Key role for constitutive cyclooxygenase-2 of MDCK cells in basal signaling and response to released ATP

Rennolds S. Ostrom, Caroline Gregorian, Ryan M. Drenan, Kathryn Gabot, Brinda K. Rana, and Paul A. Insel

Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0636


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Madin-Darby canine kidney (MDCK) cells release ATP upon mechanical or biochemical activation, initiating P2Y receptor signaling that regulates basal levels of multiple second messengers, including cAMP (J Biol Chem 275: 11735-11739, 2000). Data shown here document inhibition of cAMP formation by Gd3+ and niflumic acid, channel inhibitors that block ATP release. cAMP production is stimulated via Ca2+-dependent activation of cytosolic phospholipase A2, release of arachidonic acid (AA), and cyclooxygenase (COX)-dependent production of prostaglandins, which activate prostanoid receptors coupled to Gs and adenylyl cyclase. In the current investigation, we assessed the expression and functional role of the two known isoforms of COX, COX-1 and COX-2. Treatment of cells with either a COX-1-selective inhibitor, SC-560, or COX-2-selective inhibitors, SC-58125 or NS-398, inhibited basal and UTP-stimulated cAMP levels. COX inhibitors also decreased forskolin-stimulated cAMP formation, implying this response is in part attributable to an action of AA metabolites. These findings imply an important role for the inducible form of COX, COX-2, under basal conditions. Indeed, COX-2 expression was readily detectable by immunoblot, and treatments that induce or reduce COX-2 expression in other cells (interleukin-1beta , tumor necrosis factor-alpha , phorbol ester, or dexamethasone) had minimal or no effect on the levels of COX-2 immunoreactivity. RT-PCR using isoform-specific primers detected COX-2 mRNA. We conclude that COX-2 is constitutively expressed in MDCK-D1 cells and participates in basal and P2Y2-mediated signaling, implying a key role for COX-2 in regulation of epithelial cell function.

adenosine 3',5'-cyclic monophosphate; adenosine 5'-triphosphate release; arachidonic acid; prostaglandin E2


    INTRODUCTION
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ABSTRACT
INTRODUCTION
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CYCLOOXYGENASE (COX) catalyzes the production of eicosanoids, such as prostaglandins and thromboxanes, which are integral to the regulation of many biological responses, including platelet activation, tissue perfusion, and inflammation. Of the known isoforms of COX, COX-1 is constitutively expressed and thereby involved in ambient events in cells, while COX-2 is considered an inducible isoform, which is regulated by cytokines and hormonal mediators and thus thought to be the principal isoform that mediates inflammatory processes (28).

We have recently described a critical regulatory signaling pathway in cells that is dependent on COX (16). Using the well-differentiated renal tubular epithelial cell line Madin-Darby canine kidney (MDCK), we showed that activation of this pathway is initiated by cellular release of ATP after biochemical or mechanical stimulation. This released ATP helps to establish the basal levels of cellular second messengers via activation of P2Y receptors. MDCK-D1 cells express multiple isoforms of P2Y receptors (18), which couple to the activation of cytosolic phospholipase A2 (cPLA2) via both Ca2+- and mitogen-activated protein (MAP) kinase-dependent mechanisms (31). cPLA2 cleaves arachidonic acid (AA) from membrane phospholipids and serves as the rate-limiting step for production of eicosanoids, in particular PGE2, by COX (18, 23). In the current studies, we examined the role of COX-1 and COX-2 to infer which isoform(s) mediate the generation of products involved in regulating basal signaling. Unexpectedly, we found that both COX isoforms, but particularly COX-2, are expressed in a constitutive manner in MDCK-D1 cells and contribute to the generation of basal levels of cAMP and in modulation of cellular response to various hormonal agonists.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Cell culture reagents were obtained from Fisher. Cells were routinely cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (endotoxin free and heat inactivated), except where noted. Radiolabeled chemicals were obtained from NEN Life Science Products. Forskolin was obtained from Calbiochem. NS-398 was obtained from Cayman Biochemicals. SC-560 and SC-58125 were generous gifts from Monsanto-Searle. Primary antibody for COX-1 was obtained from Cayman Biochemicals. All other antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). All other drugs and reagents were obtained from Sigma (St. Louis, MO).

Cell culture. MDCK-D1 cells were grown in DMEM supplemented with 2.5% fetal bovine serum and 7.5% horse serum. Cells were passaged every 3-4 days by trypsinization using trypsin-EDTA. Cells were used for experiments in 24-well plates (Costar) grown to approximately 60-70% confluence. In some experiments, cells were washed and cultured in serum-free DMEM for 24-48 h before assay.

