Effect of osmolarity on LDL binding and internalization in hepatocytes

Annette Krämer-Guth1, Gillian L. Busch3, Nubia Kristen Kaba3, Susanne Schwedler2, Christoph Wanner2, and Florian Lang3

1 Department of Internal Medicine, University of Freiburg, D-79140 Freiburg; 2 Department of Internal Medicine, University of Würzburg, D-97070 Würzburg; and 3 Institute of Physiology, University of Tübingen, D-72076 Tübingen, Germany

    ABSTRACT
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Abstract
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Methods
Results
Discussion
References

The present study has been performed to elucidate a possible role of cell volume in low-density lipoprotein (LDL) binding and internalization (LDLb+i). As shown previously, increase of extracellular osmolarity (OSMe) and K+ depletion, both known to shrink cells, interfere with the formation of clathrin-coated pits and thus with LDLb+i. On the other hand, alterations of cell volume have been shown to modify lysosomal pH, which is a determinant of LDLb+i. LDLb+i have been estimated from heparin-releasable (binding) or heparin-insensitive (internalization) uptake of 125I-labeled LDL. OSMe was modified by alterations of extracellular concentrations of ions, glucose, urea, or raffinose. When OSMe was altered by varying NaCl concentrations, LDLb+i decreased (by 0.5 ± 0.1%/mM) with increasing OSMe and LDLb+i increased (by 1.2 ± 0.1%/mM) with decreasing OSMe, an effect mainly due to altered affinity; the estimated dissociation constant amounted to 20.6, 48.6, and 131.6 µg/ml at 219, 293, and 435 mosM, respectively. A 25% increase of OSMe increased cytosolic (by 0.46 ± 0.03) and decreased lysosomal (by 0.14 ± 0.02) pH. Conversely, a 25% decrease of OSMe decreased cytosolic (by 0.28 ± 0.02) and increased lysosomal (by 0.17 ± 0.02) pH. Partial replacement of extracellular Na+ with K+ had little effect on LDLb+i, although it swelled hepatocytes and increased lysosomal and cytosolic pH. Hypertonic glucose, urea, or raffinose did not exert similar effects despite a shrinking effect of hypertonic raffinose. Monensin, which completely dissipates lysosomal acidity, virtually abolished LDLb+i. In conclusion, the observations reveal a significant effect of ionic strength on LDLb+i. The effect is, however, not likely to be mediated by alterations of cell volume or alterations of lysosomal pH.

receptors; monensin; lysosomes; cell volume; ionic strength; low-density lipoprotein

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

AS SHOWN PREVIOUSLY, CELL swelling leads to alkalinization of acidic cellular compartments, whereas cell shrinkage enhances the acidity in those compartments (9, 27, 36, 37). The effect is mediated by the microtubule network (8) and probably is due to an H+ leak (7). Accordingly, any pH-dependent function of these compartments may be expected to be modified by alterations of cell volume. Among the functions sensitive to lysosomal pH is receptor recycling. Lysosomal acidification has been described to be a prerequisite for normal recycling of low-density lipoprotein (LDL) receptors (3) and transferrin receptors (12). On the other hand, an increased extracellular osmolarity, which shrinks cells and decreases lysosomal pH, has been reported to inhibit the formation of coated pits and internalization of LDL and transferrin receptors (11, 19, 23, 32). Moreover, K+ depletion, which may similarly be expected to shrink cells, has been shown to decrease receptor abundance (19, 28, 29).

The present study was conducted to test for an effect of altered extracellular osmolarity and KCl concentration on lysosomal pH, cell volume, and receptor binding and internalization to test for a correlation of cell volume and lysosomal pH with receptor recycling.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cell culture. Hep G2 cells were grown in Dulbecco's modified Eagle's medium (DMEM; GIBCO, Eggenstein, Germany) supplemented with 10% fetal calf serum (FCS; GIBCO), 15 µM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 100 U/ml penicillin-10 ng/ml streptomycin at 37°C in a humidified 5% CO2-95% O2 atmosphere.

