1 Margaret M. Dyson Vision Research Institute, Department of Ophthalmology, and 3 Department of Cell Biology, Weill Medical College of Cornell University, New York, New York 10021; and 2 Department of Physiology, Center for Research and Advanced Studies, 07000 Mexico City DF, Mexico
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ABSTRACT |
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Madin-Darby canine kidney (MDCK)
I and Fisher rat thyroid (FRT) cells exhibit transepithelial electrical
resistance (TER) values in excess of 5,000 · cm2. When these cells were
incubated in the presence of various inhibitors of sphingolipid
biosynthesis, a >5-fold reduction of TER was observed without changes
in the gate function for uncharged solutes or the fence function for
apically applied fluorescent lipids. The localization of ZO-1 and
occludin was not altered between control and inhibitor-treated cells,
indicating that the tight junction was still intact. Furthermore, the
complexity of tight junction strands, analyzed by freeze-fracture
microscopy, was not reduced. Once the inhibitor was removed and the
cells were allowed to synthesize sphingolipids, a gradual recovery of
the TER was observed. Interestingly, these inhibitors did not attenuate
the TER of MDCK II cells, a cell line that typically exhibits values
below 800
· cm2. These results
suggest that glycosphingolipids play a role in regulating the
electrical properties of epithelial cells.
lipid microdomains; caveolin; claudin; occludin
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INTRODUCTION |
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GLYCOSPHINGOLIPIDS are composed of a hydrophobic ceramide backbone covalently linked to a polar carbohydrate group. If classified solely by their carbohydrate groups, there are more than 150 different glycosphingolipids (GSL) expressed in humans (31). Approximately one-third of this number contain sialic acid and are known commonly as gangliosides (29). Although the consequences of sphingolipid accumulation in pathologies such as Gaucher's and Tay-Sach's disease are well known (3, 12), there are few reports detailing the physiological function of sphingolipids, despite the fact that the first GSLs were isolated and characterized over a century ago. In contrast, phosphatidylinositols, which have less than a dozen different headgroups, are known to be critical for such diverse functions as calcium release, protein kinase C activation, vesicle budding, and endocytic recycling (21, 25, 36, 40). The diversity of GSLs suggests they could mediate a large number of biological functions.
Interest in the cell biology of GSLs was sparked when it was theorized
that GSLs could associate with cholesterol and form a membrane domain
that was distinct from bulk phospholipids (44). Subsequently, the in vivo association of certain integral membrane proteins with these GSL- and cholesterol-rich membranes was inferred from the observation that insoluble material from cells extracted with
1% Triton X-100 was highly enriched with GSLs, cholesterol, and
glycosylphosphatidylinositol-anchored proteins (5).
Proteins such as caveolin, G proteins, and src family
kinases can be found in these detergent-insoluble membranes, which are
often referred to as Triton-insoluble floating fractions (TIFF)
(9, 18, 30, 35, 37, 46). Interestingly, two proteins of
the tight junction (TJ), ZO-1 and occludin, can also be isolated from
TIFF (26). Cholesterol depletion from T84 cells using
lovastatin caused a reduction in transepithelial electrical resistance
(TER) from 1,000 to 400 · cm2
within 24 h, as well as the mislocalization of occludin from the
lateral membrane to the cytoplasm. Moreover, disassembly of the TJ via
calcium chelation caused the dissociation of occludin and ZO-1 from
TIFF, leading to the conclusion that the TJ is a specialized lipid
microdomain. A role for cholesterol in modulating TJ electrical
properties has been demonstrated in Madin-Darby canine kidney (MDCK)
cells. In contact-naive MDCK cells, cholesterol depletion results in
accelerated Ca2+-induced TJ formation (39),
whereas in fully polarized MDCK cells, cholesterol depletion results in
a transient increase in TER, which can be reversed once cholesterol is
restored to the cells (10). In both cases, the mechanism
was attributed to altered signaling through lipid second messengers.
