange sensitive to 30 µM HOE-694 and determined as total Na+/H+ exchange minus Na+/H+ exchange in the presence of 30 µM HOE-694. NHE3 was calculated as Na+/H+ exchange remaining in the presence of 30 µM HOE-694. Under control conditions, 57 ± 5% of total BB Na+/H+ exchange was contributed by NHE2 and NHE3 contributed 43 ± 5% (Table 1).
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Glucocorticoid-Induced Increase in BB Na+/H+ Exchanger Is Due to NHE3
Total BB Na+/H+ exchange was measured, and the contributions made by NHE2 and NHE3 were determined from control and methylprednisolone-treated animals, as described above. In BB made from methylprednisolone-treated animals, BB Na+/H+ exchange was increased by 2.5-fold (Table 1). There was no significant change in the NHE2 activity (1.3 times control) with methylprednisolone treatment, although large variability could have caused us to miss a small increase. However, methylprednisolone treatment caused a significant increase (4.1 times control, P < 0.02) in NHE3 activity. In methylprednisolone-treated animals, NHE3 made up 69% of total BB Na+/H+ exchange and NHE2 contributed 31%. Studies were then performed to determine whether there were also changes in the amount of ileal BB NHE3.Ab 1380 Recognizes MBP/E3-C85 and GST/E3-C85
Quantitative Western analysis was used to measure the amount of BB NHE3. In this assay Ab 1380 was used, which was produced in rabbits by injection with affinity purified GST/E3-C85. Because Ab 1380 also recognized GST, attempts were made to remove antibodies recognizing GST but not NHE3. This was done by electrophoresis of 1 mg of GST protein and subsequently transferring the protein onto nitrocellulose. The GST protein was then identified by Ponceau S staining, and the nitrocellulose strip containing the protein was cut out. This strip was then blocked with 5% milk-TBS and was incubated with 1:50 dilution of Ab 1380 overnight at 4°C. However, this preabsorption step did not completely remove the antibody, which only recognized GST but not NHE3, as determined by Western blotting. Therefore, another fusion protein, MBP/E3-C85, was studied for potential use as an internal standard. As shown in Fig. 6A, MBP/E3-C85 was purified to homogeneity. The apparent molecular mass calculated from the mobility of MBP/E3-C85 is 48 kDa. This is very close to the calculated size of this protein based on the amino acid sequence (52.5 kDa). In addition, the relative efficiency with which Ab 1380 recognized both fusion proteins was compared by studying detection by Western analysis of various amounts of the fusion proteins with a single dilution of antibody. As shown in Fig. 6B, when studied on the same gel, Ab 1380 recognizes MBP/E3-C85 as a 48-kDa protein and GST/E3-C85 as a 36-kDa protein. Figure 6 also shows that Ab 1380, which was initially preabsorbed against GST before use for Western analysis, produces a larger signal to GST/E3-C85 than to MBP/E3-C85 even when signal is normalized per molecule of fusion protein transferred (note that molar ratio of the fusion proteins is 1.33). Because some of the signal to the GST/E3-C85 comes from antibody to the GST, all further studies to quantitate the amount of NHE3 were done using as internal standard known amounts of MBP/E3-C85. As a negative control, we compared MBP and MBP/E3-C85 on the same gel. Figure 6C shows that Ab 1380 does not recognize MBP.
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Rabbit Jejunal and Ileal BB NHE3 Is a 85-kDa Protein
To determine whether Ab 1380 recognizes NHE3 in rabbit ileal BB membranes, Western analysis was performed. As negative and positive controls, respectively, untransfected PS120 cells (a fibroblast cell line deficient in all endogenous NHEs) and PS120 cells stably transfected with NHE3V (PS120/NHE3V) were used, and they were compared with rabbit ileal and jejunal BB membranes. Figure 7 shows that Ab 1380 recognizes NHE3 as a 85-kDa protein in stably transfected PS120/NHE3 cells and in ileal and jejunal BB membranes. The untransfected PS120 cells did not show a protein of similar size. The similar size of NHE3 in PS120 cells and in rabbit small intestine suggests similar posttranslational modification in both cell types (12).