Fluorescence measurements. Calcein fluorescent intensity has been utilized for determination of cell volume changes. To this end, cells were loaded by exposure to 2 µM calcein-acetoxymethyl ester (AM; Molecular Probes, Eugene, OR) for 15 min. Calcein is insensitive to changes in intracellular pH and Ca2+ (1). Furthermore, swelling or shrinkage of cells is accompanied by a decrease or increase, respectively, in dye concentration in the cell (34). Therefore, changes in cell volume are expected to be reflected by changes in the fluorescent intensity, with decreased intensity during cell swelling and increased intensity during cell shrinkage. A linear relationship between osmolarity of the perfusing medium and fluorescent intensity has been demonstrated with this technique. The relationship is linear because dye concentration is inversely proportional to cell volume and cell volume is inversely proportional to external osmolarity for cells that exhibit osmometric behavior. Fluorescence was measured with a ×100 oil immersion lens (Zeiss). Fluorescence in the absence of fura 2 or calcein was <1% of the values in the presence of the dyes and was not significantly modified by the experimental maneuvers.

For determination of lysosomal pH, Hep G2 cells were incubated for 2 h before experiments with 70 µM fluorescein isothiocyanate (FITC)-dextran (Sigma Chemical, Deisenhofen, Germany) in extracellular fluid containing (in µM) 115 NaCl, 21 NaHCO3, 5 KCl, 1.3 CaCl2, 1 MgCl2, and 2 NaH2PO4, equilibrated to pH 7.4 by gassing with O2-CO2 (19:1), and maintained at 37°C. For determination of FITC-dextran fluorescence, light alternating between 480 and 440 nm from a monochromator light source (Uhl, Munich, Germany) was directed through gray filters (nominal transmission 0.7%; Oriel, Darmstadt, Germany) and was deflected by a dichroic mirror (515 nm; Omega Optical, Brattleboro, VT) into the microscope objective (Plan-Neofluar ×40, Zeiss). Emitted fluorescence was directed through a 520-nm cutoff filter to a photomultiplier tube (213-IP28A, Seefelder Messtechnik, Seefeld, Germany). To decrease the size of the region from which the fluorescence was collected, a plate with a pinhole (diameter of 1.6 mm) was placed in the image plane of the phototube. Fluorescence in the absence of FITC-dextran was <1% of that in the presence of the dye. Data acquisition was executed with a computer program (IMG 8, Lindemann & Meiser, Homburg, Germany). Specific vesicular pH was calculated after calibration of the dye (35). Briefly, cells were superfused with solutions containing KCl (105 mM), MgCl2 (1 mM), Na2HPO4 / NaH2PO4 (6 mM, with pH ranging from 5 to 6.5), and nigericin (10 µM). The fluorescence ratios at 480:440 were linear with vesicular pH between 5 and 6.5.

For cytosolic pH measurements, cells were incubated for 30 min with 1 µM 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM; Molecular Probes), followed by a 30-min incubation in medium without dye. The same microspectrophotometry system was utilized as described for FITC-dextran measurements, with minor alterations. Specifically, excitation was alternated between wavelengths of 485 and 440 nm, and a dichroic mirror with 535-nm cutoff (FT-535, Omega) and two emission filters of 521 nm (Ramon band pass, Omega) and 515 nm (long pass, Schott, Mainz, Germany) were used. Cytosolic pH calibration was performed according to the method of Thomas et al. (35). Briefly, cells were superfused with solutions containing KCl (105 mM), MgCl2 (1 mM), nigericin (10 µM), and HEPES (30 mM, pH varied between 6.5 and 7.5). The 485-to-440 nm fluorescence ratio was linear with cytosolic pH between 6.5 and 7.5. To reduce the region from which fluorescence was collected, a plate with a pinhole (diameter of 1.5 mm) was placed in the image plane of the phototube. Data acquisition was executed with a computer program (IMG 8, Lindemann & Meiser). Fluorescence in the absence of fluorescent dyes was <1% of the values in the presence of the dyes and was not significantly modified by the experimental maneuvers.