Studies demonstrating the effect of cholesterol depletion in mammalian
tissue culture cells are possible because of the availability of
cholesterol biosynthesis inhibitors such as compactin and lovastatin. In the case of GSLs, the other putative component of TIFF,
fumonisin-1 (FB1), an inhibitor of ceramide biosynthesis, has been
shown to reduce the steady-state levels of glucosylceramide in MDCK
cells (22). However, FB1 can also cause an increase in
sphingoid bases, potential lipid second messengers (12,
13). Other inhibitors, such as
2-amino-3,4-dihydroxy-2-hydroxymethyl-14-oxo-6-eicosenoic acid (ISP1)
and 1-phenyl-2-hexadecanoylamino-3-morphilino-1-propanol-HCl (PPMP), have been shown to reduce the levels of complex
sphingolipids in melanoma cells (16) and MDCK cells
(1), respectively. These inhibitors do not directly
inhibit the formation of ceramide and thus should not increase the
levels of sphingoid bases, making them potentially valuable tools in
determining the function of GSLs.
While studying the properties of these GSL biosynthesis inhibitors, we observed that these inhibitors attenuated the TER in MDCK I cells by nearly an order of magnitude compared with control cells. Fisher rat thyroid cells (FRT), a cell line that also displays high TER, demonstrated the same susceptibility to these inhibitors. Under these conditions, the localization of ZO-1 and occludin was not altered between control and lipid-depleted cells, indicating that the TJ was not grossly altered. The presence of claudin-2, which was shown to reduce TER when expressed in MDCK I cells, was not observed (11). Finally, loss of GSLs did not cause a dissociation of occludin from TIFF. These results show that GSLs are involved in regulating the electrical properties of epithelial cells.
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METHODS |
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Cell culture. MDCK (I and II) or FRT cells from a confluent 75-cm2 flask were trypsinized and seeded onto 12- or 25-mm-diameter Transwell filters (Corning, Rochester, NY) for TER measurements or onto 35-mm-diameter dishes for lipid analysis. Cells were cultured in DMEM supplemented with 5% fetal bovine serum. TERs were measured using an ohmeter (World Precision Instruments, Bradenton, FL).
Inhibition of sphingolipid biosynthesis. All inhibitors were added to 0.2% fat-free BSA in DMEM. PPMP (Matreya, Pleasant Gap, PA), FB1 (Sigma, St. Louis, MO), and ISP1 (Cayman, Ann Arbor, MI) were added from concentrated stocks in absolute ethanol. The final ethanol concentration did not exceed 0.1% (vol/vol). After 5 days at confluency, monolayers of MDCK or FRT cells were rinsed with DMEM and switched to media containing the indicated inhibitor. Control cells received 0.2% BSA and ethanol (0.1% vol/vol) only. In some studies, the PPMP-containing medium was replaced with 0.2% fat-free BSA in DMEM + 0.1% ethanol after 48 h.
Lipid analysis. GSLs from cultured cells were isolated following a published method (2). Briefly, cells grown on 35-mm dishes were rinsed twice with ice-cold phosphate-buffered saline and scraped into methanol/acetic acid (98:2; vol/vol, 1 ml) and transferred to a 13 × 100 mm test tube. Chloroform (0.5 ml) and water (1 ml) were added, and the solution was vortexed. Phase separation was achieved by the addition of 1 ml each of chloroform and water. The upper phase was discarded, and the organic phase evaporated under reduced pressure. Glycerophospholipids were removed by saponification in alkaline methanol followed by reextraction and evaporation. Aliquots of sphingolipids were spotted onto a thin-layer chromatography (TLC) plate (Keiselguhr 60, Merck) and developed in chloroform/methanol/water (60:35:4; vol/vol/vol). Lipids were visualized after spraying with hydroxytetralone in sulfuric acid followed by heating in a TLC oven (110°C, 20 min) using a Storm 860 phosphorimager in the red fluorescent mode.
