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HCO3− exchange (DRA, SLC26A3) in villus cells in the chronically inflamed rabbit ileum
Biochimica et Biophysica Acta 1828 (2013) 179–186
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Prostaglandins, not the leukotrienes, regulate Cl −/HCO3− exchange (DRA, SLC26A3) in villus cells in the chronically inflamed rabbit ileum Palanikumar Manoharan, Steven Coon, Walter Baseler, Shanmuga Sundaram, Ramesh Kekuda, Uma Sundaram ⁎ Section of Digestive Diseases and West Virginia Clinical and Translational Science Institute, West Virginia University, Morgantown, WV 26505, USA
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Article history: Received 20 February 2012 Received in revised form 10 July 2012 Accepted 7 August 2012 Available online 3 September 2012 Keywords: Inflammatory bowel disease Down regulated in adenoma Cyclooxygenase Lipoxygenase Arachidonic acid metabolites Regulation of Cl−/HCO3− exchange Piroxicam
a b s t r a c t Previously studies have demonstrated that Cl−/HCO3− exchange was inhibited during chronic intestinal inflammation secondary to decrease in the affinity of the exchanger for Cl − rather than the number of transporters. Arachidonic acid metabolites (AAM) are elevated in the mucosa of the chronically inflamed small intestine. However, their role in the alteration of Cl −/HCO3− during chronic enteritis was unknown. Inhibition of AAM formation with arachidonyl trifluoro methylketone (ATMK) in chronically inflamed rabbit intestine reversed the diminished Cl−/HCO3− exchange activity. Kinetics studies showed that the reversal was secondary to restoration of the altered affinity of transporter. Downstream regulation of Cl −/HCO3− inhibition by AAM was determined to be by the cyclooxygenase pathway since only inhibition of cyclooxygenase with piroxicam treatment reversed the inhibited Cl −/HCO3− exchange. Further, DRA was shown to be the primary Cl −/HCO3− exchanger in villus cells. Kinetics and molecular studies indicated that the mechanism of inhibition of Cl −/HCO3− exchange by cyclooxygenase pathway metabolites was secondary to diminished affinity of the transporter for Cl − without a change in DRA BBM expression. Thus our data indicated that cyclooxygenase pathway metabolites mediate the inhibition of DRA during chronic intestinal inflammation. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The most common and disabling morbidity of human inflammatory bowel disease is diarrhea. It results as a result of altered absorption and secretion of electrolytes and fluid by intestinal epithelial cells. Neutral NaCl absorption occurs in the mammalian small intestine through coupled Na/H and Cl−/HCO3− exchange on the brush border membrane (BBM) of absorptive villus cells. In an animal model of chronic small intestinal inflammation which has many characteristics of human IBD, we have demonstrated the inhibition of coupled NaCl absorption [1]. The mechanism is secondary to a reduction in Cl−/HCO3− exchange, without a change in Na/H exchange in the BBM of villus cells [2]. However, the identity and the specific immune regulation of Cl−/HCO3− exchange in the chronically inflamed rabbit intestine are not known. Various Cl−/HCO3− exchangers (SLC26 family), PAT1, DRA and Pendrin have been demonstrated to have selective and limited tissue distribution. Further, PAT1 (SLC26A6) and DRA (SLC26A3) have been reported in the BBM of intestinal epithelia [3–5]. Nevertheless, the major Cl−/HCO3− exchanger that is down regulated in the chronically inflamed rabbit small intestine is not known. In IBD (e.g. Crohn's disease) alterations in absorption of electrolytes and water causing diarrhea is mediated by inflammatory mediators ⁎ Corresponding author. Tel.:+1 304 2936111. E-mail address: [email protected] (U. Sundaram). 0005-2736/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbamem.2012.08.003
known to be elevated in the mucosa of the chronically inflamed intestine [6–9]. Inhibition of Cl −/HCO3− exchange and stimulation of luminal Cl− secretion is one of the major transport alterations observed during intestinal inflammation [10]. Arachidonic acid metabolites have been shown to be elevated in the mucosa of inflammatory bowel disease [11] and in ulcerative colitis [12,13] and may be important for the observed diminished Cl−/HCO3− exchange [14]. In the rabbit model of chronic intestinal inflammation multiple unique alterations in electrolyte and nutrient transport processes have been shown to occur [1,15,16]. For example, the two distinct anion exchangers, Cl−/HCO3− and SCFA/HCO3− on the BBM of villus cells in the rabbit small intestine are uniquely regulated in the chronically inflamed intestine. While, both Cl−/HCO3− and SCFA/HCO3− exchange are inhibited in the chronically inflamed intestine, their mechanisms of alteration are different. Cl−/HCO3− exchange is diminished secondary to a reduction in the affinity of the exchanger for Cl− without a change in transporter numbers during chronic enteritis. In contrast, SCFA/HCO3− exchange is reduced secondary to a reduction in exchanger numbers without a change in the affinity of the transporter. However, it is not known whether a given immune-inflammatory mediator pathway is responsible for these unique alterations of anion exchangers seen during chronic enteritis. Immune-inflammatory mediators such as metabolites of arachidonic acid are known to infiltrate intestinal mucosa during intestinal inflammation [17]. Cytosolic phospholipase A2 (PLA2) catalyzes the release of
