In vivo fluorescence measurement of Na+ concentration in the pericryptal space of mouse descending colon

J. R. Thiagarajah2,*, S. Jayaraman1,*, R. J. Naftalin2, and A. S. Verkman1

1 Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521; and 2 Division of Physiology, Centre for Vascular Biology and Medicine, King's College London, Guy's Campus, London SE1 1UL, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A method involving surgical exposure of the colonic mucosa, fluorescent dye addition, and confocal microscopy has been developed for monitoring colonic crypt function in vivo in mice. Na+ concentration in the extracellular pericryptal space of descending colon was measured using a low-affinity Na+-sensitive fluorescent indicator consisting of an Na+-sensitive chromophore (sodium red) and an Na+-insensitive chromophore (Bodipy-fl) immobilized on 200-nm-diameter polystyrene beads. The Na+ indicator beads accumulated in the pericryptal spaces surrounding the colonic crypts after a 1-h exposure of the colonic luminal surface to the bead suspension. Na+ concentration ([Na+]) in the pericryptal space was 491 ± 62 mM (n = 4). After a 70-min exposure to amiloride (0.25 mM), pericryptal [Na+] was reduced to 152 ± 21 mM. Blockage of the crypt lumen with mineral oil droplets reduced pericryptal [Na+] to 204 ± 44 mM. Exposure of the colonic mucosa to FITC-dextran (4.5 kDa) led to rapid accumulation of the dye into the crypt lumen with a half time of 19.8 ± 1.0 s, which was increased to 77.9 ± 6.0 s after amiloride treatment. These results establish an in vivo fluorescence method to measure colonic crypt function and provide direct evidence for accumulation of a hypertonic absorbate in the pericryptal space of descending colon. The pericryptal space represents the first example of a hypertonic extracellular compartment in mammals that is not created by a countercurrent amplification mechanism.

water transport; fluid absorption; colonic crypt


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SEVERAL LINES OF EVIDENCE suggest that fecal dehydration in the colon involves active salt accumulation in a pericryptal space, resulting in water movement from the crypt lumen into the pericryptal space and the generation of a cryptal suction force. Previous studies have shown that colonic absorbates are hypertonic (3, 16) and that descending colonic crypts are surrounded by a myofibroblastic pericryptal sheath (9). It is thought that the pericryptal sheath creates a barrier to Na+ movement resulting in a hypertonic compartment. In isolated colonic mucosa, the Na+-sensitive fluorescent dye sodium green accumulates in the pericryptal space in vitro to an extent related to the accumulation of Na+ in the pericryptal space (10, 11, 15). Pericryptal sheath growth in the descending colon is stimulated by angiotensin II and aldosterone during feeding of a low-Na+ diet. This correlates with an enhanced capacity to absorb fluid against a hydraulic resistance in rats fed a low-Na+ diet (11). Additionally, damage to the pericryptal sheath by ionizing radiation makes it leaky and correlates with a reduced capacity to generate hard feces (20).

