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Departments of 1Medicine and of 2Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York
ABSTRACT
During water deprivation, prostaglandin E2 (PGE2), formed by renal medullary interstitial cells (RMICs), feedback inhibits the actions of antidiuretic hormone. Interstitial PGE2 concentrations represent the net of both PGE2 synthesis by cyclooxygenase (COX) and PGE2 uptake by carriers such as PGT. We used cultured RMICs to examine the effects of hyperosmolarity on both PG synthesis and PG uptake in the same RMIC. RMICs expressed endogenous PGT as assessed by mRNA and immunoblotting. RMICs rapidly took up [3H]PGE2 to a level 5- to 10-fold above background and with a characteristic time-dependent "overshoot." Inhibitory constants (Ki) for various PGs and PGT inhibitors were similar between RMICs and the cloned rat PGT. Increasing extracellular hyperosmolarity to the range of 335485 mosM increased the net release of PGE2 by RMICs, an effect that was concentration dependent, maximal by 24 h, reversible, and associated with increased expression of COX-2. Over the same time period, there was decreased cell-surface activity of PGT due to internalization of the transporter. With continued exposure to hyperosmolarity over 710 days, PGE2 release remained elevated, COX-2 returned to baseline, and PGT-mediated uptake became markedly reduced. Our findings suggest that hyperosmolarity induces coordinated changes in COX-2-mediated PGE2 synthesis and PGT-mediated PGE2 uptake in RMICs.
hypertonicity; prostaglandin transporter; cyclooxygenase-2
PRODUCTION OF CONCENTRATED urine by the mammalian kidney is dependent on two interrelated antidiuretic hormone (ADH)-dependent processes: 1) the establishment of a hyperosmolar environment in the renal medullary interstitium by the countercurrent multiplier mechanism and 2) a selective increase in collecting duct water permeability (18). Prostaglandins (PGs), specifically PGE2, act as pharmacological antagonists of ADH on the medullary thick ascending limb of Henle (mTALH) and the collecting duct and, therefore, induce water diuresis (34). Renal medullary interstitial cells (RMICs) are important sources of PGE2 (17), and dehydration increases the expression of cyclooxygenase in RMICs (14).
For PGs to modulate cellular events over short temporal and spacial domains, there must be local and rapid signal termination. Although some PGs, such as PGI2, are inherently structurally unstable and inactivate spontaneously, PGE2 and other PGs are very stable in whole blood or plasma (31). For these latter PGs, termination of signaling involves the two-step process of carrier-mediated uptake followed by cytoplasmic oxidation (31).
The uptake step in PG signal termination is mediated by the PG carrier PGT (22, 24, 28). PGT is widely expressed in situ in cell types that synthesize and release PGs, including endothelial cells, platelets, renal collecting duct principal cells, and RMICs (1, 3, 35). PGT transports PGE2, PGF2, and PGD2 at physiological affinities, i.e., the Kms are 50100 nM range (22, 24, 28). PGT is a bidirectional lactate/PG exchanger (2, 9). Because cells of the renal medulla engage in substantial glycolysis and generate lactate (12), PGT is energetically poised to remove PGs from the renal medullary interstitium.
The present studies were designed to 1) develop cultured RMICs as a model system for studying the regulation of PGT and 2) examine the short- and long-term regulation of PGT, PG release, and cyclooxygenase (COX)-2 enzyme regulation in response to hyperosmolar media. We find that, on exposure to hyperosmolar media, COX-2 expression increases in the first 24 h but returns to control levels by 710 days. There is a sustained elevation of PGE2 release over this extended time period that is accompanied by a similarly sustained reduction of PGT function at the plasma membrane.
MATERIALS AND METHODS
Materials. Unless otherwise noted, all chemicals of analytic grade or better were obtained from Sigma (St. Louis, MO). Tracer-PGE2 ([3H]PGE2) was obtained from DuPont New England Nuclear (Boston, MA). Primary cultures of rat RMICs were obtained courtesy of Dr. E. Nord (SUNY at Stony Brook, NY) at passage 132.
