scholarly journals NKCC2A and NFAT5 regulate renal TNF production induced by hypertonic NaCl intake

2013 ◽  
Vol 304 (5) ◽  
pp. F533-F542 ◽  
Author(s):  
Shoujin Hao ◽  
Lars Bellner ◽  
Nicholas R. Ferreri
Keyword(s):  
Tap Water ◽  
Primary Cultures ◽  
Fold Increase ◽  
Thick Ascending Limb ◽  
Outer Medulla ◽  
Activated T Cells ◽  
Mrna Accumulation ◽  
Nacl Concentration ◽  
Promoter Construct ◽  
Mrna And Protein Expression

Pathways that contribute to TNF production by the kidney are not well defined. Mice given 1% NaCl in the drinking water for 3 days exhibited a 2.5-fold increase in urinary, but not plasma, TNF levels compared with mice given tap water. Since furosemide attenuated the increase in TNF levels, we hypothesized that hypertonic NaCl intake increases renal TNF production by a pathway involving the Na+-K+-2Cl− cotransporter (NKCC2). A 2.5-fold increase in NKCC2A mRNA accumulation was observed in medullary thick ascending limb (mTAL) tubules from mice given 1% NaCl; a concomitant 2-fold increase in nuclear factor of activated T cells 5 (NFAT5) mRNA and protein expression was observed in the outer medulla. Urinary TNF levels were reduced in mice given 1% NaCl after an intrarenal injection of a lentivirus construct designed to specifically knockdown NKCC2A (EGFP-N2A-ex4); plasma levels of TNF did not change after injection of EGFP-N2A-ex4. Intrarenal injection of EGFP-N2A-ex4 also inhibited the increase of NFAT5 mRNA abundance in the outer medulla of mice given 1% NaCl. TNF production by primary cultures of mTAL cells increased approximately sixfold in response to an increase in osmolality to 400 mosmol/kgH2O produced with NaCl and was inhibited in cells transiently transfected with a dnNFAT5 construct. Transduction of cells with EGFP-N2A-ex4 also prevented increases in TNF mRNA and protein production in response to high NaCl concentration and reduced transcriptional activity of a NFAT5 promoter construct. Since NKCC2A expression is restricted to the TAL, NKCC2A-dependent activation of NFAT5 is part of a pathway by which the TAL produces TNF in response to hypertonic NaCl intake.

scholarly journals Differential regulation of NFAT5 by NKCC2 isoforms in medullary thick ascending limb (mTAL) cells

2011 ◽  
Vol 300 (4) ◽  
pp. F966-F975 ◽  
Author(s):  
Shoujin Hao ◽  
Hong Zhao ◽  
Zbigniew Darzynkiewicz ◽  
Sailaja Battula ◽  
Nicholas R. Ferreri
Keyword(s):  
Protein Expression ◽  
Laser Scanning ◽  
Transcriptional Activity ◽  
Primary Cultures ◽  
Fold Increase ◽  
Thick Ascending Limb ◽  
Activated T Cells ◽  
Mrna Accumulation ◽  
Medullary Thick Ascending Limb ◽  
Nacl Concentration

The effects of Na+-K+-2Cl− cotransporter type 2 (NKCC2) isoforms on the regulation of nuclear factor of activated T cells isoform 5 (NFAT5) were determined in mouse medullary thick ascending limb (mTAL) cells exposed to high NaCl concentration. Primary cultures of mTAL cells and freshly isolated mTAL tubules, both derived from the outer medulla (outer stripe>inner stripe), express NKCC2 isoforms A and F. The relative expression of NKCC2A mRNA was approximately twofold greater than NKCC2F in these preparations. The abundance of NKCC2A mRNA, but not NKCC2F mRNA, increased approximately twofold when mTAL cells were exposed for 2 h to a change in osmolality from 300 to 500 mosmol/kgH2O, produced with NaCl. Total NKCC2 protein expression also increased. Moreover, a 2.5-fold increase in NFAT5 mRNA accumulation was observed after cells were exposed to 500 mosmol/kgH2O for 4 h. Laser-scanning cytometry detected a twofold increase in endogenous NFAT5 protein expression in response to high NaCl concentration. Pretreatment with the loop diuretic bumetanide dramatically reduced transcriptional activity of the NFAT5-specific reporter construct TonE-Luc in mTAL cells exposed to high NaCl. Transient transfection of mTAL cells with shRNA vectors targeting NKCC2A prevented increases in NFAT5 mRNA abundance and protein expression and inhibited NFAT5 transcriptional activity in response to hypertonic stress. Silencing of NKCC2F mRNA did not affect NFAT5 mRNA accumulation but partially inhibited NFAT5 transcriptional activity. These findings suggest that NKCC2A and NKCC2F exhibit differential effects on NFAT5 expression and transcriptional activity in response to hypertonicity produced by high NaCl concentration.