The purpose of this study was to develop an in vivo method to measure crypt function to test the hypothesis that pericryptal fluid is hypertonic and that the hypertonicity is created by active salt transport from the crypt lumen. A surgical procedure was developed to expose the colonic mucosa to allow for direct visualization of crypts by fluorescence confocal microscopy. A low-affinity ratiometric Na+-sensitive fluorescent indicator, which was described recently in measurements of salt concentration in the airway surface liquid (6), was introduced into the pericryptal space to report the local Na+ concentration. The indicator has been shown to be insensitive to pH and organic solutes (6). Our results provide compelling evidence for a hypertonic pericryptal space that is created by active fluid transport, representing the first example of a hypertonic extracellular compartment that is not created by a countercurrent amplification.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In vivo colon preparation. Mice (CD1 strain, 30-40 g) were anesthetized with pentobarbital sodium (5 mg/kg ip). An abdominal incision was made, and the descending colon was elevated above the incision without compromise of the mesenteric blood supply. The colon was ligated with a silk suture proximal to the section being investigated. With the use of microscissors, the colon was opened with a 0.5-cm transverse incision along the antimesenteric border, and the feces were removed. The cut edge of the colonic mucosa was immobilized using a forked clamp (Fig. 1). The fork holds the tissue, providing a flat surface for viewing the mucosa by confocal microscopy, and permits blood flow via the mesenteric vessels. The mucosal surface was maintained in buffer (120 mM NaCl, 25 mM NaHCO3, 3.3 mM KH2PO4, 0.8 mM K2HPO4, 1.2 mM MgCl2, 1.2 mM CaCl2, and 10 mM glucose, pH 7.4; 296 mosmol/kgH2O) by continuous-drip perfusion. For Na+-free experiments, the mucosa was washed thoroughly and replaced in buffer containing 124 mM choline chloride, 5.8 mM KCl, 0.3 mM CaCl2, 1.2 mM MgCl2, 10 mM glucose, and 20 mM HEPES, pH 7.4. The mouse and clamp were fixed rigidly to an aluminum plate on the stage of an upright confocal fluorescence microscope. Body temperature was maintained using a heating lamp. The surgical preparation takes ~5 min. At the end of each experiment, the mouse was killed by an overdose of pentobarbital sodium. For blood flow experiments, the carotid artery was cannulated and 0.2 ml of 200-nm rhodamine beads was injected. Rhodamine fluorescence was monitored in time in the colon by rapid image acquisition (every 0.2 s) after injection. Time of fluorescence appearance was calculated from the number of captured images before fluorescence. For oxygen experiments, a Harvard Apparatus ventilator was used to ventilate mice with 100% oxygen. All protocols were approved by the University of California, San Francisco, Committee on Animal Research.


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Fig. 1.   In vivo experimental technique.

Fluorescent indicators. Fifty micrograms of sodium red (sample provided by Molecular Probes, Eugene, OR; catalog no. 71351) and 30 µg of Bodipy-fl in 2% methanol-water were added to a 1% (vol/vol) suspension of 200-nm-diameter carboxyl latex beads (Polymer Lab, Amherst, MA) suspended in 4 ml of water. After they were shaken at room temperature for 1 h, the beads were centrifuged and repetitively redispersed by brief sonication in distilled water until no free dye was present in the supernatant. The chromophores remained quantitatively immobilized on the beads during measurements and for >2 mo of storage in water at 4°C in the dark. Before experiments, the beads were centrifuged and resuspended by brief sonication in the appropriate buffer, and the crypts were loaded with the dye with 200 µl of a 10% (vol/vol) solution by incubation for 1 h.

For calibration of the Na+ sensitivity of the indicator, beads were added to solutions containing 0-700 mM Na+ (counterion choline). Sodium red and Bodipy-fl image pairs were obtained for each calibration solution. Na+ indicator calibration was also verified by measurement of ratios on the colonic mucosal surface. The fluorescence ratio remained stable for >= 60 min.

Microscopy. Fluorescence microscopy was carried out using a Leitz epifluorescence microscope equipped with a Nipkow wheel coaxial-confocal attachment (Technical Instruments, San Francisco, CA). The colonic crypts were viewed with a Nikon CF Plan ×50 ELWD air objective (NA 0.55, working distance 8.7 mm) or Nikon CF Plan ×20 SLWD (NA 0.35, working distance 20.5 mm) air objective. Confocal fluorescence images were acquired using a cooled charge-coupled device camera (model AT200; Photometrics, Tucson, AZ) with a back-thinned 14-bit detector (model TK 512 CB, Tetronix).

Sodium red and Bodipy-fl fluorescence were observed with standard rhodamine and fluorescein filter sets. For z scanning, the microscope fine focus was driven by a microstepper motor (Compumotor, 12,200 steps/revolution, 1,400 steps/200 µm). Motor control and signal detection were controlled by custom-written LabView software.

Na+ ratio analysis. Image pairs of sodium red and Bodipy-fl were acquired (exposure time 1 s) for the same field. Background images were obtained under the same conditions but without loading with the dye. Analysis was performed using PMIS software (Photometrics). Ratio images (red-to-green fluorescence) were obtained by pixel-by-pixel division of background-subtracted images. Background values were <10% of signal. Averaged ratios were obtained by integration of red and green fluorescence intensities over specified regions of interest. Two to three crypts were measured from each set of images.