Cell culture. HeLa cells were grown on 35-mm dishes, infected with vaccinia vTF73, and transfected with a functional rat PGT cDNA as described (22). RMICs were maintained in humidified incubators with 5% CO2-air at 37°C in the RMIC media: DMEM containing 10% fetal bovine serum, 100 U/ml each of penicillin and streptomycin, and 0.28 U/ml of bovine insulin. Unless otherwise noted, RMICs were grown to confluence as monolayers on 35-mm dishes for 2 days. At that point, the medium was replaced by RMIC media without or with added NaCl (25, 50, or 100 mM, equivalent to 50, 100, or 200 mosmol/kgH2O) or urea (50, 100, or 200 mM, equivalent to 50, 100, or 200 mosmol/kgH2O). Experiments were performed between 2 h and 10 days later.
Northern blotting and immunoblotting. Total RNA was extracted from RMICs and whole rat kidney as described previously (22) using guanidinium acid phenol extraction (11). A rat PGT RNA probe was generated using a 3'-truncated PGT cDNA in the vector pGEM3Z; this was linearized with Nco1, and an anti-sense digoxigenin-labeled cRNA probe was generated with SP6 RNA polymerase (22). RNA was separated by glyoxal agarose gel electrophoresis, transferred to Hybond N, hybridized with the digoxigenin-labeled probe, and washed twice at 0.1 x SSC, 65°C, and visualized by chemiluminescence autoradiography (Amersham), 2-h exposure. The degree of lane loading was established by methylene blue staining and by probing for GAPDH.
For immunoblotting, cell monolayers were rinsed twice with ice-cold balanced salt solution (BSS) followed by ice-cold lysis buffer (25 mM Tris?HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 1 mM PMSF, 1 μg/μl leupeptin, 1 μg/μl pepstatin). Cells were incubated in lysis buffer for 20 min at 4°C. Lysates were collected by scraping and were centrifuged at 15,000 g for 5 min at 4°C. After protein concentration of the supernatant was measured as above, cell lysates (30 μg/lane) were subjected to SDS-PAGE (10% polyacrylamide). Proteins were separated by SDS-PAGE under reducing conditions (2 mM -mercaptoethanol added to loading buffer) for immunoblot of COX-1 and COX-2 and under nonreducing conditions for immunoblot of PGT (3). Proteins were transferred to nitrocellulose membranes (Optitran, Schleicher & Schuell, Keene, NH) by electroporation. Blots were blocked (5% nonfat dry milk, 1% Tween 20 in PBS) for 1 h, then immunostained for 2 h with the following primary antibodies: anti-PGT [undiluted hybridoma cell culture media, anti-rat mouse monoclonal (3)], anti-COX-2 (1:1,000, anti-sheep goat polyclonal, Cayman Chemical, Ann Arbor, MI), or anti-COX-1 (1:1,000, anti-sheep mouse monoclonal, Cayman Chemical). After three rinses with PBS containing 1% Tween 20 (PBS/Tween), blots were incubated with appropriate horseradish peroxidase (HRP)-coupled secondary antibodies (1:5,000) in PBS/Tween for 1 h. Blots were rinsed three times again with PBS/Tween, after which protein bands were detected by chemiluminescence (New England Nuclear). For quantification, blots were stained with Coomassie Blue and scanned for total protein per lane.
PGE2 influx measurements. Cell monolayers were rinsed twice with BSS (135 mM NaCl, 5 mM KCl, 13 mM [H]HEPES, 13 mM Na-HEPES, 2.5 mM CaCl2, 1.2 mM MgCl2, 0.8 mM MgSO4, 25 mM glucose). PG influx was initiated by addition of BSS containing [3H]PGE2 (0.6 nM). Influx measurements were carried out at room temperature and were terminated by aspiration of the influx solution, followed by rapid rinsing with ice-cold BSS containing 5% BSA and two additional rinses with ice-cold BSS. Monolayers were lysed with 5% SDS for 10 min at room temperature. An aliquot of each lysate was set aside to measure protein concentration by a modified Lowry method (DC Protein Assay, Bio-Rad, Hercules, CA). The remaining lysates were mixed with 10 ml of liquid scintillation solution (National Diagnostics) and radioactivity was quantified by liquid scintillation spectroscopy. Influx values were calculated as the mean of duplicate monolayers and expressed as femtomoles per milligram of protein per nanomolar tracer PGE2. In some experiments, influx measurements were performed in the presence and absence of excess unlabeled PGE2 (1 μM) or bromcresol green (BCG; 10 μM).