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Effect of Methylprednisolone Treatment on the Amount of NHE3 Present on the Ileal BB
Quantitative Western analysis to determine the amount of NHE3 present on the ileal BB was performed by using MBP/E3-C85 fusion protein as an internal standard, with control, methylprednisolone, and standard studied on the same gel (Fig. 8A). In this study it was assumed that Ab 1380 has the same affinity for the fusion protein MBP/E3-C85 as it has for NHE3 in the ileal BB. The amount of NHE3 present on the ileal BB was quantitated by densitometry, comparing the NHE3 ECL signal to that generated by applying known amounts of MBP/E3-C85 to the same gel. The standard curve was generated to yield a linear relationship between amounts of fusion protein separated by SDS-PAGE and magnitude of ECL signal. This was done by empirical repetition of the standard curve, with separation and transfer of different amounts of fusion protein, and correlation of the amount of MBP/E3-C85 with ECL signal.
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After separation of known amounts of MBP/E3-C85 fusion protein on 10% SDS-PAGE gels, the protein was transferred to nitrocellulose and Western analysis was performed with anti-NHE3 Ab 1380. The nitrocellulose membranes were then exposed to preflashed film and, on the basis of densitometric analysis, a linear relationship was obtained between fusion protein band density and the amount of fusion protein separated and transferred (Fig. 8B) for four concentrations of fusion protein (insufficient signal was generated by the lowest concentration). From this calibration curve (using the molecular mass of the MBP/E3-C85 fusion protein estimated from the SDS gel to calculate the picomoles of protein present, since the size of NHE3 used in these calculations was also estimated from the SDS gel), picomoles of NHE3 present on the ileal BB membrane could be determined by linear regression analysis. The average NHE3 for the control animals was 2.02 ± 0.05 pmol/mg BB membrane protein (0.018% of BB membrane protein was NHE3; n = 3; Table 2). Similar studies were carried out with ileal BB membrane of methylprednisolone-treated animals. For comparison, control and methylprednisolone-treated animals were studied on the same gel. In methylprednisolone-treated animals, the ileal BB NHE3 was 5.24 ± 0.04 pmol/mg BB membrane (0.042% of membrane protein was NHE3; n = 3, P < 0.02). Thus, as shown in Table 2, methylprednisolone treatment caused a 2.3-fold increase in the amount of NHE3 present on the ileal BB. The enrichment of the ileal BB, measured by performing sucrase-isomaltase assays on BB and homogenate, was 9.8 ± 4.3 times for the control and 7.9 ± 1.3 times for the methylprednisolone-treated animals (n = 3, not significant).
|
Effect of 24-h Treatment With Methylprednisolone on the Amount of NHE2 Protein Present in the Ileal BB
We have previously demonstrated that in rabbit the message for NHE2 is predominantly expressed in epithelial cells, especially in kidney and intestine. Initially two polyclonal antibodies were used to identify NHE2 in rabbit ileal BB by comparing signal detected in PS120 cells, PS120/NHE2 cells, and ileal BB. Both antibodies were raised in rabbits using the same fusion protein (GST/E2-C87). As shown in Fig. 9 and as we have reported (27), when NHE2 is expressed in PS120 cells, it is recognized as 85- and 75-kDa peptides. Also Ab 597 but not Ab 1797 recognizes a PS120 protein of 95 kDa (27). Both Ab 597 and Ab 1797 recognize rabbit ileal BB NHE2 as a 95-kDa protein. Ab 597 also recognizes a smaller amount of BB protein of 85 kDa; it is not known whether this represents NHE2 or a breakdown product of the 95-kDa protein. Thus ileal NHE2 is a 95-kDa protein. Because NHE2 expressed in PS120 cells is O-glycosylated (27), it is likely that the difference in the size of NHE2 observed in ileal BB and in PS120/NHE2 is due to differences in posttranslational modification, i.e., in amount of glycosylation. However, it is not yet known whether the 95- and 85-kDa BB proteins are the heteroglycosylated forms of rabbit ileal NHE2 or whether the 85-kDa protein is a proteolytic fragment of the 95-kDa protein.