Dextran uptake. For visualization of crypts, the colonic mucosa was exposed to an isotonic solution containing rhodamine-dextran (mol wt 500,000) for 10 min. After the field of view was reduced to one or a few crypts, a solution containing FITC-dextran (mol wt 10,000, 0.2 mg/ml; Molecular Probes) was added at the surface, and time courses and z scans were carried out during visualization with the fluorescein filter set. Time-course data were normalized to the change in fluorescence over the time course. The z-scan data were normalized to fluorescence intensity at the crypt opening and to that at the base of the crypt.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Na+ sensitivity calibration of ratiometric indicator. The Na+ indicator calibration was confirmed by recording images of the colonic surface immediately after addition of the indicator suspended in solutions of known Na+ concentrations. The fluorescence ratio vs. Na+ calibration curve showed increasing red-to-green fluorescence ratio with increasing Na+ concentration (Fig. 2A; n = 5), in agreement with results obtained for this indicator applied to the airway surface liquid (6). Indicator fluorescence ratio was sensitive to changes in Na+ concentration to >= 700 mM, and the ratio was not sensitive to pH in the range 6-8 (not shown). The calibration curve was used to deduce Na+ concentrations in the pericryptal space in vivo from measured fluorescence intensities.


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Fig. 2.   A: Na+ dye calibration curve. Data (n = 5) were obtained as described in MATERIALS AND METHODS and fitted to a saturation curve (dissociation constant = 430 mM). B: effect of amiloride (0.25 mM) on Na+ concentration in the pericryptal space. Data (n = 3) from collected images were analyzed, and Na+ concentration was calculated. C: effect of Na+-free buffer on Na+ concentration in the pericryptal space. Data (n = 3) from collected images were analyzed, and Na+ concentration was calculated. Buffer was replaced at 30 min. D: effect of mineral (paraffin) oil on Na+ concentration in pericryptal space. Data (n = 3) were analyzed as for B. ***P = 0.005 (2-tailed t-test).

Na+ concentration in the pericryptal space in vivo and effect of amiloride. The Na+ indicator beads were loaded into the pericryptal space by addition of 0.1 ml of a bead suspension in Krebs-Ringer solution onto the colonic mucosa. After the external indicator was washed off, confocal images indicated greatest fluorescence in the pericryptal spaces at a depth of 10-40 µm from the surface (Fig. 3). The fluorescence intensity ratio in the pericryptal space in mouse descending colon of 13.1 ± 0.8 gave a pericryptal Na+ concentration of 491 ± 62 mM (n = 4 mice). Addition of amiloride (0.25 mM) to the luminal bathing solution resulted in a slow decrease in fluorescence ratio to 6.9 ± 0.9, giving a pericryptal Na+ concentration of 152 ± 21 mM (n = 3) after 70 min (P = 0.005, Student's unpaired 2-tailed t-test; Fig. 2B). To confirm that pericryptal hypertonicity is dependent on active Na+ absorption, the pericryptal Na+ concentration was measured after replacement of the bathing solution with Na+-free buffer. The Na+ concentration in the pericryptal space decreased over an 80-min time course to 207 ± 26 mM (Fig. 2C). To look at the blood flow, fluorescent beads (200-nm rhodamine beads) were injected intravenously. Fluorescence was then followed in colonic capillaries in cut-and-clamped sections and unclamped sections. There was no significant difference in the time of fluorescence appearance (1.40 ± 0.12 and 1.47 ± 0.13 s in unclamped and clamped sections, respectively), showing that microvascular perfusion is unimpaired in these experiments. To further confirm that the tissue was viable, pericryptal Na+ concentration was monitored while the mouse was ventilated with 100% oxygen. The pericryptal Na+ concentration in the oxygen-ventilated preparation (504 mM) was not significantly different from that in the normal preparation. Also there was no evidence of large amounts of mucous production during the experiments.


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Fig. 3.   Ratio images of sodium red to Bodipy-fl showing Na+ concentration in murine descending colon in vivo. Greatest fluorescence is seen in pericryptal spaces, and fluorescence is reduced after treatment with 0.25 mM amiloride for 50 min. Scale bar, 50 µm. Fluorescence images are 40 µm below mucosal surface.