Confocal immunofluorescence microscopy. MDCK I cells grown on filters in the presence or absence of lipid biosynthesis inhibitors were processed for immunofluorescence microscopy. Briefly, filters were washed twice in phosphate-buffered saline containing 0.1 mM CaCl2 and 1.0 mM MgCl2 (PBS-CM) and fixed in 3% paraformaldehyde for 30 min. After quenching with 75 mM glycine and 50 mM NH4Cl in PBS-CM for 15 min, the filters were rinsed in PBS-CM and incubated in PBS-CM containing 0.2% BSA and 0.075% saponin (buffer A) for 30 min. The filters were excised from their plastic holders and incubated in buffer A containing 2.0 µg/ml of rat anti-ZO-1 (Chemicon, Temecula, CA) and 5.0 µg/ml of rabbit anti-occludin (Zymed, San Francisco, CA). After 1 h, the filters were rinsed 3 × in buffer A and incubated in buffer A containing 5.0 µg/ml each of FITC-labeled donkey anti-rat IgG and Texas Red-labeled goat anti-rabbit IgG (Jackson Immunoresearch, West Grove, PA). After 30 min, the filters were rinsed, mounted, and examined with a Zeiss LSM 510 confocal microscope. Images were acquired and processed using the Zeiss Image Browser software.
Flotation gradients. The method described by Harder et al. (15) was used with the following modifications. Briefly, MDCK or FRT cells were grown on 25-mm-diameter Transwell filters in the absence or presence of PPMP. Two filters were used for each gradient. After 2 days, the filters were rinsed with ice-cold Hanks buffer (without Mg2+ and Ca2+), and the cells were scraped into 500 µl of the same buffer and centrifuged at 200 g for 2 min at 4°C. The supernatant was removed and the cell pellet was lysed in 200 µl of buffer B (25 mM Tris · HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 10% sucrose, 2% Triton X-100, 10 µg/ml each of aprotinin, leupeptin, and pepstatin, and 1 mM PMSF) at 4°C. Once the cell pellet was dissolved, 400 µl of ice-cold 60% Optiprep was added. The mixture was agitated gently and transferred to a SW 50 centrifuge tube. Additional 600-µl gradient steps consisting of 35, 30, 25, 20, and 0% Optiprep in buffer B were overlaid. Gradients were centrifuged for 6 h at 42,000 g at 4°C. Six fractions (600 µl each) were removed from the top of each tube, and the proteins in each fraction precipitated. The precipitated proteins were analyzed by SDS-PAGE and Western blotting. For Western blotting, rabbit anti-occludin, rabbit anti-claudin-1, and rabbit anti-claudin-2 antibodies were purchased from Zymed. Rabbit anti-claudin-4 was a generous gift from S. Tsukita (Kyoto Univ., Japan).
"Fence" function: diffusion of BODIPY-sphingomyelin. Sphingomyelin/BSA complexes (5 nmol/ml) were prepared in P buffer [145 mM NaCl, 10 mM HEPES (pH 7.4), 1.0 mM Na-pyruvate, 10 mM glucose, 3 mM CaCl2] using BODIPY-FL-sphingomyelin (Molecular Probes, Eugene, OR) and defatted BSA. Filter-grown MDCK cells were labeled from the apical side with BODIPY-sphingomyelin/BSA complexes for 10 min on ice. Cells were washed with P buffer and incubated for 0 or 1 h on ice and then analyzed by confocal microscopy Z-sectioning. In wild-type MDCK cells, polar lipid staining was stable for at least 20 min, and the lateral appearance of BODIPY-sphingomyelin was preceded by internalization. All pictures shown, however, were generated within the first 5 min of analysis.
"Gate" function: paracellular flux of 3-kDa dextran. FITC-labeled dextran (10 mg/ml in P buffer) was added to the apical side of filter-grown MDCK cells and incubated at 37°C. After 1 h, the medium from the basolateral chamber was collected, and FITC-dextran fluorescence was measured with a fluorometer (excitation 492 nm; emission 520 nm).
Freeze fracture analysis.