Luciferin/luciferase detection of ATP. For luciferin/luciferase detection of ATP, we washed cells with 0.5 ml DMEM buffered with 20 mM Na-HEPES (DMEH, pH 7.4) by carefully aspirating media without tilting the tissue culture plate and then adding fresh media slowly to the side of the well. The cells were then incubated for 60 min (to allow reequilibration and hydrolysis of released ATP) before a small volume of drug (typically 50 µl) was gently applied in a dropwise fashion without agitation of the plate. We collected 100 µl of the medium 5 min later and centrifuged this material to eliminate cell contaminants. An ATP bioluminescence kit containing luciferin/luciferase reagent was used to detect ATP in each sample (ATP bioluminescence assay kit HS II, Roche Molecular Biochemicals), and luminescence was measured in a Monolight 2010 tube-reading luminometer. Bioluminescence controls were performed with each drug solution to eliminate drug effect on luciferase activity as well as to control for ATP contamination. In this and other assays, some cells were subjected to physical perturbation by agitation of the media: five 50-µl squirts of media in an up-and-down fashion with a pipetteman.

Assay of cAMP. Cells were labeled with 1 µCi/well of [3H]adenine in growth media for 90 min to allow incorporation of radiolabel into intracellular ATP pools. Growth medium was removed, and cells were washed extensively and equilibrated for 30 min at 37°C in DMEH. Cells were then incubated for 10 min with either 200 µM 3-isobutyl-1-methylxanthine or 100 µM Ro-20-1724 (to inhibit phosphodiesterases) along with various drugs of interest. Reactions were terminated by aspiration of medium and addition of 7.5% trichloroacetic acid. Approximately 1,000 cpm of [32P]cAMP internal standard was added to each sample, and the volume was brought to 1 ml with water. [3H]cAMP and [3H]ATP were separated from the supernatant fraction using a chromatography method modified from Ref. 21, as described previously (16).

[3H]AA release in intact cells. Cells were labeled with [3H]AA by incubation with 0.5 µCi [3H]AA (specific activity 100 Ci · mmol-1 · ml-1) for ~20 h in 24-well plates. Cells were washed three times with serum-free DMEM containing 20 mM HEPES buffer (DMEH, pH 7.4) supplemented with 5 mg/ml bovine serum albumin (BSA) and allowed to equilibrate at 37°C for 15 min. This equilibration medium was aspirated, and drugs of interest were added to the wells and incubated with cells for 20 min. Assays were terminated by removal of medium and transferring this medium into tubes containing 50 µl of 55 mM EDTA with 55 mM EGTA. Two-hundred fifty microliters of 0.5% Triton X-100 were added to each well to solubilize cellular membranes. Liquid scintillation counting was performed to quantitate released [3H]AA in media. The results were normalized as a percentage of incorporated radioactivity measured from detergent-solubilized cells.

Assay of PGE2 production. Cells were cultured and treated with various drugs, as described above for assay of cAMP. Fifty microliters of media were removed from cells after 10 min of drug exposure and assayed for PGE2 using a commercial monoclonal EIA kit (Cayman Chemical, Ann Arbor, MI) according to manufacturer's instructions. Results were read in a Vmax plate-reading spectrophotometer (Molecular Devices), and data are expressed as picograms of PGE2 per milliliter.

Immunoblot analysis. Whole cell lysates were prepared by scraping cells in lysis buffer (50 mM Tris · HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, protease inhibitor cocktail), homogenizing with a Polytron, and sonicating. Proteins from whole cell lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes by electroblotting. Membranes were blocked in 20 mM phosphate-buffered saline (PBS) with 3% nonfat dry milk and incubated with primary antibody (see Materials) overnight at 4°C. Bound primary antibodies were visualized using an appropriate secondary antibody with conjugated horseradish peroxidase (Santa Cruz Biotechnology) and enhanced chemiluminescence reagent (Amersham Pharmacia Biotech). The amount of protein per fraction was determined using a dye-binding protein assay (Bio-Rad). In some cases, membranes were stripped using Re-Blot reagent (Chemicon International) and reprobed with another primary antibody.

RT-PCR. Primer pairs were designed to selectively amplify either COX-1 or COX-2 based on published species-conserved sequences. Primer sequences were COX-1, 5'-CGA GCC CAG TTC CAA TAT CG-3' and 3'-ACC CCA TAG TCC CAC CAG CAT AG-5'; COX-2, 5'-AC ATC CTG ACC CAC TTC AAG-3' and 3'-CA GGT CCT CGC TTA TGA TCT-5'. Total RNA and genomic DNA was isolated from subconfluent MDCK-D1 cells using TRIzol reagent (Life Technologies) following the manufacturer's protocol. RNA was reverse transcribed using SuperScript II (Life Technologies) and poly-T priming. Twenty-five cycles of PCR were performed using isoform-specific primers using either cDNA, genomic DNA as a positive control, or non-reverse transcribed mRNA as a negative control. In the case of COX-1 RT-PCR, mouse colon cDNA served as a positive control. PCR products were visualized under UV light after gel electrophoresis in 2% agarose containing ethidium bromide.