Na+ concentration after treatment with mineral (paraffin) oil. Mineral (paraffin) oil droplets have been previously shown to block fluid uptake into the rat colonic crypts (11). A 1% (vol/vol) emulsion of mineral oil prepared by sonication in PBS containing 0.1 mM deoxycholate was layered on the mucosa for 15 min and then removed by washing with Krebs solution. By bright-field microscopy, it was observed that the crypt lumens were loaded with oil droplets. At 40 min after the oil was applied, the pericryptal Na+ concentration was reduced to 204 ± 44 mM (P = 0.017; Fig. 2D). Thus the principal route for Na+ entry into the pericryptal space is via the crypt lumen, since the oil droplets do not affect the surface mucosa (11, 24).

Time course of dextran uptake. To show that the hypertonic Na+ in the pericryptal space is generating an osmotic pressure gradient that results in fluid uptake into the crypt lumens, we measured FITC-dextran uptake into mouse descending colonic crypts in vivo. A larger osmotic gradient is predicted to increase the rate of FITC-dextran uptake (11, 12).

The uptake of FITC-dextran was measured from the time course of fluorescence change at a fixed distance of 30 µm below the crypt opening at the mucosal surface. The time course in Fig. 4 shows a rapid increase in fluorescence intensity with a half time of 19.8 ± 1.0 s. Treatment with amiloride (0.25 mM) increased the half time of dextran uptake to 77.9 ± 6.0 s (P < 0.01; Fig. 4).


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Fig. 4.   Solid line, FITC-dextran uptake in vivo in normal murine descending colon. Fluorescent intensity data (n = 4) were fitted to an exponential curve with half time of 19.8 ± 1.0 s. Dashed line, FITC-dextran uptake in vivo in murine descending colon after treatment with 0.25 mM amiloride for 30 min. Fluorescent intensity data (n = 4) were fitted to an exponential curve with half time of 77.9 ± 6.0 s.

Concentration polarization of dextran along crypt length. FITC-dextran accumulates in the crypt lumen as a result of concentration polarization (12). The accumulation results from convective uptake of dextran by solvent drag in the mass flow stream entering the crypt lumen. This mass flow is produced by the suction tension within the crypt lumen resulting from the large osmotic gradient generated across the crypt wall. Dextran accumulates in the lumen, because its diffusive mobility is much lower than its convective mobility. The extent of accumulation is a function of the rates of dextran diffusion within the crypt lumen and permeation across the crypt wall, as well as the rate of mass flow. The FITC-dextran concentration profile along the length of the crypt lumens was obtained by z scans of fluorescence intensity down the crypt. The profile (Fig. 5) shows accumulation of dextran 20-80 µm from the crypt opening above the concentration at the crypt lumen. The accumulation of FITC-dextran is abolished after amiloride treatment. This Na+ pump-dependent concentration polarization of FITC-dextran in the crypt lumen demonstrates that the osmotic gradient across the crypt wall is used to generate a mass fluid flow into the crypt lumens in vivo.


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Fig. 5.   Control line, z scans of FITC-dextran fluorescence along crypt length in normal murine descending colon. Fluorescent intensity data from z scans (n = 3, z-scan acquisition time ~2 s) show accumulation of dextran 20-80 µm from crypt opening. Amiloride line, z scan of FITC-dextran fluorescence along crypt length in murine descending colon after treatment with 0.25 mM amiloride. Fluorescent intensity data from z scans (n = 3) show no accumulation of dextran along crypt length.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hypertonic pericryptal spaces in vivo. The principal finding of this study is that murine descending colonic crypts in vivo can generate and sustain a very large osmotic gradient as a result of active transport of Na+ from the crypt lumen into the pericryptal space. Na+ concentration in the pericryptal space, as reported by Na+ indicator fluorescence intensity ratios in the pericryptal spaces, was two- to threefold higher than in the external bathing solution. The ratiometric Na+ indicator was sensitive to changes in Na+ concentration at >= 500 mM.