scholarly journals Expression and function of NFAT5 in medullary thick ascending limb (mTAL) cells

2009 ◽  
Vol 296 (6) ◽  
pp. F1494-F1503 ◽  
Author(s):  
Shoujin Hao ◽  
Hong Zhao ◽  
Zbigniew Darzynkiewicz ◽  
Sailaja Battula ◽  
Nicholas R. Ferreri
Keyword(s):  
Primary Cultures ◽  
Constitutive Expression ◽  
Transient Transfection ◽  
Pcr Analysis ◽  
Thick Ascending Limb ◽  
Rt Pcr ◽  
Activated T Cells ◽  
Mrna Accumulation ◽  
Culture Cells ◽  
Medullary Thick Ascending Limb

The contribution of nuclear factor of activated T cells 5 (NFAT5) to the regulation of tumor necrosis factor-α (TNF) production in medullary thick ascending limb (mTAL) cells is unclear. RT-PCR analysis was performed on primary cultures of mouse mTAL cells and freshly isolated mTAL tubules to determine which NFAT isoforms are present in this nephron segment. Primer pairs were designed, based on published sequences for mouse NFAT1-5, to produce fragments of ∼200 bp. Analysis of PCR products by gel electrophoresis and subsequent DNA sequencing indicated that cells and tubules contained mRNA for all five NFAT isoforms. The relative expression of NFAT isoforms was then determined using quantitative real-time RT-PCR. The data indicate that NFAT isoforms 5 ≥ 1 are the predominant isoforms present in mTAL cells and tubules. Western blot analysis demonstrated constitutive expression of NFAT5 in nuclear extracts from mTAL tubules and primary culture cells; expression in mTAL cells also was detected by immunofluorescence. Expression of NFAT5 was increased in mTAL cells transiently transfected with an NFAT5 overexpression vector (pcDNA3.1-NFAT5), resulting in increased basal and calcium-sensing receptor (CaR)-mediated TNF production. Transient transfection of mTAL cells with a small hairpin RNA vector that targeted exon 8 of NFAT5 (U6-N5 ex8) significantly inhibited TNF promoter activity. Transient transfection with U6-N5 ex8 also reduced nuclear expression of NFAT5, TNF mRNA accumulation, and attenuated CaR-mediated activation of Cl−entry into polarized mTAL cells. Collectively, these data suggest that activation of NFAT5 is part of a TNF-dependent pathway that inhibits apical Cl−influx in the mTAL after activation of CaR.

scholarly journals Fluid dilution and efficiency of Na+ transport in a mathematical model of a thick ascending limb cell

2013 ◽  
Vol 304 (6) ◽  
pp. F634-F652 ◽  
Author(s):  
Aniel Nieves-González ◽  
Chris Clausen ◽  
Mariano Marcano ◽  
Anita T. Layton ◽  
Harold E. Layton ◽  
...  
Keyword(s):  
Mathematical Model ◽  
Cell Model ◽  
Cell Volume Regulation ◽  
Mass Conservation ◽  
Thick Ascending Limb ◽  
Outer Medulla ◽  
Transport Efficiency ◽  
Nacl Concentration ◽  
Model Equations ◽  
Static Head

Thick ascending limb (TAL) cells are capable of reducing tubular fluid Na+ concentration to as low as ∼25 mM, and yet they are thought to transport Na+ efficiently owing to passive paracellular Na+ absorption. Transport efficiency in the TAL is of particular importance in the outer medulla where O2 availability is limited by low blood flow. We used a mathematical model of a TAL cell to estimate the efficiency of Na+ transport and to examine how tubular dilution and cell volume regulation influence transport efficiency. The TAL cell model represents 13 major solutes and the associated transporters and channels; model equations are based on mass conservation and electroneutrality constraints. We analyzed TAL transport in cells with conditions relevant to the inner stripe of the outer medulla, the cortico-medullary junction, and the distal cortical TAL. At each location Na+ transport efficiency was computed as functions of changes in luminal NaCl concentration ([NaCl]), [K+], [NH4+], junctional Na+ permeability, and apical K+ permeability. Na+ transport efficiency was calculated as the ratio of total net Na+ transport to transcellular Na+ transport. Transport efficiency is predicted to be highest at the cortico-medullary boundary where the transepithelial Na+ gradient is the smallest. Transport efficiency is lowest in the cortex where luminal [NaCl] approaches static head.