PGE2 release measurements. Cell monolayers were rinsed twice with BSS followed by addition of BSS without or with endothelin-1 (ET-1; 10 nM; Peptide International), calcium ionophore (A23187 , 10 μM), or arachidonic acid (AA; 10 μM) for 5 min. Release assays were performed at 37°C. Assays were terminated by collection of an aliquot of assay solution. Aliquots were stored at 70°C until measurement of PGE2 concentration. Cells were lysed in 5% SDS and an aliquot was taken for protein measurement, as above. PGE2 concentrations in assay samples were measured by enzyme immunoassay (Cayman Chemical). Release values were calculated as the mean of duplicate monolayers and expressed as nanograms per milliliters per milligram of protein per 5 min. In some experiments, RMICs were incubated for 15 min before and during PGE2 release measurements without or with a nonselective COX inhibitor [10 μM indomethacin (Indo)] or a selective COX-2 inhibitor (5 μM NS-398; Calbiochem, San Diego, CA).
Immunocytochemistry. RMICs grown on glass coverslips were blocked in 510% goat serum, incubated overnight at 4°C with mouse monoclonal antibody no. 20 as straight hybridoma supernatatant (3), washed, and incubated for 1 h in FITC-coupled goat anti-mouse IgG at 1:2,000 (Alexa Fluor 488, Molecular Probes, Invitrogen, Eugene, OR). Negative controls consisted of omission of the primary antibody. Photomicroscopy was performed using a Bio-Rad Laser Confocal Microscope.
Lactate concentration. Cell monolayers were grown overnight in media with and without 100 mosmol/kgH2O added NaCl, after which the medium was collected and assayed for lactate concentration using a commercially available kit (Sigma) that uses a colorimetric assay based on the enzymatic conversion of lactic acid to pyruvate and hydrogen peroxide by lactate oxidase. Due to the long incubation, it was assumed that lactate was in equilibrium between the intracellular and extracellular compartments and, therefore, extracellular would be proportional to intracellular .
Statistical analysis. Values represent means ± SE. Statistical analysis was performed by Student's t-test or by ANOVA followed by Newman-Kuels modified t-test as a post hoc analysis. The null hypothesis was rejected at P < 0.05.
RESULTS
Endogenous PGT expression in cultured RMICs. Rat RMICs were probed for expression of PGT mRNA and protein by Northern blotting and immunoblotting, respectively. Figure 1 shows that a single 4.4-kb RNA band from cultured rat RMICs hybridized with a rat PGT antisense RNA probe (left). This mRNA band comigrated with a PGT band from whole rat kidney RNA (right); both were in accord with previously published results on rat PGT RNA from our laboratory (22). The additional band detected in whole rat kidney may derive from PGT expressed outside of RMICs by glomeruli, endothelia, and/or collecting ducts (3).
Similarly, as shown in Fig. 2, RMIC whole cell lysates probed with an anti-rat PGT monoclonal antibody (3) revealed a single immunoreactive band at 65 kDa. This band is similar in size to that of PGT from rat kidney as reported previously (3) and is also similar in size to rat liver PGT (Fig. 2, right). The slight difference in migration between RMIC PGT and liver PGT is similar to variations we observed among various tissues or cell types (Bao Y and Schuster VL, unpublished observations). The basis for this variation is currently unknown but presumably is due to factors such as alternative RNA splicing and/or differential glycosylation.
To determine whether PGT protein in cultured RMICs is expressed functionally at the plasma membrane, we examined the timed uptake of radioactive PGs from the medium. Figure 3 demonstrates that [3H]PGE2 undergoes rapid, time-dependent uptake by cultured RMICs. In contrast, control HeLa cells, which lack PGT expression, exhibited very low [3H]PGE2 uptake. The "overshoot" pattern seen here with PGE2 uptake by cultured RMICs is characteristic of PGT-mediated PG transport and is probably due to the time-dependent dissipation of an outwardly directed lactate gradient during the uptake assay (9, 10).