One perhaps surprising finding is the relative ease with which the large 200-nm-diameter beads accumulated in the pericryptal space. It was found previously that high-molecular-weight dextrans and horseradish peroxidase readily accumulate in the pericryptal space (8). Bacterial binding to apically exposed adhesion molecules, such as beta 1-integrin, or adhesion to extracellular matrix elements increases the bacterial passage across epithelial boundaries. This acceleration in paracellular bacterial trafficking may be due to disruption of tight junctions and other adhesion molecules as a result of interactions between the surface receptors and cytoskeletal elements such as F-actin (13, 19). This type of cooperative interaction between the hydrophobic beads and elements at the colonic mucosal surface seems a probable factor in the observed rapid trafficking of the hydrophobic beads across the colon.

The high pericryptal Na+ concentration was reduced by amiloride, addition of Na+ free buffer, and blockage of the crypt lumens by mineral oil droplets, indicating that active salt pumping from the crypt lumen generates the hypertonic pericryptal space. It follows that the osmolarity of the pericryptal space is ~800-1,000 mosmol/kgH2O, which is sufficient to generate a very large suction tension across the crypt wall of >= 4-5 atm (1).

The method of in vivo confocal microscopy of colonic mucosa permits us to visually monitor blood flow in the capillaries surrounding the crypts. Rapid transits of erythrocytes through the capillary network surrounding each crypt can be observed with bright-field microscopy. The fact that blood is perfusing the mucosa means that a barrier to Na+ diffusion must exist between the pericryptal space and the extracellular fluid. This barrier has been shown to be due to the myofibroblasts and fibronexus that make up the pericryptal sheath surrounding descending colonic crypts (9, 20). The high Na+ concentration in the pericryptal space in vivo provides evidence that the pericryptal sheath is a sufficient barrier to prevent dissipation of the Na+ gradient by mucosal blood flow. The experiments on blood flow and with 100% oxygen confirmed that the in vivo preparation is viable and has unimpaired microvascular perfusion. There was also no evidence of mucous overproduction during these experiments as happens when the animal dies or when there is abnormal perfusion of the tissue, again confirming that the preparation is viable.

Dextran uptake into crypt lumen in vivo. The demonstration that FITC-labeled dextran is accumulated into murine crypts in vivo to a higher concentration in the lumen than in the external solution confirms previous work using rat mucosa in vitro (12). Amiloride is a specific inhibitor of Na+ uptake across the apical border mainly by inhibition of the epithelial Na+ conductance channel and also by inhibition of the Na+/H+ exchanger (2). Amiloride has no direct action on water transport. Hence, the only plausible explanation for the threefold slower rate of dextran accumulation into a crypt with amiloride present is that crypt fluid absorption is dependent on an Na+ pump-driven process. Hence, murine crypts in vivo absorb fluid as a consequence of the hypertonicity generated in the pericryptal spaces.

The large sustained osmotic gradient across the cryptal barrier probably requires a very low osmotic water permeability. Fluid absorption rates in murine colonic mucosa in vivo are ~30 µl · cm-2 · h-1 (10 nl · cm-2 · s-1) (17). Given ~15,000-20,000 crypts/cm2 in murine colon with an average crypt luminal radius of 2.5-5 µm (1, 5), crypt luminal surface area is ~0.4-1% of the mucosal surface area. The total crypt surface area is equal to crypt perimeter length × crypt length (absorptive surface ~150-200 µm) × number of crypts (~0.5-1.0 cm2/cm2 mucosa). Hence, water flow per square centimeter of crypt mucosa is 1 × 10-5 cm/s. Given an osmotic gradient of ~700 mosmol/kg (18,200 cmH2O) across the cryptal barrier, the computed osmotic water permeability coefficient is very low, ~2.5 × 10-7 cm/s. This low water permeability is consistent with absence of aquaporin (AQP)-4 or other water channels in the crypt (22) and suggests that the crypt barrier may have specialized membrane/structural elements that sustain a remarkably low water permeability. However, several factors will reduce the efficiency of the osmotic force, such as the low pressure in the crypt lumen, which will retard absorption, the raised pressure in the pericryptal sheath, which will also retard fluid movement across the crypt wall induced by osmotic pressure in the pericryptal sheath, and the reflection coefficient of NaCl across the paracellular pathway, which may be <1.