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To determine the effect of 24 h treatment with methylprednisolone on the amount of NHE2 protein present on the ileal BB, Western analysis using Ab 597 (1:1,000 dilution) was performed in three paired sets of animals. As shown in Fig. 10, no significant change in the amount of NHE2 (95 kDa) occurred when the same amounts of BB protein from the control and methylprednisolone-treated animals were studied. Note that study of actin on the same Western analysis controlled for amount of protein separated and for transfer efficiency.
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DISCUSSION |
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Two new approaches were developed and used to study the contribution of NHE2 and NHE3 to basal ileal BB Na+/H+ exchange and to the increase in Na+/H+ exchange caused by glucocorticoid treatment: 1) functional separation of the contribution of NHE2 and NHE3 to BB Na+/H+ exchange activity by use of HOE-694 and 2) quantitation of the amount of BB NHE3 by quantitative Western analysis.
The separate expression of NHE2 and NHE3 in cell lines having no endogenous Na+/H+ exchanger has made possible the characterization of their pharmacological profiles, including their sensitivity to the amiloride analog HOE-694. Unlike amiloride, HOE-694 is capable of discriminating between NHE2 and NHE3, based on the considerable differences in the IC50 values (5 µM and 650 µM at 1 mM Na+, respectively). Because NHE2 and NHE3 often are present in the same plasma membrane domain in vivo, the use of HOE-694 constitutes a powerful tool to dissect the respective contribution of both isoforms to Na+/H+ exchange. Nevertheless, there was the possibility that interactions occurred between NHE2 and NHE3, which might have invalidated this approach. Hence, we set out to verify whether any interaction, first structurally and then functionally, could be detected by coexpressing the transporters in PS120 cells. First, we comment on structural interactions. The NHEs are known to exist as multimers, at least as homodimers (9). Immunoprecipitating NHE1, NHE2, and NHE3 and separating the immunoprecipitates with SDS-PAGE reveal proteins of the expected NHE molecular mass but also proteins of approximately double that size (9, 12). Also, studies with NHE1 using a truncated and VSVG epitope-tagged mutant NHE1 were used to show that NHE1 existed as a dimer in PS120/NHE1 cells (9). Although NHE1 was shown not to heterodimerize with NHE3, it was not known whether the more functionally similar epithelial isoforms NHE2 and NHE3 did heterodimerize. In this study, using similar approaches to those initially used to show that NHE1 homodimerized, we show that NHE2 and NHE3 expressed in the same PS120 cells failed to coprecipitate each other (Fig. 2).
That NHE2 and NHE3 also function independently and that the presence of one isoform does not influence the sensitivity of the other epithelial isoform to HOE-694 was demonstrated by showing that PS120 cells expressing NHE2 and NHE3V have a HOE-694 sensitivity curve that is intermediate between those of PS120/NHE2 and PS120/NHE3 and that a plateau is apparent between 30 and 100 µM inhibitor, i.e., the concentration range in which NHE2 is downregulated by at least ~90% and NHE3 is largely unaffected (Fig. 3). Furthermore, examining the contribution of NHE2 to total Na+/H+ exchange in the cells that contain both NHE2 and NHE3V gave a sigmoidal response curve to HOE-694 that was not distinguishable from the concentration-dependent curve obtained for NHE2 studied alone (Fig. 4). Although our results do not support the existence of a direct association between NHE2 and NHE3, we cannot rule out this possibility. For instance, it is conceivable that the solubilization conditions disrupted the interaction or that the antibodies competed for the interaction site. Nevertheless, we believe this is unlikely because 1) two different antibodies recognizing different epitopes were used and 2) no interaction was apparent at the pharmacological level.