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arachidonic acid (AA) from membrane glycerophospholipids. In turn, AA metabolites (AAM) formed by both the cyclooxygenase (COX) and 5-lipoxygenase (LOX) pathways may contribute to the clinical diarrhea of IBD [17–20]. In fact, AAM have been reported to stimulate electrolyte secretion and inhibit NaCl absorption in animals with intestinal inflammation [9,21]. But, it is not known whether prevention of AA biosynthesis or the subsequent formation of AAM via COX and/or LOX pathways may reverse the alteration in NaCl absorption during chronic intestinal inflammation. Therefore, in this study we first studied the in vivo effect of arachidonyl trifluoro methylketone (ATMK) a potent inhibitor of PLA2 biosynthesis of AA [22]. Then to delineate whether the AAM of COX and/or LOX pathways regulate Cl −/HCO3−exchange, we treated rabbis with chronically inflamed with piroxicam and MK-886 to inhibit the COX and LOX pathways respectively. Finally, we also determined the molecular identity of the villus cell Cl −/HCO3−exchanger in the rabbit ileum. 2. Materials and methods 2.1. Induction of chronic inflammation Chronic intestinal inflammation was induced in pathogen-free New Zealand White male rabbits as described earlier [1]. Briefly, rabbits weighing 1.8–2.0 kg were intragastrically inoculated with Eimeria magna oocytes (~ 10,000 per animal) or sham inoculated with 0.9% NaCl (control animals). Animals inoculated with oocytes developed chronic intestinal inflammation on 13–15 days post inoculation. Only enterocytes from those animals that were histologically confirmed for chronic ileal inflammation were utilized for experiments. All the sham inoculated animals did not show any evidence of intestinal inflammation. 2.2. Drug treatment Normal and inflamed rabbits were treated with ATMK (3 mg/kg), piroxicam (10 mg/kg), or MK-886(0.5 mg/kg) (on days 12 and 13 post inoculation) by intramuscular injection. Animals were euthanized with 100 mg/kg of pentobarbital sodium through the ear vein according to protocol approved by the West Virginia University Animal Care Use Committee. ATMK, piroxicam and MK-886 were obtained from Sigma-Aldrich, St. Louis, MO, USA. 2.3. Cell isolation Villus and crypt cells were isolated from the intestine by a calcium chelation technique as previously described [23,24]. Previously established criteria were utilized to validate separation of villus and crypt cells. Some of these criteria included marker enzymes (e.g., thymidine kinase, alkaline phosphatase levels), transporter specificity (e.g., Na/H on the BBM of villus, but not crypt cells), intracellular pH (e.g., intracellular pH is higher in crypt cells compared with villus), and morphological differences (e.g., villus cells are larger and with better developed BBM compared with crypt cells). Viability of the isolated villus cells was determined by trypan blue exclusion. Isolated cells were then used for BBM vesicle (BBMV) preparation. 2.4. BBMV preparation BBMV from rabbit ileal villus cells were prepared by CaCl2 precipitation and differential centrifugation as previously described [25]. BBMV were resuspended in a medium appropriate to each experiment. BBMV purity was assured with marker enzyme enrichment (e.g., alkaline phosphatase).
2.5. Uptake studies in villus cell BBMV BBMV uptake studies were performed by the rapid-filtration technique as previously described [1,23]. Cl −/HCO3− exchange experiments were performed by resuspending BBMV in vesicle medium containing 105 mM N-methyl-D-glucamine (NMG) gluconate, 50 mM HEPES-Tris pH7.5 and either 50 mM KHCO3− gassed with 5%CO2-95%N2 or 50 mM potassium gluconate gassed with 100% N2. The reaction was started by adding 5 μl of vesicle to 95 μl reaction mix containing 5 mM NMG 36Cl −, 149.7 mM potassium gluconate, 50 mM MES-Tris pH 5.5 and either 0.3 mM KHCO3− gassed with 5% CO2-95% N2 or 150 mM potassium gluconate gasses with 100% N2. 1 mM 4,4-Diisothiocyanatostilbene-2,2-disulfonic acid disodium salt (DIDS), a potent anion exchange inhibitor, was added to the reaction mix when relevant. The uptake was stopped at desired time with ice cold stop solution containing 50 mM HEPES-Tris buffer (pH7.5), 0.10 mM MgSO4, 50 mM potassium gluconate and 100 mM NMG gluconate. The mixture was filtered on 0.45 μm Millipore (HAWP) filters and washed twice with 5 ml ice-cold stop solution. Filters with BBMV were dissolved in 5 ml scintillation fluid (Ecoscint, National Diagnostics), and radioactivity was determined in a Beckman 6500 Beta Scintillation Counter. Results were calculated as the HCO3 dependent DIDS sensitive Cl− uptake. 2.6. Immunohistochemistry for DRA Normal and chronically inflamed rabbit intestinal tissue was fixed in 10% (vol/vol) neutral-buffered formalin (Richard-Allan Scientific, Kalamazoo, MI) and embedded in paraffin. Sections (4 μm) were obtained by a microtome and were mounted on glass slides. Paraffin was removed from the sections by incubating the slides with xylene, and sections were hydrated gradually by incubating with graded ethanol. Antigen retrieval was performed by incubating the sections with 10 mM sodium citrate buffer, pH 6, at 95 °C for 5 min. Nonspecific binding sites in the tissue sections were blocked by incubation with goat serum for 1 hour at room temperature. The tissue sections were then incubated overnight with DRA primary antibody. Excess antibody was removed by washing with phosphate buffered saline (PBS) and incubated with goat anti-chicken IgG secondary antibody coupled to FITC (Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 1 hour. Excess secondary antibody was removed by washing with PBS and the tissue sections were mounted by use of ProLong Gold Antifade Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). Finally, the signal generated by FITC was observed under Zeiss LSM510 confocal microscope and photographed. 2.7. Real time PCR (RTQ-PCR) for DRA Total RNA was isolated from normal, inflamed and treated rabbit intestinal villus cells using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and RTQ-PCR was performed by a two step method. First–strand cDNA synthesis from total RNA was performed using Superscript™ III from Invitrogen Life Technologies using an equal mixture of oligo (dT) primer and random hexamer. The cDNAs generated were used as templates for RTQ- PCR. The reactions were performed using TaqMan universal PCR master mix from Applied Biosystems on an ABI 7300 Real Time PCR system according to the manufacturer- recommended protocol. The primer and probe sequences used for DRA RTQ-PCR are given below. Forward primer — 5′-TGGCACTTCTAAACACATCTCTG-3′ Reverse primer — 5′-TCGTCTGGGGCTAATCTCAC-3′ TaqMan probe — 5′ FAM-TCCATTTCCGGTTCTGAGCATGATGGTG‐ TAMRA 3′ RTQ-PCR for β-actin, using specific primers for rabbit gene, was run along with the DRA-specific RTQ-PCR as an endogenous control under