Abstract 553: Prostaglandin E2 EP3 Receptor Downregulates COX2 Expression in the Medullary Thick Ascending Limb (mTAL) Induced by Hypertonic NaCl Intake

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Shoujin Hao ◽  
Carlos P Vio ◽  
Carlos Cespedes ◽  
Mariana Quiroz-Munoz ◽  
Nicholas R Ferreri
Keyword(s):  
Protein Expression ◽  
Tap Water ◽  
Receptor Expression ◽  
Free Access ◽  
Thick Ascending Limb ◽  
Mrna Levels ◽  
Mrna Accumulation ◽  
Cox 2 ◽  
Ep3 Receptor ◽  
In Cells

We recently showed that a novel negative feedback mechanism involving PGE 2 acting on EP3 receptors regulates cyclooxygenase-2 (COX-2) expression in the thick ascending limb (TAL) induced by the selective COX-2 inhibitors, celecoxib and rofecoxib. In the present study we tested the hypothesis that inhibition of EP3 facilitates COX-2 expression in the TAL induced by ingestion of hypertonic NaCl or exposure of mTAL cells to hypertonic media. COX-2 protein expression in the outer medulla (OM) increased 2.2±0.3 fold in mice given free access to 1% NaCl in the drinking water for 3 days compared with tap water; p<0.05. The increase was associated with a 4-fold elevation in COX-2 mRNA accumulation (tap water: 0.5±0.1 vs 1% NaCl: 1.9±0.4; p<0.05), and higher PGE 2 production by freshly isolated mTAL tubules (tap water: 87.6±9.4 vs 1% NaCl: 203.3±19.3 pg/mg protein; p<0.05). EP3 mRNA levels also increased approximately 2-fold in OM of mice ingesting 1% NaCl (tap water: 0.7±0.2 vs 1% NaCl: 1.3±0.2). Administration of a selective EP3 receptor antagonist (L-798106: 20μg/kg/day) for 2 days increased COX-2 mRNA accumulation in mTAL tubules (L-798106: 2.1±0.1 fold change vs control; p<0.05), and the elevation in COX-2 protein expression induced by 1% NaCl was increased an additional 50% in mice given L-798106. COX-2 mRNA accumulation in primary cultures of mTAL cells increased 2-fold in response to media made hypertonic by addition of NaCl (400 mosmol/kg H 2 O), compared with cells incubated in isotonic media. L-798106 increased COX-2 mRNA 2-fold in isotonic media and 4-fold in cells exposed to 400 mosmol/kg H 2 O. PGE 2 production by mTAL cells increased approximately 4-fold in response to challenge with 400 mosmol/kg H 2 O for 9 hr, and was inhibited in cells transiently transfected with a lentivirus shRNA construct (EGFP-C2-ex5) to silence COX-2 (286.1±14.8 to 64.3±5.2 pg/mg protein; p<0.05). Collectively, the data suggest that local hypertonicity in the mTAL is associated with an increase in COX-2 expression concomitant with elevated EP3 receptor expression, which attenuates COX-2 activity in this segment of the nephron. Moreover, PGE 2 signaling via EP3 receptors in the TAL may be part of a mechanism that regulates mTAL COX-2 expression and function in response to diverse stimuli.

Regulation by glucocorticoids and osmolality of expression of ROMK (Kir 1.1), the apical K channel of thick ascending limb

2003 ◽  
Vol 284 (5) ◽  
pp. F977-F986 ◽  
Author(s):  
Morgan Gallazzini ◽  
Amel Attmane-Elakeb ◽  
David B. Mount ◽  
Steven C. Hebert ◽  
Maurice Bichara
Keyword(s):  
Protein Expression ◽  
In Vivo Studies ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Rt Pcr ◽  
Immunoblotting Analysis ◽  
Medullary Thick Ascending Limb ◽  
Nacl Concentration ◽  
Mrna And Protein Expression