For freeze fracture analysis, cell monolayers grown in flasks (Falcon
Plastics, Cockeysvillen, MD) were fixed with 2.5% glutaraldehyde in
PBS for 30 min at 37oC, washed three times with PBS, and
cryoprotected by successive incubations in 10, 20, and 30% glycerol,
for 30, 30, and 60 min, respectively. They were then detached from the
substratum as a sheet by gently scraping with a rubber policeman,
placed on gold specimen holders, and rapidly frozen in the liquid phase
of partially solidified Freon 22 cooled with liquid nitrogen. Freeze
fractures were performed in a Blazers BAF 400 (Balzers, Liechtenstein)
at 150°C and 5 × 10
9 bar. Fractured faces were
shadowed with platinum and carbon at 45 and 90°, respectively.
Replicas were cleaned with chromic mixture and washed in distilled
water, placed on 300-mesh copper grids, and examined in an electron
microscope (JEM-2000EX: JEOL, Tokyo, Japan).
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RESULTS |
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Sphingolipid biosynthesis inhibitors reduce TER in MDCK I.
Several commercially available inhibitors of sphingolipid biosynthesis,
ISP1, PPMP, and FB1 (Fig. 1), were tested
for their ability to reduce the amounts of GSLs in MDCK cells. For PPMP and ISP1, we determined the minimum concentration of each inhibitor that inhibited the activity of glucosylceramide or serine
palmitoyltransferase by at least 90%, respectively (data not shown).
In the case of FB1, we used the previously reported concentration
(22). Unexpectedly, these inhibitors reduced the TER in
MDCK I cells, but not in MDCK II cells (Fig. 2, A and
B). Although all three
inhibitors were effective in reducing the amount of glucosylceramide
and lactosylceramide in MDCK I cells (Fig. 2C), PPMP (20 µM) and ISP1 (12 µM) were the most effective in reducing TER. After
48 h, the resistance values were ~1,000
· cm2. While significantly lower
than in control MDCK I cells, the TER in drug-treated cells was still
higher than that in MDCK II cells. In MDCK II cells, the same
inhibitors had little effect on the TER or the level of
lactosylceramide, but there was a small decrease in the amount of
glucosylceramide (Fig. 2D).
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TJ remains intact while TER is reduced.
To determine if the loss of lipids may have altered the TJ structure,
we stained control and drug-treated cells for two TJ proteins, ZO-1 and
occludin (Fig. 4). Treatment with 20 µM
PPMP resulted in a fourfold decrease in TER of MDCK I cells, but
occludin and ZO-1 were still localized to the apical-most portion of
lateral plasma membrane. The inhibitors ISP1 and FB1 also reduced TER without altering the overall appearance of the TJ proteins. In control
and drug-treated cells, the TJ was located between 2 and 3 µm from
the apical membrane plasma membrane. Drug-treated cells retained their
cuboidal shape and were similar from control cells. A small amount of
intracellular occludin can be seen in the PPMP-treated cells, but this
is absent in the ISP1- and FB1-treated cells, leading us to conclude
that the appearance of this small pool of occludin in PPMP is not the
cause of attenuated TER. In conclusion, the inhibitors reduced the TER
without causing significant alterations to the appearance of the TJ at
the level of light microscopy.
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Effect of PPMP on the number of TJ strands.
Because the previous experiments ruled out gross structural alteration
of the TJ as a mechanism for the action of PPMP, we examined the
possibility that the TJ ultrastructure might have been subtly modified.
In freeze-fracture replicas, TJs appear as a network of interconnected
strands. In some tissues, a high TER is correlated with a higher number
of strands, leading to the hypothesis that increased electrical
resistance is proportional to the number of strands (6).
Therefore, one possible mechanism for the action of PPMP we considered
was the reduction of the number of strands in MDCK I cells. When we
examined freeze-fracture replicas of MDCK I cells grown under control
and PPMP treatment, the mean number of strands in PPMP-treated cells
were not lower than those of control cell. PPMP slightly alters the
complexity of the TJ network compared with control cells (Fig.
6), increasing the mean strand number
(2.77 for control vs. 3.06 for PPMP). Therefore, PPMP does not reduce
TER by reducing the complexity of the strands.
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Effect of PPMP on the expression of claudins 2 and 4.