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similar conditions. The expression of β-actin was used to normalize the expression levels of DRA between the individual samples. The sequences of the rabbit β-actin primers and probes are given below: Forward primer — 5′ GCTATTTGGCGCTGGACTT 3′ Reverse primer — 5′ GCGGCTCGTAGCTCTTCTC 3′ TaqMan Probe — 5′ FAM-AAGAGATGGCCACGGCCGCGAAC-TAMRA 3′ Final concentrations of primer and probe for both DRA and β-actin were 500 nM and 100 nM respectively. The parameters of Real-time PCR were 95 °C for 15 seconds and 62 °C for 1 minute. All experiments were performed in triplicate and repeated at least thrice. 2.8. Western blot studies for DRA Polyclonal antibody against rabbit DRA was raised in chicken by using the custom antibody services provided by Invitrogen Life Technologies. Western blotting for DRA was performed according to the standard protocols. Briefly, BBM from villus cells of normal, inflamed and treated rabbit ileum were solubilized in RIPA buffer (50 mM Tris–HCl pH 7.4, 1% Igepal, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF) containing protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Equal volume of 2× SDS/sample buffer (100 mM Tris, 25% glycerol, 2%SDS, 0.01% bromophenol blue, 10% 2- mercapto ethanol, pH 6.8) was added and the proteins were separated on a 4–20% Ready Gel (Bio-Rad Laboratories, Hercules, CA, USA). The separated proteins were transferred on to polyvinylidenedifluoride membrane and probed with the primary antibody (Ab) against rabbit DRA Ab at a dilution of 1:2000 and incubated overnight at 4 °C. Rabbit anti-chicken secondary antibody coupled to horseradish peroxidase was used to monitor the binding of the primary antibody. ECL western blotting detection reagent (GE Healthcare Biosciences, Piscataway, NJ, USA) was used to detect the DRA specific signal. The chemiluminescent signal was quantitated by using a Molecular Dynamics (Sunnyvale, CA, USA) densitometric scanner. All experiments were performed in triplicate. 2.9. Data presentation Results presented represent means ± SE of experiments performed and calculated using GraphPadInstat program. All uptakes were done in triplicate. Student t-test was performed for statistical analysis and is significant if the p value is b 0.05. The n number in each figure represents the number of animals with respective conditions that were studied.
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3. Results 3.1. Effect of inhibition of AA release on Cl −/HCO3− exchange Cl −/HCO3− exchange, defined as HCO3−-dependent and DIDSsensitive Cl uptake, is diminished in villus cell BBMV from the chronically inflamed rabbit intestine. In order to determine whether arachidonic acid metabolites are responsible for the decrease in Cl −/HCO3− activity during chronic inflammation, rabbits were treated in vivo with ATMK. ATMK treatment reversed Cl −/HCO3− exchange inhibition in villus cell BBMV (756 ± 55 pmol/mg protein/min in control, 159 ± 22 in inflamed, and 678 ± 47 in inflamed treated with ATMK, p b 0.01, n = 5; Fig. 1A). Interestingly, ATMK treatment stimulated Cl−/HCO3− exchange in villus cell BBMV from normal rabbit intestine (1438 ± 114 pmol/mg protein/min in ATMK treated, p b 0.05, n = 5; and Fig. 1B). These data indicate that blocking the release of AA reverses the inhibition of Cl −/HCO3− activity during chronic inflammation. 3.2. Kinetic studies for Cl −/HCO3− exchange during inhibition of AA release Previous studies from our lab have shown that HCO3−-dependent and DIDS-sensitive 36Cl uptake in BBMV was linear for at least 10 seconds, hence uptake studies for various concentrations were carried out at 3 seconds. Fig. 2A shows 36Cl uptake in BBMV as a function of varying concentrations of extra-vesicular Cl. As the concentration of extra-vesicular Cl was increased, 36Cl uptake was stimulated and subsequently became saturated in all conditions. Kinetic parameters demonstrated that in the chronically inflamed intestine inhibition of Cl −/HCO3− exchange activity occurs as a result of an increase in the Km, and in vivo ATMK treatment revered this change (Km for Cl − uptake in villus cell BBMV was 20.6 ± 1.0 mM in normal, 51.3 ± 1.4 in inflamed, 21.7 ± 1.2* inflamed + ATMK, n = 3, p b 0.05). The maximal rate of uptake (Vmax) was unaffected in all conditions (Vmax was 43.5 ± 1.5 nmol/mg protein min in normal, 45.4 ± 1.3 inflamed, 46.2 ± 1.4 inflamed + ATMK; n = 3). In the normal intestine, kinetic parameters demonstrated that ATMK treatment increased the maximal rate of uptake of Cl (Vmax for Cl− - uptake in BBMV was 98.6 ± 2.7 nmol/mg protein min in normal + ATMK, n = 3, p = 0.01 (Fig. 2B), without a change in the affinity of the transporter for Cl. These data indicate that the mechanism of reversal of inhibition of Cl −/HCO3− exchange by ATMK during chronic intestinal inflammation was secondary to a restoration in the affinity for Cl − rather than an alteration in the number of BBM of Cl −/HCO3− exchangers.