Mechanisms of regulation of ROMK channel mRNA and protein expression in medullary thick ascending limb (MTAL) were assessed in rat MTAL fragments incubated for 7 h. ROMK mRNA was quantified by quantitative RT-PCR and ROMK protein by immunoblotting analysis of crude membranes. Medium hyperosmolality (450 mosmol/kgH2O; NaCl plus urea added to isoosmotic medium) increased ROMK mRNA ( P < 0.04) and protein ( P < 0.006), and 10 nM dexamethasone also increased ROMK mRNA ( P < 0.02). Hyperosmolality and dexamethasone had no additive effects on ROMK mRNA. NaCl alone, but not urea or mannitol, reproduced the hyperosmolality effect on ROMK mRNA. 1-Deamino-(8-d-arginine) vasopressin (1 nM) or 0.5 mM 8-bromo-cAMP had no effect per se on ROMK mRNA and protein. However, 8-bromo-cAMP abolished the stimulatory effect of dexamethasone on ROMK mRNA in the isoosmotic but not in the hyperosmotic medium ( P < 0.004). In in vivo studies, the abundance of ROMK protein and mRNA increased in adrenalectomized (ADX) rats infused with dexamethasone compared with ADX rats ( P < 0.02). These results establish glucocorticoids and medium NaCl concentration as direct regulators of MTAL ROMK mRNA and protein expression, which may be modulated by cAMP-dependent factors.

CaR-mediated COX-2 expression in primary cultured mTAL cells

2001 ◽  
Vol 281 (4) ◽  
pp. F658-F664 ◽  
Author(s):  
Dairong Wang ◽  
Shao-Jian An ◽  
Wen-Hui Wang ◽  
John C. McGiff ◽  
Nicholas R. Ferreri
Keyword(s):  
Protein Expression ◽  
Intracellular Signaling ◽  
Primary Cultures ◽  
Extracellular Fluid ◽  
Thick Ascending Limb ◽  
Dependent Manner ◽  
Mrna Accumulation ◽  
Beneficial Effects ◽  
Cox 2 ◽  
Salt And Water Balance

Primary cultures of medullary thick ascending limb (mTAL) cells retain the capacity to express calcium-sensing receptor (CaR) mRNA and protein. Increases in cyclooxygenase-2 (COX-2) mRNA accumulation, protein expression, and PGE2 synthesis were observed in a dose- and time-dependent manner after exposure of these cells to extracellular calcium (Ca[Formula: see text]). Moreover, transfection of mTAL cells with a CaR overexpression vector significantly enhanced COX-2 expression and PGE2 production in response to calcium compared with cells transfected with an empty vector. Challenge with the CaR-selective agonist poly-l-arginine (PLA) also increased COX-2 mRNA accumulation, protein expression, and PGE2 synthesis. Furthermore, Ca[Formula: see text]- and PLA-mediated PGE2production was abolished in the presence of NS-398 or nimesulide, two different COX-2-selective inhibitors. These data suggest that intracellular signaling mechanisms initiated via activation of CaR contribute to COX-2-dependent PGE2 synthesis in the mTAL. Because Ca[Formula: see text] concentration varies along Henle's loop, calcium may contribute to salt and water balance via a COX-2- and CaR-dependent mechanism. Thus novel calcimimetics might be useful in conditions such as hypertension in which manipulation of extracellular fluid volume provides beneficial effects.

CaR activation increases TNF production by mTAL cells via a Gi-dependent mechanism

2008 ◽  
Vol 294 (2) ◽  
pp. F345-F354 ◽  
Author(s):  
Huda Ismail Abdullah ◽  
Paulina L. Pedraza ◽  
John C. McGiff ◽  
Nicholas R. Ferreri
Keyword(s):  
Extracellular Fluid ◽  
Inhibitory Effect ◽  
Dominant Negative ◽  
Thick Ascending Limb ◽  
Dependent Manner ◽  
Activated T Cells ◽  
Calcium Sensing ◽  
Promoter Construct ◽  
Gf 109203X ◽  
20 Nm