In addition, the HOE-694 concentration-dependent inhibition of ileal BB vesicle Na+/H+ exchange closely duplicated the response in PS120 cells stably transfected with NHE2-NHE3V (compare Figs. 3 and 5). As in the PS120 cells, in the presence of 1 mM Na+, the HOE-694 inhibition curve of ileal BB Na+/H+ exchange had two components, at least the first being sigmoidal. The concentration-dependent inhibition curve had a plateau at 10-50 µM, and this component of the curve had a Ki of 3 µM, which is consistent with the contribution of NHE2. The second component was sensitive to HOE-694 starting at 100 µM, and 50% of this residual Na+/H+ exchange activity was inhibited by ~650 µM HOE-694, consistent with a contribution of NHE3. Thus we suggest that, both in cell culture models containing both NHE2 and NHE3 and in ileal BB that contains both NHE2 and NHE3 at the apical surface, 30 µM HOE-694 in the presence of 1 mM Na+ can be used to separate functionally the contributions of NHE2 and NHE3 to total BB Na+/H+ exchange.
This pharmacological approach was used to determine the contribution of NHE2 and NHE3 to basal and glucocorticoid-stimulated Na+/H+ exchange in BB of rabbit ileum. BB from Na+-absorbing cells from human, rabbit, and rat jejunal and ileal villus and human and rat ascending and descending colon and rabbit ascending but not descending colonic surface cells contain NHE2 and NHE3, based on immunocytochemistry (12). However, it was previously assumed that all small intestinal BB Na+/H+ exchange was due to NHE3, since 1) the small intestinal BB Na+/H+ exchanger is relatively resistant to amiloride, which is most similar to NHE3 (NHE3 has a Ki amiloride of ~39-100 µM), whereas NHE2 is similarly sensitive to amiloride as is NHE1 (Ki 1-3 µM) (28, 30, 33); 2) the small intestinal BB Na+/H+ exchanger is inhibited by phorbol esters, which is also true of NHE3, whereas phorbol esters stimulate NHE2 (33); 3) in dog ileum, concentrations of amiloride and MPA that inhibit NHE2 and NHE1 but not NHE3 do not alter basal Na+ absorption or the postprandial increase in ileal water and Na+ absorption, whereas concentrations that also block NHE3 block basal intestinal Na+ absorption and the postprandial increase in ileal absorption (17, 18). Nonetheless, until now, no studies have attempted to specifically assess the contribution to rabbit ileal BB Na+/H+ exchange of NHE2 and NHE3. The results using the HOE-694 approach are somewhat surprising. Both NHE2 and NHE3 contribute approximately equally to basal rabbit ileal BB Na+/H+ exchange. This preparation of BB is not enriched in the basolateral membrane marker Na+-K+-ATPase (1.0-1.7 times) (5), but there is some basolateral membrane present. Could we be including NHE1 in the component of BB Na+/H+ exchange attributed to NHE2? NHE1 would be expected to be almost entirely inhibited at 1 µM HOE-694 (Ki 0.15 µM), and 1 µM HOE-694 only inhibited 30% of the component of Na+/H+ exchange thought to be NHE2. Thus NHE2 significantly contributes to basal Na+/H+ exchange. Thus, unlike dog ileum (17, 18), basal rabbit ileal BB Na+/H+ exchange is made up of approximately equal contributions of NHE2 and NHE3, indicating species differences in the contribution of NHE2 and NHE3 to basal ileal BB Na+/H+ exchange. A similar approach using HOE-694 was recently reported in rat kidney cortex, in which all BB Na+/H+ exchange was found to be due to NHE3 (31).