Fig. 1. Effect of ATMK treatment on Cl−/HCO3− exchange in villus cell BBMV from the normal and chronically inflamed rabbit ileum. Cl−/HCO3− exchange was significantly inhibited during chronic intestinal inflammation. A: Inhibited Cl−/HCO3− exchange in chronically inflamed rabbit BBMV was reversed back to normal levels by ATMK treatment, n = 5, *p b 0.05. B: ATMK treatment significantly stimulated Cl−/HCO3− exchange in normal rabbit BBMV, n = 5, *p b 0.05.
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Fig. 3. Effect of piroxicam treatment on Cl−/HCO3− exchange in villus cell BBMV from the normal and chronically inflamed rabbit ileum. Cl−/HCO3− exchange that is inhibited during chronic intestinal inflammation was reversed back to normal levels by piroxicam treatment (1147±73 pmol/mg protein min in normal; 324±16* in inflamed and 1100±34* in inflamed+piroxicam, n=3, *pb 0.001). Piroxicam treatment did not have any effect on the normal rabbit BBMV (1037±33 pmol/mg protein min, n=3).
3.5. Kinetic studies for Cl −/HCO3− exchange during inhibition of COX pathway
Fig. 2. Kinetics of Cl−/HCO3− exchange in villus cell BBMV from normal, inflamed and ATMK treated rabbit ileum. HCO3−-dependent and DIDS-sensitive 36Cl uptake is shown as a function of varying concentrations of extra vesicular Cl− at 3 secs. As the concentration of extra-vesicular Cl− was increased, 36Cl uptake was stimulated and subsequently became saturated in all conditions. Analysis of the data yielded kinetic parameters. A: In chronically inflamed BBMV the Cl−/HCO3− exchange was inhibited by altering the affinity of the transporter for Cl− and ATMK treatment significantly reversed the inhibited Cl−/HCO3− exchange in chronically inflamed villus cell BBMV by restoring the affinity of the transporter (Km) without an alteration in the maximal rate of uptake (Vmax) of Cl, n = 3. B: In normal rabbit villus cell BBMV ATMK treatment stimulated Cl−/HCO3− exchange by altering the number of transporters (Vmax), n = 3.
To determine the mechanism of piroxicam-mediated reversal of inhibition of Cl−/HCO3− exchange in the chronically inflamed intestine, kinetics studies were performed. These data demonstrated that, piroxicam did not alter the Vmax for Cl− uptake (Vmax for Cl− uptake in BBMV was 47.5 ± 2.2 nmol/mg protein. Min in normal, 42.8 ±2.8 in normal + piroxicam, 42.4 ±1.0 in inflamed and 45.1 ± 1.8 in inflamed + piroxicam; n = 3). However, the affinity for Cl− uptake, which was reduced in the chronically inflamed rabbit intestine, was restored by piroxicam treatment (Km for Cl− uptake in BBMV was 19.0 ± 1.0 mM in normal, 43.3 ±1.8*inflamed, 20.5 ± 2.5 normal+ piroxicam, and 23.1 ± 1.2*inflamed+ piroxicam; n = 3, *p b 0.05). Thus, these data indicated that the mechanism of reversal of inhibition of Cl −/HCO3− exchange by piroxicam during chronic intestinal inflammation was secondary to a restoration of the affinity for Cl− rather than an increase in transporter numbers. 3.6. Identification of the Cl −/HCO3− exchanger in rabbit ileum BBMV–DRA Different members of SLC26A protein family may mediate Cl−/HCO3− exchange depending upon their tissue distribution. In order to identify
3.3. Effect of inhibition of COX pathway on Cl −/HCO3− exchange Piroxicam, was utilized to inhibit cyclooxygenase (COX1 and COX2) pathway in vivo in the chronically inflamed intestine. Piroxicam reversed the inhibition of Cl−/HCO3− exchange in villus cell BBMV (1147 ± 73 pmol/mg protein min in normal; 324 ±16 in inflamed; 1037± 33 in normal + piroxicam and 1100 ±34* in inflamed+ piroxicam, n = 3, *p b 0.01; Fig. 3). These data indicate that the COX pathway likely mediates the inhibition of Cl −/HCO3− exchange during chronic intestinal inflammation.
3.4. Effect of inhibition of LOX pathway on Cl −/HCO3− exchange Since AA can be metabolized by the LOX pathway as well, MK-886 was utilized to inhibit this pathway. In vivo treatment of rabbits with chronic intestinal inflammation with MK-886 did not reverse the diminished Cl −/HCO3−exchange in villus cell BBMV (Fig. 4). Thus, these data indicate that inhibition of Cl −/HCO3− exchange during chronic intestinal inflammation is not mediated by the LOX pathway.
Fig. 4. Effect of MK-886 treatment on Cl−/HCO3− exchange in villus cell BBMV from chronically inflamed rabbit ileum. Cl−/HCO3− exchange that was inhibited during chronic intestinal inflammation was not reversed by MK-886 treatment (324 ± 16 in inflamed and 411 ± 46 in inflamed + MK-886, n = 3, p = not significant).