Data presentation and analysis. Data were obtained in triplicate, averaged for each condition in an experiment, and are presented as means ± SE of at least three experiments. A paired t-test was used to determine statistical significance. For concentration-response relationships, the data were fit by nonlinear regression analysis (with variable slope) using Prism by GraphPad (San Diego, CA). EC50 and maximal response are reported as means ± SE of at least three individual experiments. For immunoblot data, a representative image is shown of two or three individual experiments.


    RESULTS
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have previously reported that cultured cells release ATP and other nucleotides during media change or other mechanical manipulation, resulting in increased levels of multiple second messengers in MDCK-D1 cells (16). In addition, we found that addition of various agonists, but most prominently UTP, also could enhance ATP release (16). In the present studies, we tested two anion channel inhibitors as possible inhibitors of the ATP release channel: gadolinium chloride (Gd3+) and niflumic acid (20, 22). Gentle squirting of cells with media delivered with a pipetteman activated ATP release from MDCK-D1 cells, increasing levels of extracellular ATP from 0.3 ± 0.1 to 10.0 ± 1.1 nM. Addition of 2 mM Gd3+ or 0.5 mM niflumic acid blocked both this stimulated release and basal release, reducing extracellular ATP concentrations roughly in half (Fig. 1, A and B). Basal and forskolin-stimulated cAMP accumulation was reduced by inclusion of either Gd3+ or niflumic acid (Fig. 1, A and C). We have previously demonstrated that released nucleotides activate P2Y receptors coupled to an indomethacin-sensitive (cyclooxygenase-dependent) production of prostaglandins, which act via Gs to synergize with forskolin in stimulating adenylyl cyclase activity (16, 17). Therefore, Gd3+ and niflumic acid appear to inhibit this synergy with forskolin by blocking the release of nucleotides to the extracellular space.


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Fig. 1.   Blockade of basal and stimulated ATP release and cAMP production in Madin-Darby canine kidney (MDCK)-D1 cells by Gd3+ and niflumic acid. A: unstimulated levels of ATP release (solid bars) and cAMP accumulation (hatched bars) in control cells or cells treated with 2 mM Gd3+, 0.5 mM niflumic acid, or 1 µM indomethacin. B: extracellular ATP concentrations in basal conditions and after media disturbance (see MATERIALS AND METHODS) in cells incubated with vehicle (control), 2 mM Gd3+, or 500 µM niflumic acid. C: cAMP accumulation in basal conditions and after media disturbance along with 10 µM forskolin in cells incubated with vehicle (control), 2 mM Gd3+, or 500 µM niflumic acid. Each bar represents mean ± SE of 3-6 experiments. * P < 0.05, ** P < 0.01 by paired t-test compared with control condition.

Consistent with this conclusion, 2 mM Gd3+ or 0.5 mM niflumic acid also inhibited basal levels of AA release and PGE2 production (data not shown), implying that the resting levels of these second messengers are also determined, in large part, by the release of ATP via membrane channels. Moreover, the ATP release inhibitors were able to reduce basal cAMP levels to the same extent as did the cyclooxygenase inhibitor indomethacin (1 µM, Fig. 1A) (16).