The affinities of the RMIC uptake step for several prostanoids are compared with those of the cloned, recombinant rat PGT in Fig. 4 (21, 22, 32). Figure 4A shows the uptake of [3H]PGE2 in RMICs and in HeLa cells transiently transfected with recombinant rat PGT ("HeLa-PGT") and the competition for this uptake by unlabeled PGE2 or thromboxane B2 (TxB2) as a function of their concentrations in the uptake solution. Although the dose-response curves are offset from each other, unlabeled PGE2 and unlabeled TxB2 result in similar competition curves in RMICs and HeLa-PGT cells. Double-reciprocal plots (not shown) indicated that the inhibitor constants (Ki, nM) for PGE2 were 178 and 176 nM in RMIC and HeLa-PGT cells, respectively, and for TxB2 were 709 and 682 nM in RMIC and HeLa-PGT cells, respectively (P = NS for all RMIC vs. HeLa-PGT comparisons). Similarly, Fig. 4B shows that PGE2 had a similar affinity for the transporter in RMICs and in HeLa-PGT cells; the relevant Ki values were 462 nM for RMICs and 362 nM for HeLa-PGT cells. Figure 4C demonstrates similar results for the prostacyclin agonist U46619 , for which the Ki values were 823 nM for RMICs and 1,059 nM for HeLa-PGT cells (P = NS for all RMIC vs. HeLa-PGT comparisons in Fig. 4, B and C).
We extended this analysis by examining the inhibition of tracer PGE2 uptake by a single fixed dose of each of several other eicosanoids that exhibit lower affinities for rat PGT (21, 32). As shown in Table 1, PGE2 isopropylester, 15-keto PGE2, and 13,14 dihydro 15-keto PGE2 showed comparable inhibition, or lack thereof, in RMICs compared with HeLa-PGT cells.
To complete the characterization of endogenous PGT in RMICs, we compared the affinities of several organic anion transport inhibitors that block rat PGT. Figure 5 shows the dose-dependent inhibition of [3H]PGE2 uptake by BCG (22), DIDS (10), and indocyanine green (ICG) (22). As with competition by the unlabeled eicosanoids, these organic anions blocked PG uptake in RMICs and HeLa-PGT over comparable dose-response ranges. Double-reciprocal plots indicated that the relevant Ki values were as follows. For BCG: 8.7 μM for RMICs and 12.3 μM for HeLa-PGT cells; for DIDS: 32.2 μM for RMICs and 42 μM for HeLa-PGT cells; and for ICG: 2.7 μM for RMICs and 1.6 μM for HeLa-PGT cells (P = NS for all RMIC vs. HeLa-PGT comparisons).
Effect of increased osmolarity on PGT function in RMICs. We exposed confluent cultures of RMICs to media containing additional NaCl, an important osmolyte in the renal medulla. Increasing osmolarity by an amount equal to 50 to 200 mosmol/kgH2O (final osmolarity 335485 mosmol/kgH2O) for 24 h resulted in a concentration-dependent decrease in PGE2 uptake by RMICs (Fig. 6). PGE2 uptake in cells exposed to 200 mosmol/kgH2O of added NaCl for 24 h was indistinguishable from uptake by cells coincubated with tracer PGE2 plus a large excess of unlabeled PGE2 (data not shown), indicating complete suppression of PGT-mediated uptake by NaCl. A similar, albeit less intense, effect on PGE2 uptake was elicited by urea (Fig. 7).
As shown in Fig. 8A, PGT function began to decrease rapidly after exposure to increased NaCl. The effect was maximal after 24 h and persisted for at least 120 h. The NaCl-induced decrease in PGT function was reversible on returning the cells to isosmolar media (Fig. 8B), although the return of function was somewhat slower than the previous decline.
One possible explanation for the change in tracer PG uptake was that the driving force for PG uptake by PGT, i.e., the cell-to-medium lactate concentration gradient (9), was altered by hyperosmolarity. However, cellular lactate concentrations at the start of the timed uptake assay were not different in control cells and cells incubated with high salt (32.6 ± 6.2 vs. 36.8 ± 5.9 mM, for cells grown in media without or with 100 mosmol/kgH2O additional NaCl, respectively).
We examined whether the change in PGT transport was associated with a parallel change in PGT cell-surface protein expression. Figure 9A indicates that in control RMICs, PGT was distributed primarily in punctate cytoplasmic vesicles. Despite the clear presence of PGT at the plasma membrane by uptake assays, we were unable to adequately quantify cell-surface PGT using biotinylation approaches, suggesting that most of the PGT resides within cytoplasmic vesicles. Importantly, exposure of cultured RMICs to hyperosmolar medium for 24 h caused a dramatic internalization of PGT to an apparently nuclear distribution (Fig. 9A). Immunoblotting revealed that this redistribution was not accompanied by a change in total cellular PGT protein expression (Fig. 9B).