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with kinetic studies that Cl−/HCO3− exchange inhibition during chronic inflammation is not secondary to a decrease in protein expression. 3.8. RTQ-PCR studies for DRA Next to determine the molecular mechanism of COX mediated inhibition of DRA, we determined its mRNA abundance in villus cells by RTQ-PCR. DRA specific mRNA abundance was not significantly altered in any condition (Fig. 7). 3.9. Western blot studies for DRA
Fig. 5. Relative abundance of anion exchangers in normal rabbit ileum by RTQ-PCR. mRNA abundance is expressed relative to PAT1. Expression of mRNA levels of DRA were 10 fold higher compared to PAT1 and Pendrin, n = 3, *p b 0.05.
the major anion exchanger responsible for Cl−/HCO3− in the rabbit ileal villus cells, RTQ-PCR for DRA (SLC26A3), PAT1 (SLC26A) and Pendrin (SLC26A4) were performed. The results show that expression of DRA was 10 fold higher than PAT1 or Pendrin in rabbit villus cells (Fig. 5).
Since mRNA level may not necessarily correlate with immunoreactive protein in the BBM, we quantitated DRA specific immunoreactive protein. There was no significant difference in the immunoreactive levels of DRA in any of the conditions tested (Fig. 8A). Densitometric quantitation confirmed these observations (Fig. 8B). These results, in conjunction with kinetic parameters and RTQ-PCR data demonstrate that the mechanism of inhibition of DRA by the COX pathway during chronic intestinal inflammation was secondary to a decrease in the affinity of the transporter for Cl− without a change in the transporter numbers.
3.7. Immunocytochemistry studies of DRA
4. Discussion
In order to localize DRA in the rabbit ileum and to investigate if it is affected by chronic intestinal inflammation, immunocytochemistry was performed. Fig. 6 demonstrates that DRA is present in the BBM of rabbit small intestinal villus cells. Further, DRA protein expression was unaltered during chronic intestinal inflammation. This is consistent
This study demonstrated that in a mammalian animal model of IBD, inhibition of coupled NaCl absorption occurs secondary to the inhibition of Cl−/HCO3− but not, Na/H exchange in the BBM of absorptive villus cells. This rabbit ileal villus cell BBM Cl−/HCO3− exchanger was identified to be DRA. The inhibition of DRA in the chronically inflamed
Fig. 6. Immunofluorescence localization of DRA in rabbit ileum. A & B: Immunofluorescent detection of DRA in rabbit ileum was performed using anti-DRA antibody following a pre-incubation of the antibody with antigenic peptide. C & D: Immunofluorescence of DRA expression compared between normal and chronically inflamed rabbit ileum.
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Fig. 7. RTQ-PCR analysis for DRA in rabbit ileum. DRA specific mRNA abundance was not changed between normal, inflamed and piroxicam treated rabbit ileum, n=3, p= insignificant.
intestine was mediated by arachidonic acid metabolites, specifically by the metabolites of the cyclooxygenase, but not the lipoxygenase pathway. The most common and disabling morbidity of IBD is diarrhea which is caused by malabsorption and secretion of electrolytes and water These electrolyte transport abnormalities are likely mediated by immune-inflammatory mediators known to be elevated in the mucosa of the chronically inflamed intestine [26–28]. Given that treatments for IBD are few and fraught with numerous side effects, it is necessary to more clearly define which immune inflammatory mediators affect electrolyte absorption so a more precise treatment can be developed with fewer side effects. Therefore, this study was designed to begin to determine which immune inflammatory mediators regulate coupled NaCl absorption, specifically Cl −/HCO3−exchange, in the chronically inflamed intestine. In previous studies we had demonstrated that Cl −/HCO3−exchange was inhibited in the BBM of villus cells from the chronically inflamed rabbit intestine. The mechanism of inhibition was secondary to a reduction in the affinity of the transporter for Cl without a change in the number of transporters in the BBM. However, the molecular identity of this Cl −/HCO3−exchanger in the rabbit intestine was unknown. This study demonstrated that DRA mediates Cl −/HCO3−
Fig. 8. Western blot analysis for DRA in rabbit ileum. A: DRA specific protein expression was not altered between normal, inflamed and piroxicam treated rabbit ileum. B: Densitometric analysis shows the relative expression of DRA protein levels in all the different experimental condition with reference to its level in villus cell BBM from normal intestine, n = 3, p = insignificant.
exchange in rabbit ileal villus cells. This observation is different from that in mouse where PAT1 is the major Cl −/HCO3− exchanger expressed in BBM of the small intestine [3,37], while DRA is abundantly expressed in apical membranes of colonocytes [3,38]. Despite the low expression of DRA in the small intestine, functional studies have shown that DRA is the major Cl −/HCO3− exchanger coupled with NHE3 for electro neutral NaCl absorption in murine small intestine [39]. In contrast to mouse, RTQ-PCR studies showed that expression of mRNA of DRA was 10 fold higher compared to PAT1 and Pendrin in the rabbit small intestine (Fig. 5). Consistent with RTQ-PCR data, immunocytochemistry and Western blot studies showed that DRA was indeed the predominant anion exchanger in the BBM of villus cells in the rabbit ileum (Fig. 6). Thus, these data clearly indicate that DRA mediates Cl −/HCO3− exchange in villus cells in the rabbit small intestine. Other studies have demonstrated the role of DRA in the causation of diarrhea. Mutation in DRA causes congenital chloride diarrhea both in patients and experimental animal models [40,41] and loss of transporter function of DRA plays a crucial role in the pathogenesis of diarrhea in