Experimental solutions. Extracellular bath solution was composed of a 1:1 mixture of DMEM with either 45 mM NaCl (hypotonic, 219 mosM), 125 mM NaCl (isotonic, 293 mosM), 275 mM NaCl (hypertonic, 435 mosM), or 125 mM KCl (high KCl, 292 mosM).

Solutions with varying osmolarities (see Figs. 3 and 4) were obtained by a 1:1 mixture of DMEM with a solution of 50-300 mmol/l urea, glucose, raffinose, NaCl, or KCl, as indicated.

Lipoprotein isolation and separation. Blood from healthy donors was drawn into tubes containing 10% sodium citrate after a 12-h overnight fast. The lipoproteins were isolated by sequential ultracentrifugation (21). After separation, LDL was dialyzed against 5 mM tris(hydroxymethyl)aminomethane (Tris) · HCl (pH 7.4), 154 mM NaCl, and 250 mM EDTA and sterilized by passage through a 0.45-µm Millipore filter.

Preparation of lipoprotein-deficient serum. Human plasma was rendered lipoprotein deficient by a two-step ultracentrifugation procedure (150,000 g for 48 h at 10°C), adjusting the plasma to a density of 1.250 g/ml by the addition of solid KBr. After all lipoprotein fractions were removed, the lipoprotein-deficient serum (LDS) was dialyzed against 154 mM NaCl, 250 mM EDTA, and 5 mM HEPES (pH 7.4), heat inactivated at 54°C for 1 h, and sterilized by passage through a 0.45-µm Millipore filter. The protein content was adjusted to 40 mg/ml.

Radioiodination of LDL. Radioiodination of LDL was performed by the method of McFarlane (30) as modified for lipoproteins by Bilheimer et al. (5). For each milligram of lipoprotein, 100 µl glycine buffer (1 M, pH 10), 25 mCi [125I]sodium iodide (Amersham Buchler, Braunschweig, Germany), and 30 µl iodine monochloride (10 µM) were added. Most unbound iodine was removed by passage through a Sephadex G-25 column and by dialyzing against buffer containing 154 mM NaCl-250 mM EDTA (pH 7.4). The final preparation of 125I-labeled LDL was sterilized by passage through a 0.45-µm Millipore filter, and protein content was estimated with the Lowry method.

Assay for binding, internalization, and degradation of lipoproteins. To induce apolipoprotein B and E receptor activity, Hep G2 cells were incubated in medium that contained 10% LDS instead of FCS. After a 48-h incubation, monolayers were washed with phosphate-buffered saline and fresh LDS-containing medium was added together with 125I-LDL with or without a 25-fold excess of unlabeled lipoproteins. The cultures were incubated for 2 h at 37°C, and assays for binding and internalization were performed by standard techniques (16). Care was taken to conduct the experiments at constant temperature, since, in single experiments, the ratio of binding over internalization was observed to increase with decreasing temperature. The medium was removed, and the cell suspensions were placed on ice. The culture dishes were then washed five times with cold buffer containing 154 mM NaCl, 50 mM Tris (pH 7.4), and 2 mg/ml bovine fatty acid-free serum albumin, as described by Goldstein et al. (16). The heparin-releasable activity represents binding at 37°C. After the release of cell surface bound activity by heparin, monolayers were dissolved in 1 ml of 0.1 N NaOH, and radioactivity and protein binding and internalization were measured. The radioactivity in the pellet was taken as a measure for internalization. Nonspecific binding and internalization was defined as the amount of lipoprotein taken up in the presence of a 25-fold excess of unlabeled ligand. All experiments were performed in triplicate.

Calculations. Data are expressed as arithmetic means ± SE. Statistical analysis was made by paired or unpaired t-test, where applicable. Statistically significant differences were assumed when P < 0.05.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Cell volume. As evidenced from calcein fluorescence (Fig. 1), a 25% decrease of extracellular osmolarity significantly decreased calcein fluorescent intensity in hepatocytes by 12 ± 1% (n = 7) and a 25% increase of extracellular osmolarity significantly increased it by 12 ± 2% (n = 7). An increase of extracellular K+ concentration at the expense of extracellular NaCl concentration decreased calcein fluorescent intensity significantly by 15 ± 3% (n = 5).