Recent reports indicate that COX-2, the inducible form of COX, is constitutively expressed in certain regions of the kidney (6). To investigate which COX isoform(s) might play a role in cAMP generation in response to activation by endogenous ATP or exogenous nucleotides acting via P2Y receptors, we utilized COX isoform-selective inhibitors. Concentration dependence and extent of inhibition of three isoform-selective COX inhibitors were examined. Cells were treated with various concentrations of SC-560, SC-58125, or NS-398, and the cAMP response to a maximal concentration of UTP (100 µM) was measured. The COX-1-selective inhibitor, SC-560, maximally inhibited UTP-stimulated cAMP accumulation 51% with an IC50 of 0.68 µM (Fig. 2A). The reported IC50 values for SC-560 are 9 nM for COX-1 and 6.3 µM for COX-2 (26). The COX-2-selective inhibitor, SC-58125, maximally inhibited UTP-stimulated cAMP accumulation 84% with an IC50 of 46 nM (Fig. 2A). The reported IC50 values for SC-58125 are >10 µM for COX-1 and 50 nM for COX-2 (25). The commercially available COX-2-selective inhibitor, NS-398, inhibited UTP-stimulated cAMP with similar extent of inhibition as SC-58125 but with higher potency [IC50 of 2.1 nM, published IC50 values are 75 µM for COX-1 and 1.8 µM for COX-2 (1)]. The relative concentration dependence and extent of inhibition of both SC-560 and SC-58125 in inhibiting UTP-stimulated PGE2 production (Fig. 2B) were similar to those for inhibition of cAMP formation (Fig. 2A). UTP (100 µM) stimulated 82.26 pg/ml of PGE2, and these levels were inhibited a maximal 69% and 88% by SC-560 and SC-58125, respectively. Indomethacin inhibited UTP-stimulated PGE2 production by 97% (Fig. 2B, dashed line). Therefore, the COX-2-selective inhibitor inhibits a much larger proportion of nucleotide-mediated responses than the COX-1 inhibitor. SC-560 and SC-58125 also reduced both basal and mechanically-stimulated PGE2 production (Fig. 2C).


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Fig. 2.   Stimulated cAMP accumulation and prostaglandin (PG) E2 production is sensitive to both cyclooxygenase-1 (COX-1)- and COX-2-selective inhibitors. A: inhibition of UTP-stimulated cAMP accumulation by increasing concentrations of isoform-selective COX inhibitors SC-560 (COX-1, ), SC-58125 (COX-2, ), and NS-398 (COX-2, black-triangle). Dashed line indicates level of inhibition by indomethacin (Indo; 1 µM). B: inhibition of UTP-stimulated PGE2 production by increasing concentrations of isoform-selective COX inhibitors SC-560 (COX-1, ) and SC-58125 (COX-2, ). Dashed line indicates level of inhibition by indomethacin (1 µM). C: inhibition of PGE2 levels in control cells and cells activated by media disturbance (see MATERIALS AND METHODS) by isoform-selective COX inhibitors SC-560 (COX-1, hatched bars) and SC-58125 (COX-2, cross-hatched bars). Each point or bar represents mean ± SE of 3-4 experiments. * P < 0.05 by paired t-test compared with basal condition.

cAMP accumulation stimulated by forskolin (0.1 µM) or forskolin plus the P2Y2 agonist UTP (100 µM) was sensitive to the pretreatment with either the nonselective COX inhibitor indomethacin or the selective inhibitors SC-560 or SC-58125 (Fig. 3A). Bradykinin and alpha -adrenergic receptors, which also stimulate cAMP production in these cells in a COX-dependent manner (16), were also inhibited by both nonselective and COX-1- and COX-2-selective inhibitors (data not shown).


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Fig. 3.   COX-1- and COX-2-dependent cAMP accumulation in control and serum-starved MDCK-D1 cells. A: inhibition of cAMP stimulated by 0.1 µM forskolin (Fsk) and forskolin plus 100 µM UTP in control cells (open bars) or cells treated with indomethacin (1 µM, solid bars), SC-560 (1 µM, hatched bars), or SC-58125 (1 µM, cross-hatched bars). B and C: repeat of the study in A in 24-h (B) and 48-h (C) serum-starved MDCK-D1 cells. Each bar represents mean ± SE of 3-6 experiments. * P < 0.05, ** P < 0.01 by paired t-test compared with control condition.

Because these data implied a potentially important role for basally expressed COX-2 in MDCK-D1 cells, we investigated whether serum in standard growth medium (DMEM supplemented with 10% fetal bovine serum) might induce the expression of COX-2. We thus replaced growth medium on MDCK-D1 cells with serum-free medium for either 24 or 48 h before assaying for cAMP accumulation (longer periods of serum withdrawal negatively impacted cell viability). In 24- and 48-h serum-starved cells, cAMP accumulation stimulated by forskolin plus UTP (100 µM) was sensitive to both COX-1 (SC-560) and COX-2 (SC-58125) inhibitors, although forskolin-promoted increases in cAMP lost the sensitivity to COX inhibitors at 48 but not 24 h (Fig. 3, B and C). The loss in sensitivity to COX inhibitors of the forskolin response likely does not reflect alterations in ATP release or P2Y receptor expression in cells grown for 48 h in serum-free conditions, since UTP-stimulated cAMP was not decreased, and mechanically stimulated ATP release was unaltered (data not shown). Overall, the data imply that the COX-2 isoform plays the predominant role in the autocrine/paracrine signaling pathway by which nucleotides, in particular UTP active at P2Y2 receptors, regulate levels of cAMP in MDCK-D1 cells.