intestinal inflammation [42]. The inhibition of DRA in the chronically inflamed rabbit intestine is likely mediated by immune-inflammatory mediators known to be elevated in the mucosa. We previously reported that a variety of immune-inflammatory mediators including prostaglandins and leukotrienes are markedly increased in the mucosa of the chronically inflamed rabbit intestine [35]. Further, previous studies have demonstrated that these agents likely mediate multiple unique alterations in electrolyte, Na-nutrient and Na-bile acid transporters during chronic intestinal inflammation [1,15,16,29,30]. For example Na-glucose co-transporter was inhibited by a decrease in the number of cotransporter [16] while Na-alanine co-transporter was inhibited by a reduction in the affinity for amino acid [15]. Our in vitro studies have demonstrated that prostaglandin E2 (PGE2), a cyclooxygenase pathway metabolite, inhibits Na-glucose co-transport while leukotriene D4 (LTD4), a 5-lipoxygenase pathway metabolite, was responsible for inhibition Na-alanine co-transport in IEC-18 cells [36]. That DRA inhibition in the chronically inflamed rabbit intestine is likely mediated by immune inflammatory mediators as well is supported by our previous observation that broad spectrum immune modulation alleviated this inhibition. Glucocorticoid ethylprednisolone is a common treatment for IBD and as a nonspecific immune modulator known to block multiple pathways of the immune system including the arachidonic acid metabolite cascade. Further, as a broad spectrum immune modulator, glucocorticoid treatment of IBD has been shown to alleviate the inhibition of Cl−/HCO3−exchange as well as other transporters [31–34]. Nevertheless, more specific immune mechanism of regulation of Cl −/HCO3−exchange in the chronically inflamed intestine was not well understood. In chronically inflamed rabbit intestine, specific inhibition of the formation arachidonic acid reversed the inhibition of Cl −/HCO3− exchange activity, Kinetic studies showed that the mechanism of reversal of Cl −/HCO3− exchange by ATMK treatment was secondary to restoration in the affinity of the transporter for Cl − without affecting transporter number (Vmax) in chronically inflamed rabbit ileum. These results indicate that AAM mediate the inhibition of Cl −/HCO3− exchange during chronic enteritis Interestingly, ATMK treatment stimulated Cl −/HCO3− exchange in the normal intestine and the mechanism of stimulation was secondary to an increase in exchanger numbers in the BBM. This mechanism of regulation of Cl −/HCO3− exchange appears to be lost in the chronically inflamed intestine. It may be that AAM produced in the normal intestine have a baseline inhibitory effect on this exchanger which is alleviated by ATMK. Future studies will need to further elucidate this finding. Since AAM produced by both the COX and/or LOX pathways are known to be elevated in the chronically inflamed intestine, each pathway was selectively inhibited to determine which specifically mediated
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the inhibition of Cl−/HCO3− exchange. It was demonstrated that the cyclooxygenase inhibitor piroxicam reversed the Cl−/HCO3− exchange while 5-lipoxygenase inhibitor MK-886 did not have an effect on Cl−/HCO3− exchange during chronic intestinal inflammation. This indicated that during chronic intestinal inflammation, Cl−/HCO3− exchange is regulated by immune inflammatory mediators produced by the cyclooxygenase but not, the 5-lipoxygenase pathway. Other investigators have also shown the importance of DRA regulation by immune inflammatory mediators. For example, Saksena et al., have demonstrated that IFN-γ significantly inhibited DRA gene expression and promoter activity in Caco-2 cells. The inhibition of DRA was mediated by signal transducer and activator of transcription factor 1 (STAT1) [43,44]. Therefore, delineating the pathway responsible for alteration of coupled NaCl absorption by altering DRA may yield precise targeted therapies for patients with IBD. The COX pathway mediated alteration of Cl−/HCO3− exchange at the molecular level appears to be post translational. RTQ-PCR and Western blot studies showed that there was no change in DRA specific mRNA abundance and protein expression respectively in the chronically inflamed intestine(Figs. 7 and 8). Immunocytochemistry studies also showed there was no difference in expression of DRA between normal and inflamed rabbit villus cell BBM (Fig. 6C and D). Further, both ATMK and piroxicam treatment reversed the inhibited Cl−/HCO3− exchange by restoring the affinity of the transporter. Thus, together our functional and molecular data demonstrate that the mechanism of inhibition of DRA is post translational during chronic enteritis. The alteration in the affinity of the DRA for Cl− during chronic ileitis may be due to changes in glycosylation and/or phosphorylation of the transporter since they are the most common post translational modifications observed in membrane proteins. In conclusion, this study demonstrates that DRA is the primary villus cell BBM Cl−/HCO3− exchanger in the rabbit intestine. It is inhibited secondary to a reduction in the affinity of the transporter for Cl in the chronically inflamed intestine. This inhibition of DRA is mediated by arachidonic acid metabolites during chronic enteritis. Finally, cyclooxygenase but not the 5-lipoxygenase pathway metabolites mediate the inhibition of DRA during chronic intestinal inflammation. References [1] U. Sundaram, A.B. West, Effect of chronic inflammation on electrolyte transport in rabbit ileal villus and crypt cells, Am. J. Physiol. 272 (1997) G732–G741. [2] U. Sundaram, R.G. Knickelbein, J.W. Dobbins, Mechanism of intestinal secretion. Effect of serotonin on rabbit ileal crypt and villus cells, J. Clin. Invest. 