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Fig. 1.   Effect of a 25% decrease (hypotonic, A), a 25% increase (hypertonic, A), and replacement of KCl for NaCl (high K+, B) on cell volume as evidenced from calcein fluorescence. Calcein fluorescence is given in arbitrary units. Original tracings are representative of at least 4 similar experiments.

Cytosolic pH. The mean cytosolic pH of hepatocytes bathed in isotonic extracellular fluid was 7.06 ± 0.06 (n = 18), as shown with BCECF fluorescence (Fig. 2). A 25% decrease of extracellular osmolarity decreased cytosolic pH significantly by 0.28 ± 0.02 pH units (n = 7), and a 25% increase of extracellular osmolarity increased cytosolic pH significantly by 0.46 ± 0.03 pH units (n = 5). An increase of extracellular K+ concentration increased cytosolic pH significantly by 0.16 ± 0.05 (n = 6).


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Fig. 2.   Effect of a 25% decrease (hypotonic, A), a 25% increase (hypertonic, B), and replacement of KCl for NaCl (high K+, C) on cytosolic pH as evidenced from 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) fluorescence. Values for cytosolic pH have been obtained according to the calibration procedure described in METHODS. Original tracings are representative of at least 5 similar experiments.

Lysosomal pH. The mean lysosomal pH of hepatocytes bathed in isotonic extracellular fluid was 4.75 ± 0.07 (n = 16), as shown with FITC-dextran fluorescence (Fig. 3). A 25% decrease of extracellular osmolarity increased lysosomal pH significantly by 0.17 ± 0.02 pH units (n = 6), and a 25% increase of extracellular osmolarity decreased lysosomal pH significantly by 0.14 ± 0.02 pH units (n = 4). An increase of extracellular K+ concentration increased lysosomal pH significantly by 0.05 ± 0.01 (n = 6).


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Fig. 3.   Effect of a 25% decrease (hypotonic, A), a 25% increase (hypertonic, B), and replacement of KCl for NaCl (high K+, C) on lysosomal pH as evidenced from fluorescein isothiocyanate fluorescence. Values for lysosomal pH have been obtained according to the calibration procedure described in METHODS. Original tracings are representative of at least 4 similar experiments.

Binding and internalization of 125I-LDL. As shown in Fig. 4, binding and internalization of LDL increased with the 125I-LDL concentration in the medium. As shown previously (26), half-maximal saturation is obtained at ~30-40 µg protein/ml medium. Therefore, the maximal concentration chosen in this study was 50 µg protein/ml medium to exceed maximal saturation. Monensin (10 µM) significantly reduced binding and internalization of LDL. Monensin inhibited LDL binding and internalization irrespective of extracellular osmolarity (Fig. 5).


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Fig. 4.   Influence of monensin on 125I-labeled low-density lipoprotein (LDL) binding (A) and internalization (B). Binding and internalization of 125I-LDL are plotted as a function of 125I-LDL concentration in the presence (black-square) or absence (bullet ) of 10 µM monensin. Arithmetic means ± SE are given; bars are missing if data point is larger than SE. * Significant difference (P < 0.05) between presence and absence of monensin.


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Fig. 5.   Effect of varying osmolarity due to NaCl on 125I-LDL binding (A) and internalization (B) with or without presence of monensin (10 µM). At all osmolarities tested [isotonic = 293 mosM (open bars), hypertonic = 435 mosM (hatched bars), hypotonic = 219 mosM (crosshatched bars)], monensin decreases the binding and internalization of 125I-LDL. Arithmetic means ± SE are given. * Significant difference (P < 0.05) between hypertonic vs. isotonic solution or hypotonic vs. isotonic solution.