Effect of increased osmolarity on net PGE2 release by RMICs. To determine whether exposure of RMICs to increased osmolarity affected PG synthesis and release, we measured net PGE2 release as stimulated by ET-1, the calcium ionophore A23187 , or AA. As expected, under isosmolar conditions, all three agonists greatly stimulated net PGE2 release from RMICs (in units of ng?5 min1?mg protein1: 3.1 ± 0.2 for ET-1, 9.1 ± 0.4 for A23187 , 44.6 ± 2.3 for AA vs. basal release of 0.83 ± 0.04).
As shown in Fig. 10, overnight incubation in media containing 50, 100, or 200 mosmol/kgH2O excess NaCl further increased net PGE2 release in response to these agonists. The increases were greater as osmolarity increased. The induction of ET-1- and A23187 -stimulated net release was comparable at each level of hyperosmolarity, whereas the induction of AA-stimulated net PGE2 release was less at the highest osmolarity. The time course shown in Fig. 11 illustrates that, after exposure of RMICs to hyperosmolar media, A23187 -stimulated net PGE2 release increased rapidly in a fashion similar to the osmolarity-induced decrease in PGT function.
To determine the contribution of COX-2 to the synthesis of PGs by RMICs, we compared net PGE2 release in the absence and presence of indomethacin or NS-398. As shown in Table 2, both COX inhibitors greatly decreased basal and A23187 -induced net PGE2 release from RMICs grown in isosmolar and hyperosmolar media. The proportional effect of NS-398 was similar in RMICs grown in isosmolar and hyperosmolar media. Thus, as reported by others (1416, 25, 39), PGE2 synthesis in RMICs is mediated primarily by COX-2 under both iso- and hyperosmotic conditions.
We also examined the effects of hyperosmolarity on COX-2 protein expression using immunoblot analysis. As illustrated in Fig. 12, A and C, COX-2 increased approximately threefold after 24-h exposure to 100 and 200 mosmol/kgH2O added NaCl. A concentration-dependent increase in COX expression between 100 and 200 mosmol/kgH2O NaCl was not discernible.
Long-term effects of increased osmolarity. Whereas most studies on hyperosmolarity focus on a short experimental time frame, water deprivation in nature can be sustained over time, e.g., during drought. To more closely mimic these events, we grew RMICs in media without or with 100 mosmol/kgH2O added NaCl for 710 days. The cells grew well with no apparent differences in doubling rate, viability, or subculturing efficiency compared with cultures grown in isosmolar media.
As illustrated in Fig. 12, B and C, COX-2 immunoreactivity did not remain elevated in lysates from cells grown in hyperosmolar media for 710 days. In separate studies, we found that COX-1 protein was also not elevated at these late time points (data not shown).
After 710 days in hyperosmolar media, basal and AA-stimulated net PGE2 release from RMICs were no longer higher than control (basal; 0.83 ± 0.10 vs. 1.04 ± 0.16: AA-stimulated; 44.6 ± 2.5 vs. 45.1 ± 2.3 ng?ml1?mg protein1, isosmolar vs. hyperosmolar, respectively). On the other hand, both ET-1- and A23187 -stimulated net PGE2 release remained elevated (6-fold over control; Fig. 13).
Because this increase in net agonist-induced PGE2 release occurred in the face of a return of COX-2 levels to control values, we examined PGT expression. As shown in Fig. 14, PGT function remained markedly suppressed in RMICs grown for 710 days in hyperosmolar media.
DISCUSSION
We demonstrated here that cultured rat RMICs take up PGE2 from the extracellular medium in a time-dependent and concentrative fashion. The substrate selectivity and inhibitor sensitivity of the transport, and the presence of the appropriate hybridizing mRNA and immunoreactive protein, indicate that the prostaglandin transporter PGT is mediating the uptake. Exposure of cultured rat RMICs to hyperosmolarity, either in the form of NaCl or urea, causes a sustained increase in the release of PGE2. This increase is accompanied by a transient increase in COX-2 expression and a sustained reduction in cell-surface PGT expression. The data are consistent with a model in which pericellular PG concentrations are controlled at the levels of both release and uptake.