In contrast to roles for both NHE2 and NHE3 in basal ileal Na+/H+ exchange, only an increase in NHE3 contributed to the increase in ileal BB Na+/H+ exchange caused by methylprednisolone. In this study, ileum from methylprednisolone-treated animals had a 2.5-fold increase in BB Na+/H+ exchange, as we previously reported (32). Again, based on sensitivity to HOE-694, this increase in BB Na+/H+ exchange was entirely due to an increase in NHE3 activity (NHE3 activity increased by 4.1 times), with no significant change in Na+/H+ exchange by NHE2 (see Table 1). This is consistent with our previous demonstration that methylprednisolone treatment increased mRNA for rabbit ileal NHE3 but not for NHE2 or NHE1 (32).
Western analysis demonstrated that methylprednisolone increased the amount of BB NHE3. However, to determine the magnitude of the increase, a quantitative technique had to be developed to determine the amount of BB NHE3. The technique developed was quantitative Western analysis. The requirements for a quantitative Western analysis for NHE3 are 1) an antibody that specifically recognizes NHE3, 2) an internal standard that is recognized by the anti-NHE3 antibody with the same affinity as it recognizes NHE3, 3) a linear relationship between the amount of standard studied and signal generated, and 4) BB NHE3 studied in the linear part of the curve.
The quantitative Western technique we used is based on using a fusion protein as a standard that contains the epitope to which the anti-NHE3 polyclonal antibody was raised. In this case, anti-NHE3 antibody was developed using a GST fusion protein, whereas quantitation was done with a MBP fusion protein; both fusion proteins contained the last 85 COOH-terminal amino acids of NHE3. The rationale was that some of the polyclonal antibodies produced were directed against the GST and that even preabsorption against the GST would not give a true representation of the amount of NHE3. We made the assumption in quantitating the amount of NHE3 that Ab 1380 has the same affinity for NHE3 as it has for the MBP/E3-C85 fusion protein. Although this seems reasonable since both NHE3 and MBP/E3-C85 contain the identical epitope for the antibody, it remains an assumption. Note that NHE3 in ileal BB contains these same COOH-terminal amino acids. This is based on the recognition by anti-VSVG antibody of a COOH-terminal epitope tag in NHE3V cells and the fact that ileal BB NHE3 and NHE3V from PS120 cells are indistinguishable in size.
We quantitated the amount of NHE3 present on the ileal BB by Western analysis by comparing the density of the bands of a series of dilutions of known amounts of the fusion protein MBP/E3-C85 with the density of the bands of NHE3 protein in the control and the methylprednisolone-treated ileal BB. The amount of NHE3 present on the ileal BB under basal conditions was ~0.02% of total ileal BB membrane protein. This is less than for another characterized BB membrane transport protein. In rabbit jejunum, the Na+-glucose cotransporter SGLT1 makes up 0.3% of BB protein (E. Wright, personal communication).
By determining the amount of ileal BB NHE3, we were able to calculate
an approximate turnover number for NHE3, based on the following
formula: Vmax of
Na+/H+
exchange (in pmol · mg
protein1 · s
1) = turnover number (in
cycles · s
1 · NHE3
molecule
1) × amount of active NHE3 in the BB (in pmol/mg
protein).