To confirm the presence of both COX isoforms, immunoblot analysis was used to detect COX protein in lysates of control and serum-starved MDCK-D1 cells. COX-2 and COX-1 immunoreactivity was readily detectable in equal amounts in control cells, 24-h serum-starved cells, 48-h serum-starved cells, and 48-h serum-starved cells that been returned to serum for 24 h (Fig. 4A, left). Therefore, withdrawal of serum in the growth media did not result in decreased COX-2 expression. Incubation of cells with 10 µM dexamethasone for 24 h also had virtually no effect on the amount of COX-2 immunoreactivity (Fig. 4A, right). COX-2 expression in various cells is induced on exposure to interleukin (IL)-1beta , tumor necrosis factor (TNF)-alpha , or the phorbol ester, phorbol 12-myristate 13-acetate (PMA; Refs. 4 and 13). Therefore, we exposed MDCK-D1 cells to either 1 µM IL-1beta , 1 µM TNF-alpha , or both (Fig. 4B, left) or 1 µM PMA (Fig. 4B, right) for 24 h. Cells exposed to TNF-alpha , IL-1beta , both, or PMA did not display increased COX-2 immunoreactivity. COX-1 immunoreactivity was also detectable in these conditions but was also not induced (Fig. 4B). In contrast, both inducible nitric oxide synthase (iNOS) and cPLA2 immunoreactivity were induced by the above treatments. These data indicate that COX-2 is constitutively expressed and, apparently, not readily subject to induction or deinduction by the types of factors and hormones that regulate COX-2 in other cells.


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Fig. 4.   Immunoblot analysis of COX isoform expression in control, serum-free, and -induced conditions. A: COX-2 and COX-1 immunoreactivity in lysates from MDCK-D1 cells cultured without serum for 24 or 48 h and in cells returned to serum after 48-h serum starvation (A, left) and in cells treated for 24 h with vehicle or 10 µM dexamethasone (A, right). B: COX-2, COX-1, cytosolic phospholipase A2 (cPLA2), and inducible nitric oxide synthase (iNOS) immunoreactivity in lysates from MDCK-D1 cells treated for 24 h in either 1 µM interleukin (IL)-1beta , 1 µM tumor necrosis factor (TNF)-alpha , both IL-1beta and TNF-alpha (B, left), or 1 µM phorbol 12-myristate 13-acetate (PMA; B, right). Equal total protein was loaded in each lane in a given experiment (7-20 µg/lane). Representative blots of at least 3 experiments are shown.

To confirm expression of COX isoforms in MDCK-D1 cells, we performed RT-PCR using specific primers designed to amplify species-conserved COX-2 and COX-1 sequences. Using COX-2-specific primers, a prominent product of the expected size from both cDNA and DNA (but only minimally noted in the negative control) was evident after 25 cycles of PCR; sequence analysis of the product confirmed the expression of COX-2 mRNA (Fig. 5A, left). The sequence of the COX-2 PCR product is shown aligned with the published human sequence in Fig. 5B. The homology of the canine COX-2 sequence to published COX-2 sequences from other species (including human, rat, and sheep) ranged from 86 to 91%. COX-1-specific primers were unable to amplify any MDCK-D1 cell COX-1 product but did amplify COX-1 from murine colon cDNA (Fig. 5A, right). Three other COX-1-specific primers, including a degenerate primer pair, also could not amplify canine COX-1 (data not shown), likely reflecting sequence divergence among species.


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Fig. 5.   RT-PCR analysis of COX isoforms and cDNA sequence of COX-2 expressed in MDCK-D1 cells. A: agarose gel electrophoresis of PCR products from reactions using either COX-2- or COX-1-specific primers to amplify either cDNA, non-reverse transcribed RNA (-RT), or genomic DNA (gDNA) as template. For COX-1, a product was only amplified from murine colon cDNA (mouse cDNA). Representative gel of 3 experiments is shown. B: sequence of PCR product from COX-2 RT-PCR (canine) is shown aligned with the human sequence. Underlined segments indicate the sites of primer annealing.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our previous work has shown that ATP is released on mild mechanical or biochemical activation and that basal levels of cAMP are substantially determined by this ATP release. The fact that cells can release ATP on activation by specific signals and subsequently respond to extracellular nucleotides implicates this phenomenon as a means of autocrine/paracrine regulation. ATP can be an important physiological signal, influencing ion conductance and volume regulation and contributing to chloride conductance by the cystic fibrosis transmembrane conductance regulator (CFTR) (3, 11, 24). However, the mechanism for cellular ATP release, which is separate from vesicular exocytosis at synapses and by platelets, remains undefined (24).