At present, we can only speculate on the physiological role of PG uptake by RMICs. Medullary interstitial PGE2 suppresses osmotic water reabsorption by the collecting duct (7, 30) and solute resorption by the mTALH (33) and induces vasodilation of medullary vasa rectae (27, 40). The latter buffers vasoconstriction so that blood flow is not completely abrogated in this region. PGE2 uptake by RMICs would presumably lower medullary interstitial PGE2 concentrations, increasing ADH action on the collecting duct and mTALH and decreasing medullary blood flow, all of which would decrease urinary salt and water excretion.
Alternatively or additionally, PGT may play a role in controlling RMIC survival. PGE2 generated by COX-2 in RMICs, particularly in response to hypertonicity, prevents RMIC apoptosis (4, 8, 1416, 25, 29, 36, 37, 39, 41, 42). To the extent that PGT expression at the plasma membrane of these cells would lower pericellular PGE2 concentrations, PGT would be predicted to be proapoptotic in RMICs. The reduction of cell-surface PGT expression in response to hyperosmolarity, along with induction of COX-2, would be predicted to protect RMICs from osmolarity-induced apoptosis.
Hyperosmolarity appears to regulate PGT by altering cell-surface protein expression. Aquaporins (23), epithelial sodium channels (13), glucose transporters (19, 20), and other transporters reside in a vesicular reservoir that can cycle through the plasma membrane (5, 6, 26). Immunocytochemistry revealed that PGT in cultured RMICs is expressed in cytoplasmic vesicles, a pattern that mimics PGT expression in RMICs in situ (3). Although plasma membrane PGT was below the limit of detection using biochemical (biotinylation) detection methods, it is clearly present at the plasma membrane as demonstrated by tracer uptake measurements. In response to hyperosmolarity, PGT function at the cell surface decreases and immunoreactive protein is further internalized, suggesting that PGT, like other plasma membrane transporters, is regulated by membrane insertion and/or retrieval.
Incubation of RMICs in hyperosmolar media greatly increased the net release of PGE2. Early on, basal as well as ET-1-, A23187 -, and AA-stimulated PGE2 release were augmented, an effect accompanied by an increase in COX-2 expression. These data are in accord with several other studies in which dehydration of rats resulting in medullary hyperosmolarity (38), and exposure of cultured rabbit renal interstitial cells to a hyperosmolar environment (15), resulted in increased expression of COX-2 and increased PGE2 synthesis/release.
A new feature of our studies is the observation that, after 710 days exposure to hyperosmolarity, both A23187 and endothelin could still stimulate increased net PGE2 release in the hyperosmolar state despite low COX-2 expression. Changes in cell-surface receptors cannot explain the endothelin results, as ET-A receptors are downregulated after exposure to hyperosmolarity (36). It is possible that phospholipase-mediated release of arachidonic acid from membrane stores might be upregulated by long-term hyperosmolarity. However, a more likely explanation for the increase in net PGE2 release is the dramatic reduction in cell-surface activity of PGT at 710 days.
These studies add to our understanding of the coordinate control of COX and PGT. Work from our laboratory demonstrated that serum coordinately induces both COX-2 and PGT in cultured Swiss 3T3 fibroblasts (R. Lu and V. L. Schuster, unpublished observations). Additionally, a recent report on the bovine corpus luteum showed that PGT and COX-2 were coordinately regulated during the estrus cycle (2). Presumably, a system that regulates both PG synthesis and PG uptake permits finer tuning of PGE2 signaling at cell-surface receptors compared with induction of either alone.
In summary, cultured RMICs take up PGE2 from the extracellular fluid via the prostaglandin transporter PGT. COX-2 and PGT are coordinately regulated in these cells by extracellular osmolarity. Hypertonicity acutely increases COX-2 expression, but over 710 days continued exposure COX-2 returns toward baseline. Despite the reduction in COX expression, PGE2 release remains elevated. Hypertonicity acutely lowers PGT cell-surface expression, which remains suppressed during several days' exposure to hypertonicity. The combined regulation of COX and PGT by hypertonicity suggests that pericellular PG concentrations are controlled at the levels of both release and uptake in these cells.
ACKNOWLEDGMENTS
This work was supported by National Institutes of Health Grants R01-DK-049688 and P50-DK-064236 to V. L. Schuster. We thank the Einstein Analytical Imaging Facility for use of the confocal microscope.
FOOTNOTES
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.
M. L. Pucci and S. Endo contributed equally to this work.
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