Binding and internalization of 125I-LDL were enhanced in hypotonic and decreased in hypertonic extracellular fluid, if extracellular osmolarity was modified by variations of either NaCl or KCl (Fig. 6). A Scatchard plot analysis yields that for binding maximal velocity (Vmax) was 185, 171, and 131 ng/mg cell protein and the dissociation constant (Kd) was 20.8, 33.3, and 91.6 µg/ml medium for hypotonic, isotonic, and hypertonic NaCl, respectively. For internalization, the respective values were 1,665, 1,364, and 917 ng/mg cell protein for Vmax and 20.6, 48.6, and 131.6 ng/ml medium for Kd. Thus alterations of extracellular NaCl concentrations mainly modified the affinity of LDL binding and internalization.


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Fig. 6.   Effect of osmolarity on 125I-LDL binding (A) and internalization (B). Binding and internalization of 125I-LDL are plotted as a function of 125I-LDL concentration in isotonic (293 mosM, black-square), hypotonic (219 mosM, bullet ), and hypertonic (435 mosM, black-triangle) media. Arithmetic means ± SE are given; bars are missing if data point is larger than SE. * Significant difference (P < 0.05) between hypertonic vs. isotonic solution or hypotonic vs. isotonic solution.

Partial replacement of extracellular NaCl by KCl (high-K+ concentration) slightly decreased binding and internalization of 125I-LDL, a difference that reached statistical significance only in one of five (Fig. 7) or one of six (Fig. 8) concentrations. The difference remained small irrespective of whether the replacement was made in isotonic (Fig. 7) or anisosmotic (Fig. 8) extracellular fluid.


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Fig. 7.   Effect of KCl on 125I-LDL binding (A) and internalization (B). Binding and internalization of 125I-LDL are plotted as a function of 125I-LDL concentration in isotonic NaCl (bullet ) or KCl (black-square) medium. Arithmetic means ± SE are given; bars are missing if data point is larger than SE. * Significant difference (P < 0.05) between KCl vs. NaCl.


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Fig. 8.   Effects of NaCl and KCl of varying osmolarity on 125I-LDL binding (A) and internalization (B). Binding and internalization of 10 µg/ml 125I-LDL are plotted as a function of medium osmolarity due to different concentrations of NaCl (bullet ) or KCl (black-square) medium. Arithmetic means ± SE are given; bars are missing if data point is larger than SE. * Significant difference (P < 0.05) between KCl vs. NaCl.

The effects from alterations of extracellular osmolarity using different concentrations of urea, glucose, or raffinose on binding and internalization of 125I-LDL were not similar to the effects from different NaCl or KCl concentrations (Fig. 9). Neither substance altered binding at any concentration applied. Internalization was slightly but significantly decreased by an increase of raffinose concentration.


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Fig. 9.   Effects of nonelectrolytes of varying osmolarity on 125I-LDL binding (A) and internalization (B). Binding and internalization of 10 µg/ml 125I-LDL are plotted as a function of medium osmolarity due to different concentrations of urea (square ), glucose (triangle ), and raffinose (star ). Arithmetic means ± SE are given; bars are missing if data point is larger than SE. * Significant difference (P < 0.05) between hypertonic vs. isotonic solution.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Cell shrinkage increases and cell swelling decreases cytosolic pH, as reported earlier (10, 15, 36). The cytosolic acidification is thought to be secondary to exit of HCO<SUP>−</SUP><SUB>3</SUB> through anion channels (38), by release of H+ from acidic intracellular compartments (see below) and by enhancement of Cl-/HCO<SUP>−</SUP><SUB>3</SUB> exchange due to decreasing cellular Cl- activity. The alkalinization following cell shrinkage is in large part due to activation of Na+/H+ exchange (13, 25). The present observations further confirm previous reports on the influence of cell volume on lysosomal pH, which increases with cell swelling and decreases with cell shrinkage (9, 27, 36, 37). The alkalinization following cell swelling is secondary to an H+ leak (7).