This calculation used a literature value for
Vmax for BB
Na+/H+
exchange in the same tissue (rabbit ileum) prepared by techniques similar to those we used in this study (16). NHE3
Vmax was 924.5 pmol · mg
protein1 · s
1,
since the Vmax
for basal
Na+/H+
exchange in rabbit ileum was 2,150 pmol · mg
protein
1 · s
1
(16), of which 43% is NHE3 under basal conditions. The amount of NHE3
in ileal BB was 2.02 pmol/mg protein (Table 2). Therefore, the turnover
number for NHE3 is 458 cycles · s
1 · NHE3
molecule
1 (2,150 × 0.43 / 2.02), with the assumption that all NHE3 molecules detected
by Western analysis are active. The calculated turnover number of 458 cycles · s
1 · NHE3
molecule
1 is similar in
magnitude to those of other cotransporters and exchangers previously
described, including a glucose transporter in human erythrocytes (1,000 s
1) and multiple
transporters in which the turnover number was calculated by electrical
methods, including a H+/amino
transporter from the plant Arabidopsis
thaliana with an estimated turnover
number of 350-800 s
1,
H+/peptide cotransporter from
rabbit intestine (110 s
1),
Na+-glucose cotransporter SGLT1
from human intestine (60 s
1), and
Na+-K+-2Cl
cotransporter from mouse medullary thick ascending limb cells (255 s
1) (1, 7, 10, 11, 13).
In contrast, one group has reported that the turnover number for
mammalian NHE1 ranges from 2,000 to 7,500 s
1 in rat thymic
lymphocytes and lymphoblasts, respectively (19, 22-24).
Results from this study are also relevant to the way methylprednisolone increases the amount of NHE3. Methylprednisolone treatment increases the amount of NHE3 protein present on the ileal BB by 2.6-fold. This differs from our findings that the mRNA level (standardized to other NHE isoforms, NHE1 and NHE2, which did not change with methylprednisolone treatment) increases four- to sixfold 24 h after the methylprednisolone injection. There are multiple possible explanations for this discrepancy, including that not all the mRNA gets translated into protein or that all the mRNA gets translated into protein but that only a part of the increased NHE3 is present on the BB and that a large amount of the NHE3 protein is present in a cytoplasmic pool, perhaps available for movement to the ileal BB as a part of kinase regulation. The actual explanation is unknown.
The results of these studies can be used to suggest whether the Vmax stimulation of NHE3 by glucocorticoids is entirely due to more BB protein or due to a change in turnover number of each exchanger. The fact that the increase in rate of ileal BB Na+/H+ exchange due to NHE3 (4.1 times) was greater than the methylprednisolone-induced increase in amount of BB NHE3 (2.6 times) (with no change in amount of NHE2) leads to the conclusion that the increase in NHE3 Vmax is due both to an increase in the number of BB Na+/H+ exchangers and, to a lesser extent, to an increase in the turnover number of each BB exchanger. A very crude calculation can be made of the turnover number of NHE3 in BB from methylprednisolone-treated ileum by assuming that the increase in Vmax determined here, 2.5 times, can be extrapolated from the Vmax determination used in calculation of basal ileal BB Na+/H+ exchange (16). The calculated turnover number of NHE3 in BB from methylprednisolone-treated animals would then be estimated to be 708 cycles/s (2,150 × 2.5 × 0.69 / 5.24). These data are consistent with the methylprednisolone treatment increasing both the turnover number and amount of BB NHE3. Of relevance, it was recently reported that 5 days of metabolic acidosis increased the amount of NHE3 in rat renal cortical BB by approximately threefold (31).
We found that methylprednisolone treatment had no effect on the amount of NHE2 protein present on the ileal BB. This corresponds with the finding of Yun et al. (32) that the NHE2 mRNA is not affected by methylprednisolone treatment. Thus NHE2 appears to contribute to basal ileal Na+/H+ exchange, but any further role as a "reserve" system in stimulated small intestinal absorption has not been identified.
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ACKNOWLEDGEMENTS |
---|
We thank Helen McCann for assistance in manuscript preparation and editorial assistance.
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FOOTNOTES |
---|
This work was partially supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1-DK-26523, PO1-DK-44484, R29-DK-43778, and T32-DK-0763205 and the Meyerhoff Digestive Diseases Center.
Address for reprint requests: M. Donowitz, Johns Hopkins University School of Medicine, G.I. Division, 925 Ross Research Bldg., 720 Rutland Ave., Baltimore, MD 21205-2195.
Received 1 April 1997; accepted in final form 27 January 1998.
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