We evaluated the contribution of calcium-sensing receptor (CaR)-mediated Gi-coupled signaling to TNF production in medullary thick ascending limb (mTAL) cells. A selective Gi inhibitor, pertussis toxin (PTX), but not the inactive B-oligomer binding subunit, abolished CaR-mediated increases in TNF production. The inhibitory effect of PTX was partially reversed by using an adenylate cyclase inhibitor. CaR-mediated TNF production also was partially reversed by a cAMP analog, 8-Br-cAMP. IP1 accumulation was CaR dependent and blocked by PI-PLC; partial inhibition also was observed with PTX. CaR increased calcineurin (CaN) activity by approximately threefold, and PTX prevented CaR-mediated increases in CaN activity, an nuclear factor of activated T cells (NFAT)- cis reporter construct, and a TNF promoter construct. The interaction between Gi and PKC was determined, as we previously showed that CaR-mediated TNF production was CaN and NFAT- mediated and Gq dependent. CaR activation increased PKC activity by twofold, an effect abolished by transient transfection with a dominant negative CaR construct, R796W, or pretreatment with PTX. Inhibition with the pan-specific PKC inhibitor GF 109203X (20 nM) abolished CaR-mediated increases in activity of CaN, an NFAT reporter, and a TNF promoter construct. Collectively, the data suggest that Gi-coupled signaling contributes to NFAT-mediated TNF production in a CaN- and PKC-dependent manner and may be part of a CaR mechanism to regulate mTAL function. Moreover, concurrent Gq and Gi signaling is required for CaR-mediated TNF production in mTAL cells via a CaN/NFAT pathway that is PKC dependent. Understanding CaR-mediated signaling pathways that regulate TNF production in the mTAL is crucial to defining novel mechanisms that regulate extracellular fluid volume and salt balance.

scholarly journals pG2 gene expression and its regulation in human adrenocortical and medullary tumors

1999 ◽  
pp. 590-596
Author(s):  
J Liu ◽  
R Voutilainen ◽  
P Heikkila ◽  
AI Kahri
Keyword(s):  
Kinase Inhibitor ◽  
Primary Cultures ◽  
Control Level ◽  
Fold Increase ◽  
Zona Glomerulosa ◽  
Adrenal Tumors ◽  
Specific Marker ◽  
Mrna Accumulation ◽  
Adrenal Cells ◽  
Pheochromocytoma Cells

The cDNA clone pG2 was originally isolated from a human pheochromocytoma. The respective gene was found to be strongly expressed in normal adrenal zona glomerulosa and medulla, as well as in Conn's adenomas and pheochromocytomas. To shed more light on the expression and regulation of the pG2 gene, we investigated its expression in a wide variety of different adrenal neoplasms and cultured adrenal cells. Northern blot analysis was used to determine the steady state level of pG2 mRNA. Besides normal adrenals, Conn's adenomas and pheochromocytomas, we found abundant expression of pG2 mRNA in Cushing's, virilizing and nonfunctional adrenocortical adenomas and carcinomas, as well as in hyperplastic adrenals. The relative levels of pG2 mRNA in various adrenocortical tumors were not significantly different from those in normal adrenals and pheochromocytomas. In primary cultures of normal adrenal cells, treatment with adrenocorticotropin induced a 3- to 15-fold increase in the expression of pG2 mRNA (P<0.01), and this effect was reproduced by incubation with (Bu)2cAMP. In cultured pheochromocytoma cells, treatment with (Bu)2cAMP and a protein kinase inhibitor, staurosporine, increased pG2 mRNA accumulation (2- to 4-fold over the control level, P<0.01, and 3- to 8-fold, P<0.01, respectively). These results indicate that pG2 is widely expressed in normal and pathological adrenal tissues from both cortical and medullary origin, which eliminates its usefulness as a specific marker for zona glomerulosa or medullary adrenal tumors. Accumulation of pG2 mRNA is regulated by multiple differentiating factors through different pathways in primary cultures of normal adrenal and pheochromocytoma cells.

scholarly journals Tumor necrosis factor-α is an endogenous inhibitor of Na+-K+-2Cl− cotransporter (NKCC2) isoform A in the thick ascending limb

2011 ◽  
Vol 301 (1) ◽  
pp. F94-F100 ◽  
Author(s):  
Sailaja Battula ◽  
Shoujin Hao ◽  
Paulina L. Pedraza ◽  
Charles T. Stier ◽  
Nicholas R. Ferreri
Keyword(s):  
Urine Osmolality ◽  
Thick Ascending Limb ◽  
Endogenous Inhibitor ◽  
Outer Medulla ◽  
Mrna Accumulation ◽  
Fourfold Increase ◽  
Medullary Thick Ascending Limb ◽  
Before And After ◽  
Tnf Gene