The data do not support the hypothesis that the changes of LDL binding and internalization following cell swelling or cell shrinkage are secondary to lysosomal pH changes. Whereas monensin led to the expected decrease of LDL binding and internalization, a decrease of extracellular osmolarity increased LDL binding and internalization despite concomitant cell swelling and lysosomal alkalinization. Partial replacement of extracellular Na+ with K+, a maneuver that swells the cells and alkalinizes the lysosomes, rather decreased LDL binding and internalization. This small effect may be secondary to lysosomal alkalinization but does not explain the marked opposite effect of decreased extracellular osmolarity. Obviously, cell volume is not an important determinant of LDL binding and internalization. Although an increase of lysosomal pH by addition of monensin effectively inhibited binding as shown previously for LDL (3) and transferrin (12), the alkalinization during cell swelling is obviously not strong enough to significantly interfere with receptor trafficking. Changes in cytosolic pH, similar to lysosomal pH and cell volume, similarly cannot explain the changes of LDL binding and internalization during alterations of extracellular osmolarity. Cytosolic acidosis has been shown to interfere with endocytosis from coated pits (19, 22, 33). A decrease of extracellular osmolarity increased LDL binding and internalization despite parallel cytosolic acidosis.

Extracellular ionic strength appears to be a decisive parameter determining LDL binding and internalization. The receptor binding experiments confirm previous observations on the effect of osmolarity on LDL and transferrin binding (11, 19, 23, 32). As shown in this study, NaCl and KCl are similarly effective, whereas nonelectrolytes such as urea, glucose, and raffinose do not mimic the effects of NaCl and KCl. Ionic strength mainly modifies the affinity of LDL binding. Because alterations of ionic strength lead to proportional alterations of LDL binding and internalization, the altered binding of LDL subsequently affects recycling. However, lipoprotein binding kinetics do not serve as a reliable model to estimate the amount of receptor recycling according to the amount of radioactivity found in the sample or pellet.

The formation of coated pits and internalization of LDL and transferrin receptors are inhibited not only by an increase of extracellular osmolarity (11, 19, 23, 32) but also by K+ depletion (19, 28, 29). Although the effect of osmolarity is explained by the altered ionic strength, the reason for the interference of K+ depletion with the formation of coated pits (29) remains unexplained.

The liver, unlike the kidney medulla, is not usually exposed to marked alterations of extracellular ionic strength, which is thus not a major determinant of hepatocyte LDL binding and internalization in vivo. Nevertheless, the osmolarity in portal venous blood may be significantly modified during intestinal absorption (17). Moreover, several conditions may lead to hyponatremia or hypernatremia and thus to altered extracellular ionic strength. Conditions associated with hyponatremia include diabetes mellitus, hepatic failure, cardiac failure, vomiting, diarrhea, burns, pancreatitis, salt-loosing nephritis, diuretic treatment, adrenal insufficiency, and antidiuretic hormone excess (4). Hypernatriemic conditions include diabetes insipidus, osmotic diuresis, sweating, excess intake of Na+, hypodipsia, hyperaldosteronism, and Cushing's syndrome (24). A decrease of extracellular Na+ concentration below 130 mM, a condition that is encountered in 1-2% of hospitalized patients (4), would be expected to increase LDL binding by >20%. Alterations of extracellular osmolarity due to glucose (in diabetes mellitus) or urea (in renal insufficiency), on the other hand, are not expected to appreciably modify LDL binding and internalization, even though they are prone to alter cell volume and lysosomal pH (6, 14, 18, 20, 31, and unpublished observations).

In conclusion, the present observations indicate that alterations of extracellular NaCl and KCl concentrations strongly modify LDL binding and internalization, an effect that correlates with ionic strength but not with changes in cell volume or cytosolic or lysosomal pH.

    ACKNOWLEDGEMENTS

We acknowledge the valuable technical support of Uta Hammacher and the meticulous preparation of the manuscript by Tanja Schweickert and Sandra Holzherr.

    FOOTNOTES

This study was supported by the Deutsche Forschungsgemeinschaft (grant nos. La 315/4-2 and Bu 1089/1-1).

Address for reprint requests: F. Lang, Physiologisches Institut der Universität Tübingen, Gmelinstr. 5, D-72076 Tübingen, Germany.

Received 5 November 1996; accepted in final form 6 June 1997.

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Abstract
Introduction
Methods
Results
Discussion
References

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AJP Cell Physiol 273(4):C1409-C1415
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