The recent widespread clinical usage of COX-2-selective inhibitors raises the importance of understanding the role of this isozyme in the regulation of cell function. Some data have suggested that COX-2 may be constitutively expressed in certain cell types, implying that this isoform may be involved in signaling pathways regulating other cellular functions (2, 5, 14, 27). The present studies demonstrate that COX-2 is expressed in what appears to be a constitutive, relatively nonregulated fashion in MDCK-D1 cells and that COX-2 plays a critical role in regulating cAMP levels. cAMP initiates both rapid actions, such as regulation of ion channels and effects on carbohydrate, protein, and lipid metabolism, as well as more delayed effects, such as changes in gene expression, cell growth, and proliferation. Therefore, we conclude that COX-2 likely participates in the regulation of such key functions in MDCK-D1 cells and, by inference, other cells and tissues in which the enzyme is expressed. Responses in vivo that require induction of COX-2 also depend on availability of COX substrate (i.e., AA) (7). Increased cellular ATP release, as well as increases in the ability of cells to respond to extracellular nucleotides, likely represent an important mechanism for increased AA release in settings such as inflammation. Inflammation is characterized by increases in cytokines, such as IL-1beta and TNF-alpha , and such cytokines can increase expression of both PLA2s and COX-2 (19, 29, 32).

Many studies have implicated a role for COX-2 expression, particularly in the kidney (5, 8, 12, 14, 30), but we believe ours is the first report of constitutive expression of COX-2 in a cell culture model removed from the influences of circulating hormones and growth factors. While not all possible regulators of gene expression were tested, our data show that COX-2 expression in MDCK-D1, in contrast with results in other cell systems, is only minimally influenced by common regulators of this gene. IL-1beta alone failed to increase expression of COX-2, while TNF-alpha produced a very modest increase in COX-2 expression. In contrast, levels of iNOS were increased by treatment with these cytokines. Glucocorticoid treatment, which is well known to inhibit COX-2 expression (15, 32), failed to decrease COX-2 immunoreactivity in MDCK-D1 cells. Yang and coworkers (33) recently demonstrated that COX-2 immunoreactivity was undetectable in MDCK cells but dramatically induced on incubation in hypertonic media. Our results contrast with these findings, perhaps because of the use of different clonal variants of the parental MDCK cells. These data challenge the prevailing concept that COX-2 is only an inducible isoform and indicate that the differentiated state of some cells may allow the constitutive expression of this isoform.

Renal expression of COX-2 may be important in various physiological functions and pathophysiological states. COX-2 expression increases in macula densa and inner medulla cells on dietary salt restriction, implying a role for this isozyme in salt and water excretion (9, 34). Anti-Thy-1 glomerulonephritis, an in vivo model of mesangial cell injury, is associated with marked increases in COX-2 expression, particularly in glomerular epithelial cells (10). Such findings, together with the current data, implicate an important role for COX-2 in regulation of renal function. We conclude that the ATP release/P2Y receptor/COX-2 signaling pathway we describe here is a key autocrine/paracrine regulator of cell function, particularly in renal epithelia under basal conditions as well as in pathophysiological settings.


    ACKNOWLEDGEMENTS

The authors thank Dr. R. Tukey for use of the luminometer, Dr. L. Brunton for iNOS antibody, Dr. L. Eckman for mouse cDNA, J. Truong and L. Pan for technical assistance with cell culture, and K. Hultgren for technical assistance with cAMP assays.


    FOOTNOTES

This work was supported by research and training grants from the National Institutes of Health and the Cystic Fibrosis Foundation.

Address for reprint requests and other correspondence: P. A. Insel, Dept. of Pharmacology, Univ. of California, San Diego, La Jolla, CA 92093-0636 (E-mail: pinsel{at}ucsd.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.

Received 9 January 2001; accepted in final form 2 April 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Barnett, J, Chow J, Ives D, Chiou M, Mackenzie R, Osen E, Nguyen B, Tsing S, Bach C, Freire J, Chan H, Sigal E, and Ramesha C. Purification, characterization and selective inhibition of human prostaglandin G/H synthase 1 and 2 expressed in the baculovirus system. Biochim Biophys Acta 1209: 130-139, 1994[ISI][Medline].

2.   Callejas, NA, Casado M, Boscá L, and Martín-Sanz P. Requirement of nuclear factor kappa B for the constitutive expression of nitric oxide synthase-2 and cyclooxygenase-2 in rat trophoblasts. J Cell Sci 112: 3147-3155, 1999[Abstract/Free Full Text].