The effects of TNF gene deletion on renal Na+-K+-2Cl− cotransporter (NKCC2) expression and activity were determined. Outer medulla from TNF−/− mice exhibited a twofold increase in total NKCC2 protein expression compared with wild-type (WT) mice. This increase was not observed in TNF−/− mice treated with recombinant human TNF (hTNF) for 7 days. Administration of hTNF had no effect on total NKCC2 expression in WT mice. A fourfold increase in NKCC2A mRNA accumulation was observed in outer medulla from TNF−/− compared with WT mice; NKCC2F and NKCC2B mRNA accumulation was similar between genotypes. The increase in NKCC2A mRNA accumulation was attenuated when TNF−/− mice were treated with hTNF. Bumetanide-sensitive O2 consumption, an in vitro correlate of NKCC2 activity, was 2.8 ± 0.2 nmol·min−1·mg−1 in medullary thick ascending limb tubules from WT, representing ∼40% of total O2 consumption, whereas, in medullary thick ascending limb tubules from TNF−/− mice, it was 5.6 ± 0.3 nmol·min−1·mg−1, representing ∼60% of total O2 consumption. Administration of hTNF to TNF−/− mice restored the bumetanide-sensitive component to ∼30% of total O2 consumption. Ambient urine osmolality was higher in TNF−/− compared with WT mice (2,072 ± 104 vs. 1,696 ± 153 mosmol/kgH2O, P < 0.05). The diluting ability of the kidney, assessed by measuring urine osmolality before and after 1 h of water loading also was greater in TNF−/− compared with WT mice (174 ± 38 and 465 ± 81 mosmol/kgH2O, respectively, P < 0.01). Collectively, these findings suggest that TNF plays a role as an endogenous inhibitor of NKCC2 expression and function.

scholarly journals Prostaglandin E2 EP3 receptor regulates cyclooxygenase-2 expression in the kidney

2012 ◽  
Vol 303 (3) ◽  
pp. F449-F457 ◽  
Author(s):  
Carlos P. Vio ◽  
Mariana Quiroz-Munoz ◽  
Catherina A. Cuevas ◽  
Carlos Cespedes ◽  
Nicholas R. Ferreri
Keyword(s):  
Protein Expression ◽  
Negative Feedback ◽  
Fold Increase ◽  
Treated Group ◽  
Cyclooxygenase 2 ◽  
Thick Ascending Limb ◽  
Sprague Dawley ◽  
Mrna Accumulation ◽  
Cox 2 ◽  
Ep3 Receptor

Cyclooxygenase-2 (COX-2) is constitutively expressed and highly regulated in the thick ascending limb (TAL). As COX-2 inhibitors (Coxibs) increase COX-2 expression, we tested the hypothesis that a negative feedback mechanism involving PGE2 EP3 receptors regulates COX-2 expression in the TAL. Sprague-Dawley rats were treated with a Coxib [celecoxib (20 mg·kg−1·day−1) or rofecoxib (10 mg·kg−1·day−1)], with or without sulprostone (20 μg·kg−1·day−1). Sulprostone was given using two protocols, namely, previous to Coxib treatment (prevention effect; Sulp7-Coxib5 group) and 5 days after initiation of Coxib treatment (regression effect; Coxib10-Sulp5 group). Immunohistochemical and morphometric analysis revealed that the stained area for COX-2-positive TAL cells (μm2/field) increased in Coxib-treated rats (Sham: 412 ± 56.3, Coxib: 794 ± 153.3). The Coxib effect was inhibited when sulprostone was used in either the prevention (285 ± 56.9) or regression (345 ± 51.1) protocols. Western blot analysis revealed a 2.1 ± 0.3-fold increase in COX-2 protein expression in the Coxib-treated group, an effect abolished by sulprostone using either the prevention (1.2 ± 0.3-fold) or regression (0.6 ± 0.4-fold vs. control, P < 0.05) protocols. Similarly, the 6.4 ± 0.6-fold increase in COX-2 mRNA abundance induced by Coxibs ( P < 0.05) was inhibited by sulprostone; prevention: 0.9 ± 0.3-fold ( P < 0.05) and regression: 0.6 ± 0.1 ( P < 0.05). Administration of a selective EP3 receptor antagonist, L-798106, also increased the area for COX-2-stained cells, COX-2 mRNA accumulation, and protein expression in the TAL. Collectively, the data suggest that COX-2 levels are regulated by a novel negative feedback loop mediated by PGE2 acting on its EP3 receptor in the TAL.

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