Clostridium perfringens enterotoxin (CPE) has been shown to
specifically remove claudin-4 from the TJ of MDCK I cells,
resulting in a reduction of TER within 48 h (38).
Although ZO-1 and occludin were still localized to the TJ, there was a
twofold reduction in the mean strand number of the TJ as seen by freeze
fracture EM. After the toxin was washed out, the TER returned to near
normal values. Sonoda et al. (38) speculated that
epithelial cells may regulate the flow of ions through the paracellular
space by controlling the expression of TJ components. Recently, it was shown that the expression of claudin-2 in MDCK I cells caused a
decrease in TER. Ordinarily, claudin-2 is expressed in MDCK II, but not
MDCK I (11). With this in mind, we tested whether the GSL
inhibitors caused claudin-2 expression in MDCK I cells (Fig. 7,
top). Confluent, filter-grown
MDCK cells were treated with GSL inhibitors for 2 days, resulting in a
reduction in TER. The cells were lysed and examined for the presence of
claudin-2 by Western blot. In MDCK II cells, claudin-2 is quite
prevalent, but the protein is absent in control and drug-treated MDCK I
cells. Therefore, the attenuated TER is not due to the expression of claudin-2 in MDCK I. At the same time, we did not observe a reduction in the levels of claudin-4 by Western blot (data not shown). As mentioned earlier, claudin-4 removal results in a decrease in the
complexity of the TJ, an effect not seen with PPMP.
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Lipid microdomains and the TJ. Recently, it was reported that reduction of cholesterol levels in T84 cells (using lovastatin, an inhibitor of cholesterol biosynthesis), causes a reduction of TER (26). At the same time, hyperphosphorylated occludin and ZO-1 were removed from TIFF, leading the authors to conclude that the TJ is composed of cholesterol-rich, detergent-resistant membrane microdomains. These microdomains are thought to result from the hydrophobic interaction between the saturated fatty acyl chains of sphingolipids and cholesterol and are hypothesized to recruit specific membrane-associated proteins, such as caveolin, a protein that acts as a scaffold for several kinases involved in signal transduction (28, 34, 37). Nusrat et al. (26) went on to demonstrate that occludin and caveolin-1 could be colocalized by immunofluorescence microscopy and suggested the possibility that caveolin was playing a role in regulating TJ function.
Given that GSLs are putative components of lipid microdomains, it could be that PPMP reduces the levels of a lipid essential for caveolin function, and this in turn alters TER. We examined this possibility by testing the effect of PPMP on FRT cells, a cell line that does not express caveolin-1. Although FRT cells do express caveolin-2, it is located at the Golgi and not at the plasma membrane (24). Control FRT cells exhibit TER greater than 5,000
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DISCUSSION |
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Our results demonstrate that reducing GSLs in MDCK I and FRT cells
reduces TER in a novel, specific manner. The inhibitor of
glucosylceramide synthase, PPMP, significantly reduces TER without
1) rearranging the strands, 2) impairing the
barrier to small uncharged solutes, or 3) disrupting the
fence function of the TJ. Other procedures have previously been
reported to reduce TER, but none of them have the same properties as
PPMP. Cholesterol removal in T-84 cells (~1,000
· cm2) causes a drastic (10-fold)
reduction of TER but induces redistribution of occludin to an
intracellular pool (26). In MDCK II cells, the rapid
removal of cholesterol caused a reversible decrease in TER that was
accompanied by a decrease in the occludin and ZO-1 at the TJ
(10). The actin depolymerization agents cytochalasin B and
mycalolide B both reduce TER in MDCK II cells (23, 41), and in the latter case, mycalolide B also caused increased permeability of the TJ to inulin, a small, nonionic molecule (41).
Energy (ATP) depletion with 2-deoxyglucose in MDCK II cells also
doubles the permeability to inulin and causes a slight modification in the structure of the strands (19). Incubation with CPE
causes removal of claudin 4 from MDCK I, which is accompanied by loss of gate function for small solutes and reduction in the complexity of
the strands (38). Addition of claudin-2 to MDCK I cells, which normally lacks it, has a more drastic effect than PPMP on the TER
(reduction to MDCK II levels) but like PPMP has no effect on uncharged
solute permeability or fence function (11). However, like
ATP depletion, exogenous claudin-2 expression introduces slight changes
in the morphology of the strands such as the appearance of particles in
the E face and small discontinuities in the P face. Sonoda et al.