3.   Clarke, LL, Chinet T, and Boucher RC. Extracellular ATP stimulates K+ secretion across cultured human airway epithelium. Am J Physiol Lung Cell Mol Physiol 272: L1084-L1091, 1997[Abstract/Free Full Text].

4.   Crofford, LJ, Wilder RL, Ristimäki AP, Sano H, Remmers EF, Epps HR, and Hla T. Cyclooxygenase-1 and -2 expression in rheumatoid synovial tissues. Effects of interleukin-1beta , phorbol ester, and corticosteroids. J Clin Invest 93: 1095-1101, 1994[ISI][Medline].

5.   Ferguson, S, Hébert RL, and Laneuville O. NS-398 upregulates constitutive cyclooxygenase-2 expression in the M-1 cortical collecting duct cell line. J Am Soc Nephrol 10: 2261-2271, 1999[Abstract/Free Full Text].

6.   Guan, Y, Chang M, Cho W, Zhang Y, Redha R, Davis L, Chang S, DuBois RN, Hao CM, and Breyer M. Cloning, expression, and regulation of rabbit cyclooxygenase-2 in renal medullary interstitial cells. Am J Physiol Renal Physiol 273: F18-F26, 1997[Abstract/Free Full Text].

7.   Hamilton, LC, Mitchell JA, Tomlinson AM, and Warner TD. Synergy between cyclo-oxygenase-2 induction and arachidonic acid supply in vivo: consequences for nonsteroidal anti-inflammatory drug efficacy. FASEB J 13: 245-251, 1999[Abstract/Free Full Text].

8.   Hao, CM, Kömhoff M, Guan Y, Redha R, and Breyer MD. Selective targeting of cyclooxygenase-2 reveals its role in renal medullary interstitial cell survival. Am J Physiol Renal Physiol 277: F352-F359, 1999[Abstract/Free Full Text].

9.   Harris, RC, McKanna JA, Akai Y, Jacobson HR, Dubois RN, and Breyer MD. Cyclooxygenase-2 is associated with the macula densa of rat kidney and increases with salt restriction. J Clin Invest 94: 2504-2510, 1994[ISI][Medline].

10.   Hirose, S, Yamamoto T, Feng L, Yaoita E, Kawasaki K, Goto S, Fujinaka H, Wilson CB, Arakawa M, and Kihara I. Expression and localization of cyclooxygenase isoforms and cytosolic phospholipase A2 in anti-Thy-1 glomerulonephritis. J Am Soc Nephrol 9: 408-416, 1998[Abstract].

11.   Knowles, MR, Olivier K, Noone P, and Boucher RC. Pharmacologic modulation of salt and water in the airway epithelium in cystic fibrosis. Am J Respir Crit Care Med 151: S65-S69, 1995[ISI][Medline].

12.   Kömhoff, M, Grone HJ, Klein T, Seyberth HW, and Nüsing RM. Localization of cyclooxygenase-1 and -2 in adult and fetal human kidney: implication for renal function. Am J Physiol Renal Physiol 272: F460-F468, 1997[Abstract/Free Full Text].

13.   Kujubu, DA, Fletcher BS, Varnum BC, Lim RW, and Herschman HR. TIS10, a phorbol ester tumor promoter-inducible mRNA from Swiss 3T3 cells, encodes a novel prostaglandin synthase/cyclooxygenase homologue. J Biol Chem 266: 12866-12872, 1991[Abstract/Free Full Text].

14.   McKanna, JA, Zhang MZ, Wang JL, Cheng H, and Harris RC. Constitutive expression of cyclooxygenase-2 in rat vas deferens. Am J Physiol Regulatory Integrative Comp Physiol 275: R227-R233, 1998[Abstract/Free Full Text].

15.   O'Banion, MK, Winn VD, and Young DA. cDNA cloning and functional activity of a glucocorticoid-regulated inflammatory cyclooxygenase. Proc Natl Acad Sci USA 89: 4888-4892, 1992[Abstract].

16.   Ostrom, RS, Gregorian C, and Insel PA. Cellular release of and response to ATP as key determinants of the set-point of signal transduction pathways. J Biol Chem 275: 11735-11739, 2000[Abstract/Free Full Text].

17.   Post, SR, Jacobson JP, and Insel PA. P2 purinergic receptor agonists enhance cAMP production in Madin-Darby canine kidney epithelial cells via an autocrine/paracrine mechanism. J Biol Chem 271: 2029-2032, 1996[Abstract/Free Full Text].

18.   Post, SR, Rump LC, Zambon A, Hughes RJ, Buda MD, Jacobson JP, Kao CC, and Insel PA. ATP activates cAMP production via multiple purinergic receptors in MDCK-D1 epithelial cells. Blockade of an autocrine/paracrine pathway to define receptor preference of an agonist. J Biol Chem 273: 23093-23097, 1998[Abstract/Free Full Text].