Recent estimates of the permeability coefficient of murine colon are macroscopic measurements that include the leaky surface mucosa and the crypt (22). The surface mucosa contributes ~99% of the hydraulic conductivity of the whole tissue (1). The very large difference between the hydraulic conductivity of the surface mucosa and that of the crypt wall explains why the colonic absorbate can still be hypertonic in the presence of such a high hydraulic conductivity of the surface mucosa. Approximately 50% of the absorbate passing via the crypt wall is very hypertonic, ~1,000 mosM, whereas the other 50% of the absorbate crossing the surface mucosa is nearly isotonic. The evidence for this is based on experiments with mineral oil on rat descending colonic absorption in vivo. The mineral (paraffin) oil blocks fluid absorption by 50% when absorption occurs from agarose gels of low hydraulic resistance, but it inhibits absorption by ~100% when agarose gels of high hydraulic resistance are present in the lumen (11, 24). This suggests that, with low hydraulic resistance, 50% of fluid absorption occurs via the surface mucosa, inasmuch as the paraffin droplets prevent access of fluid to the crypts but not the surface mucosa. In the case of a luminal solution with a high hydraulic resistance, the surface mucosal hydraulic resistance is too low to permit fluid absorption, so that absorption occurs through the crypts, resulting in the paraffin droplets being much more effective in preventing fluid absorption.

The very low hydraulic resistance of crypt wall epithelium is corroborated by the recent findings using conductance scanning techniques and impedance analysis. It has been shown that the crypt resistance (429 ± 86 Omega  · cm2) exceeded that of the surface epithelium (132 ± 15 Omega  · cm2). The subepithelial tissue contributed 15% to the transmural resistance of 118 ± 9 Omega  · cm2 (5). These findings demonstrate the heterogeneity of electrolyte permeability between surface and crypt mucosa. A similar heterogeneous permeability pattern of crypt and surface mucosal water and salt permeability has also been demonstrated in rabbit descending colon in vitro (1). The electrical and the hydraulic conductance of descending colonic crypts are much lower than those of the surface mucosa and are consistent with the crypt mucosa having a capacity to generate a hypertonic absorbate sufficient to generate a luminal suction tension to dehydrate feces.

Only a small degree of hypertonicity can be achieved in absorptive epithelia with a high hydraulic conductivity, such as the renal proximal tubule. Proximal tubules normally have a near-isotonic absorbate; however, deletion of the proximal tubule water channel, AQP1, reduced murine tubular water permeability fivefold and near-isotonic absorption twofold (18). Recently, it was found that the transtubular osmotic gradient increased from 12 mosmol/kgH2O in control to ~40 mosmol/kgH2O in AQP1-null mice (21). These results confirmed that functional water channels are required to maintain the near isotonicity of proximal tubular absorption.

The method of generating hypertonicity in colonic crypts contrasts with that in the renal medulla. In the renal medulla, a hypertonic interstitium is generated by countercurrent multiplication and countercurrent exchange mechanisms, which rely on a complex spatial distribution of active and passive salt/water transporters in the nephron and capillary cells, which retards solute dissipation from the medulla. The hypertonicity in the colonic pericryptal space results from active salt transport into a tissue space isolated from the capillary circulation by the pericryptal barrier.


    ACKNOWLEDGEMENTS

We thank Dr. Zhenjun Diwu (Molecular Probes) for synthesis of sodium red.


    FOOTNOTES

* J. R. Thiagarajah and S. Jayaraman contributed equally to this work.

This work was supported by the Wellcome Trust, a Peter Baker Travelling Award to J. Thiagarajah, National Institutes of Health Grants HL-60288, HL-58198, DK-35124, and DK-43840, and grants from the Cystic Fibrosis Foundation.

Address for reprint requests and other correspondence: R. J. Naftalin, Div. of Physiology, Centre for Vascular Biology and Medicine, King's College London, Guy's Campus, London SE1 1UL, UK (E-mail: richard.naftalin{at}kcl.ac.uk).

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 17 November 2000; accepted in final form 6 August 2001.


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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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