(38) have shown that CPE specifically binds to an
extracellular loop of claudin-4 in MDCK I cells, causing internalization and degradation of the claudin. This resulting decrease
in TER, however, was accompanied by an increase in paracellular flux,
as well as a reduction in the size and complexity of the TJ strands
(38).
There are several scenarios that could explain how inhibitors of GSL biosynthesis might attenuate TER. Sphingolipids such as sphinganine and ceramide act as second messengers (13, 27, 45), and altering their concentration might result in the activation of signaling pathways, leading to a loss of TER. As discussed in RESULTS, PPMP may increase the concentration of ceramide, but its concentration is reduced by ISP1 and FB1, which argues against the involvement of ceramide in the loss of TER. By the same logic, we consider it unlikely that the TER attenuation is caused by an increase in sphinganine (in the cases of PPMP and FB1) because ISP1 will reduce the concentration of sphinganine.
Another scenario is that GSLs are structural components of the TJ. We tested this hypothesis by analyzing the lipid composition of occludin immunoprecipitates from MDCK I cells. However, we were not able to detect any GSLs in these preparations. In addition, we attempted immunofluorescence microscopy of MDCK I cells after paraformaldehyde fixation to visualize specific lipids at the TJ but were not successful. These negative results are not conclusive because the lipids could have been extracted during the washing steps in the immunoprecipitation. Furthermore, unlike proteins, GSLs are not fixed with paraformaldehyde and are still capable of diffusing after the cells are chemically fixed (32, 33).
Because sphingolipid depletion has been shown to alter the transport of some transmembrane proteins in MDCK cells to the plasma membrane (22), another possibility it that the inhibitors altered the steady-state localization of TJ protein components. Although this does not appear to be the case for ZO-1 and occludin (Fig. 3), we cannot exclude the possibility that other TJ proteins are mislocalized as a result of sphingolipid depletion. Work by Tsukita and colleagues (42, 43) indicates that TER depends on the presence of specific claudins at the TJ. Removal of claudin-4 using CPE caused a loss of TER in MDCK I cells, accompanied by a reduction in the number of strands in the TJ, as well as an increase in permeability to dextran. While the loss of TER is similar to our results, it is unlikely that PPMP is acting directly on claudin-4 because we do not observe similar alterations in the TJ morphology or an increase in paracellular flux. Recent work by Furuse et al. (11) shows that the addition of claudin-2 to MDCK I cells (which do not normally express claudin-2) causes a loss of TER without altering the fence role or permeability to noncharged solute gate properties, but the reduction of GSLs in our hands does not cause expression of claudin-2 in MDCK I cells.
Finally, it is possible that the reduction in TER is mediated by increased flow through ion channels located in the plasma membrane. Recent work in amphibian A6 cells demonstrates that the amiloride-sensitive epithelial sodium channel (ENaC) is located in TIFF (17). Furthermore, various potassium channels have also been found in these lipid microdomains (4, 7, 20). Although expression of rat ENaC in MDCK cells shows that the protein is not located in TIFF (14), it is conceivable that the loss of GSLs altered the location and/or the activity of one or more ion channels. In summary, we observe that a loss of GSLs causes a loss of TER in high-resistance epithelial cells but cannot determine if this effect is due to a subtle alteration of the TJ or an alteration of ion transport activity at the plasma membrane.
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FOOTNOTES |
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Address for reprint requests and other correspondence: E. Rodriguez-Boulan, Margaret M. Dyson Vision Research Institute, Box 233, Weill Medical College of Cornell Univ., 1300 York Ave., New York, NY 10021 (E-mail: boulan{at}med.cornell.edu).
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.
First published December 21, 2002;10.1152/ajpcell.00149.2002
Received 3 April 2002; accepted in final form 6 December 2002.
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