19.   Pruzanski, W, Stefanski E, Vadas P, Kennedy BP, and van den Bosch H. Regulation of the cellular expression of secretory and cytosolic phospholipases A2, and cyclooxygenase-2 by peptide growth factors. Biochim Biophys Acta 1403: 47-56, 1998[ISI][Medline].

20.   Roman, RM, Feranchak AP, Davison AK, Schwiebert EM, and Fitz JG. Evidence for Gd3+ inhibition of membrane ATP permeability and purinergic signaling. Am J Physiol Gastrointest Liver Physiol 277: G1222-G1230, 1999[Abstract/Free Full Text].

21.   Salomon, Y, Londos C, and Rodbell M. A highly sensitive adenylate cyclase assay. Anal Biochem 58: 541-548, 1974[ISI][Medline].

22.   Sauer, H, Hescheler J, and Wartenberg M. Mechanical strain-induced Ca2+ waves are propagated via ATP release and purinergic receptor activation. Am J Physiol Cell Physiol 279: C295-C307, 2000[Abstract/Free Full Text].

23.   Schaefers, HJ, Haselmann J, and Goppelt-Struebe M. Regulation of prostaglandin synthesis in Madin Darby canine kidney cells: role of prostaglandin G/H synthase and secreted phospholipase A2. Biochim Biophys Acta 1300: 197-202, 1996[ISI][Medline].

24.   Schwiebert, EM. ABC transporter-facilitated ATP conductive transport. Am J Physiol Cell Physiol 276: C1-C8, 1999[Abstract/Free Full Text].

25.   Seibert, K, Zhang Y, Leahy K, Hauser S, Masferrer J, Perkins W, Lee L, and Isakson P. Pharmacological and biochemical demonstration of the role of cyclooxygenase 2 in inflammation and pain. Proc Natl Acad Sci USA 91: 12013-12017, 1994[Abstract/Free Full Text].

26.   Smith, CJ, Zhang Y, Koboldt CM, Muhammad J, Zweifel BS, Shaffer A, Talley JJ, Masferrer JL, Seibert K, and Isakson PC. Pharmacological analysis of cyclooxygenase-1 in inflammation. Proc Natl Acad Sci USA 95: 13313-13318, 1998[Abstract/Free Full Text].

27.   Smith, TJ, Jennings TA, Sciaky D, and Cao HJ. Prostaglandin-endoperoxide H synthase-2 expression in human thyroid epithelium. Evidence for constitutive expression in vivo and in cultured KAT-50 cells. J Biol Chem 274: 15622-15632, 1999[Abstract/Free Full Text].

28.   Smith, WL, DeWitt DL, and Garavito RM. Cyclooxygenases: structural, cellular, and molecular biology. Annu Rev Biochem 69: 145-182, 2000[ISI][Medline].

29.   Stella, N, Estellés A, Siciliano J, Tencé M, Desagher S, Piomelli D, Glowinski J, and Prémont J. Interleukin-1 enhances the ATP-evoked release of arachidonic acid from mouse astrocytes. J Neurosci 17: 2939-2946, 1997[Abstract/Free Full Text].

30.   Vio, CP, Cespedes C, Gallardo P, and Masferrer JL. Renal identification of cyclooxygenase-2 in a subset of thick ascending limb cells. Hypertension 30: 687-692, 1997[Abstract/Free Full Text].

31.   Xing, M, Firestein BL, Shen GH, and Insel PA. Dual role of protein kinase C in the regulation of cPLA2-mediated arachidonic acid release by P2U receptors in MDCK-D1 cells: involvement of MAP kinase-dependent and -independent pathways. J Clin Invest 99: 805-814, 1997[Abstract/Free Full Text].

32.   Xue, S, Slater DM, Bennett PR, and Myatt L. Induction of both cytosolic phospholipase A2 and prostaglandin H synthase-2 by interleukin-1beta in WISH cells is inhibited by dexamethasone. Prostaglandins 51: 107-124, 1996[Medline].

33.   Yang, T, Schnermann JB, and Briggs JP. Regulation of cyclooxygenase-2 expression in renal medulla by tonicity in vivo and in vitro. Am J Physiol Renal Physiol 277: F1-F9, 1999[Abstract/Free Full Text].

34.   Yang, T, Singh I, Pham H, Sun D, Smart A, Schnermann JB, and Briggs JP. Regulation of cyclooxygenase expression in the kidney by dietary salt intake. Am J Physiol Renal Physiol 274: F481-F489, 1998[Abstract/Free Full Text].


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