87 (1991) 743–746. [3] Z. Wang, S. Petrovic, E. Mann, M. Soleimani, Identification of an apical Cl(−)/HCO3(−) exchanger in the small intestine, Am. J. Physiol. Gastrointest. Liver Physiol. 282 (2002) G573–G579. [4] D.B. Mount, M.F. Romero, The SLC26 gene family of multifunctional anion exchangers, Pflugers Arch. 447 (2004) 710–721. [5] J. Kere, H. Lohi, P. Hoglund, Genetic disorders of membrane transport III. Congenital chloride diarrhea, Am. J. Physiol. 276 (1999) G7–G13. [6] T.R. Traynor, D.R. Brown, S.M. O'Grady, Effects of inflammatory mediators on electrolyte transport across the porcine distal colon epithelium, J. Pharmacol. Exp. Ther. 264 (1993) 61–66. [7] M.W. Musch, J.F. Kachur, R.J. Miller, M. Field, J.S. Stoff, Bradykinin-stimulated electrolyte secretion in rabbit and guinea pig intestine. Involvement of arachidonic acid metabolites, J. Clin. Invest. 71 (1983) 1073–1083. [8] S.A. Hojvat, M.W. Musch, R.J. Miller, Stimulation of prostaglandin production in rabbit ileal mucosa by bradykinin, J. Pharmacol. Exp. Ther. 226 (1983) 749–755. [9] C.S. Hyun, H.J. Binder, Mechanism of leukotriene D4 stimulation of Cl- secretion in rat distal colon in vitro, Am. J. Physiol. 265 (1993) G467–G473. [10] H. Zhang, N. Ameen, J.E. Melvin, S. Vidyasagar, Acute inflammation alters bicarbonate transport in mouse ileum, J. Physiol. 581 (2007) 1221–1233. [11] M. Romero, R. Artigiani, H. Costa, C.T. Oshima, S. Miszputen, M. Franco, Evaluation of the immunoexpression of COX-1, COX-2 and p53 in Crohn's disease, Arq. Gastroenterol. 45 (2008) 295–300. [12] E. Carty, C. Nickols, R.M. Feakins, D.S. Rampton, Thromboxane synthase immunohistochemistry in inflammatory bowel disease, J. Clin. Pathol. 55 (2002) 367–370. [13] T. Nishida, H. Miwa, A. Shigematsu, M. Yamamoto, M. Iida, M. Fujishima, Increased arachidonic acid composition of phospholipids in colonic mucosa from patients with active ulcerative colitis, Gut 28 (1987) 1002–1007. [14] U. Seidler, H. Lenzen, A. Cinar, T. Tessema, A. Bleich, B. Riederer, Molecular mechanisms of disturbed electrolyte transport in intestinal inflammation, Ann. N. Y. Acad. Sci. 1072 (2006) 262–275.
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Dobbins, Mechanism of intestinal secretion: effect of cyclic AMP on rabbit ileal crypt and villus cells, Proc. Natl. Acad. Sci. U. S. A. 88 (1991) 6249–6253. [25] U. Sundaram, S. Coon, S. Wisel, A.B. West, Corticosteroids reverse the inhibition of Na-glucose cotransport in the chronically inflamed rabbit ileum, Am. J. Physiol. 276 (1999) G211–G218. [26] C.M. Surawicz, Mechanisms of diarrhea, Curr. Gastroenterol. Rep. 12 (2010) 236–241. [27] O. Martinez-Augustin, I. Romero-Calvo, M.D. Suarez, A. Zarzuelo, F.S. de Medina, Molecular bases of impaired water and ion movements in inflammatory bowel diseases, Inflamm. Bowel Dis. 15 (2009) 114–127. [28] M. Field, Intestinal ion transport and the pathophysiology of diarrhea, J. Clin. Invest. 111 (2003) 931–943. [29] T. Manokas, J.J. Fromkes, U. Sundaram, Effect of chronic inflammation on ileal short-chain fatty acid/bicarbonate exchange, Am. J. Physiol. Gastrointest. Liver Physiol. 278 (2000) G585–G590. [30] U. Sundaram, S. Wisel, S. 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Prostaglandins, not the leukotrienes, regulate Cl −/HCO3− exchange (DRA, SLC26A3) in villus cells in the chronically inflamed rabbit ileum Palanikumar Manoharan, Steven Coon, Walter Baseler, Shanmuga Sundaram, Ramesh Kekuda, Uma Sundaram ⁎ Section of Digestive Diseases and West Virginia Clinical and Translational Science Institute, West Virginia University, Morgantown, WV 26505, USA
a r t i c l e
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Article history: Received 20 February 2012 Received in revised form 10 July 2012 Accepted 7 August 2012 Available online 3 September 2012 Keywords: Inflammatory bowel disease Down regulated in adenoma Cyclooxygenase Lipoxygenase Arachidonic acid metabolites Regulation of Cl−/HCO3− exchange Piroxicam
a b s t r a c t Previously studies have demonstrated that Cl−/HCO3− exchange was inhibited during chronic intestinal inflammation secondary to decrease in the affinity of the exchanger for Cl − rather than the number of transporters. Arachidonic acid metabolites (AAM) are elevated in the mucosa of the chronically inflamed small intestine. However, their role in the alteration of Cl −/HCO3− during chronic enteritis was unknown. Inhibition of AAM formation with arachidonyl trifluoro methylketone (ATMK) in chronically inflamed rabbit intestine reversed the diminished Cl−/HCO3− exchange activity. Kinetics studies showed that the reversal was secondary to restoration of the altered affinity of transporter. Downstream regulation of Cl −/HCO3− inhibition by AAM was determined to be by the cyclooxygenase pathway since only inhibition of cyclooxygenase with piroxicam treatment reversed the inhibited Cl −/HCO3− exchange. Further, DRA was shown to be the primary Cl −/HCO3− exchanger in villus cells. Kinetics and molecular studies indicated that the mechanism of inhibition of Cl −/HCO3− exchange by cyclooxygenase pathway metabolites was secondary to diminished affinity of the transporter for Cl − without a change in DRA BBM expression. Thus our data indicated that cyclooxygenase pathway metabolites mediate the inhibition of DRA during chronic intestinal inflammation. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The most common and disabling morbidity of human inflammatory bowel disease is diarrhea. It results as a result of altered absorption and secretion of electrolytes and fluid by intestinal epithelial cells. Neutral NaCl absorption occurs in the mammalian small intestine through coupled Na/H and Cl−/HCO3− exchange on the brush border membrane (BBM) of absorptive villus cells. In an animal model of chronic small intestinal inflammation which has many characteristics of human IBD, we have demonstrated the inhibition of coupled NaCl absorption [1]. The mechanism is secondary to a reduction in Cl−/HCO3− exchange, without a change in Na/H exchange in the BBM of villus cells [2]. However, the identity and the specific immune regulation of Cl−/HCO3− exchange in the chronically inflamed rabbit intestine are not known. Various Cl−/HCO3− exchangers (SLC26 family), PAT1, DRA and Pendrin have been demonstrated to have selective and limited tissue distribution. Further, PAT1 (SLC26A6) and DRA (SLC26A3) have been reported in the BBM of intestinal epithelia [3–5]. Nevertheless, the major Cl−/HCO3− exchanger that is down regulated in the chronically inflamed rabbit small intestine is not known. In IBD (e.g. Crohn's disease) alterations in absorption of electrolytes and water causing diarrhea is mediated by inflammatory mediators ⁎ Corresponding author. Tel.:+1 304 2936111. E-mail address: [email protected] (U. Sundaram). 0005-2736/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbamem.2012.08.003
known to be elevated in the mucosa of the chronically inflamed intestine [6–9]. Inhibition of Cl −/HCO3− exchange and stimulation of luminal Cl− secretion is one of the major transport alterations observed during intestinal inflammation [10]. Arachidonic acid metabolites have been shown to be elevated in the mucosa of inflammatory bowel disease [11] and in ulcerative colitis [12,13] and may be important for the observed diminished Cl−/HCO3− exchange [14]. In the rabbit model of chronic intestinal inflammation multiple unique alterations in electrolyte and nutrient transport processes have been shown to occur [1,15,16]. For example, the two distinct anion exchangers, Cl−/HCO3− and SCFA/HCO3− on the BBM of villus cells in the rabbit small intestine are uniquely regulated in the chronically inflamed intestine. While, both Cl−/HCO3− and SCFA/HCO3− exchange are inhibited in the chronically inflamed intestine, their mechanisms of alteration are different. Cl−/HCO3− exchange is diminished secondary to a reduction in the affinity of the exchanger for Cl− without a change in transporter numbers during chronic enteritis. In contrast, SCFA/HCO3− exchange is reduced secondary to a reduction in exchanger numbers without a change in the affinity of the transporter. However, it is not known whether a given immune-inflammatory mediator pathway is responsible for these unique alterations of anion exchangers seen during chronic enteritis. Immune-inflammatory mediators such as metabolites of arachidonic acid are known to infiltrate intestinal mucosa during intestinal inflammation [17]. Cytosolic phospholipase A2 (PLA2) catalyzes the release of
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arachidonic acid (AA) from membrane glycerophospholipids. In turn, AA metabolites (AAM) formed by both the cyclooxygenase (COX) and 5-lipoxygenase (LOX) pathways may contribute to the clinical diarrhea of IBD [17–20]. In fact, AAM have been reported to stimulate electrolyte secretion and inhibit NaCl absorption in animals with intestinal inflammation [9,21]. But, it is not known whether prevention of AA biosynthesis or the subsequent formation of AAM via COX and/or LOX pathways may reverse the alteration in NaCl absorption during chronic intestinal inflammation. Therefore, in this study we first studied the in vivo effect of arachidonyl trifluoro methylketone (ATMK) a potent inhibitor of PLA2 biosynthesis of AA [22]. Then to delineate whether the AAM of COX and/or LOX pathways regulate Cl −/HCO3−exchange, we treated rabbis with chronically inflamed with piroxicam and MK-886 to inhibit the COX and LOX pathways respectively. Finally, we also determined the molecular identity of the villus cell Cl −/HCO3−exchanger in the rabbit ileum. 2. Materials and methods 2.1. Induction of chronic inflammation Chronic intestinal inflammation was induced in pathogen-free New Zealand White male rabbits as described earlier [1]. Briefly, rabbits weighing 1.8–2.0 kg were intragastrically inoculated with Eimeria magna oocytes (~ 10,000 per animal) or sham inoculated with 0.9% NaCl (control animals). Animals inoculated with oocytes developed chronic intestinal inflammation on 13–15 days post inoculation. Only enterocytes from those animals that were histologically confirmed for chronic ileal inflammation were utilized for experiments. All the sham inoculated animals did not show any evidence of intestinal inflammation. 2.2. Drug treatment Normal and inflamed rabbits were treated with ATMK (3 mg/kg), piroxicam (10 mg/kg), or MK-886(0.5 mg/kg) (on days 12 and 13 post inoculation) by intramuscular injection. Animals were euthanized with 100 mg/kg of pentobarbital sodium through the ear vein according to protocol approved by the West Virginia University Animal Care Use Committee. ATMK, piroxicam and MK-886 were obtained from Sigma-Aldrich, St. Louis, MO, USA. 2.3. Cell isolation Villus and crypt cells were isolated from the intestine by a calcium chelation technique as previously described [23,24]. Previously established criteria were utilized to validate separation of villus and crypt cells. Some of these criteria included marker enzymes (e.g., thymidine kinase, alkaline phosphatase levels), transporter specificity (e.g., Na/H on the BBM of villus, but not crypt cells), intracellular pH (e.g., intracellular pH is higher in crypt cells compared with villus), and morphological differences (e.g., villus cells are larger and with better developed BBM compared with crypt cells). Viability of the isolated villus cells was determined by trypan blue exclusion. Isolated cells were then used for BBM vesicle (BBMV) preparation. 2.4. BBMV preparation BBMV from rabbit ileal villus cells were prepared by CaCl2 precipitation and differential centrifugation as previously described [25]. BBMV were resuspended in a medium appropriate to each experiment. BBMV purity was assured with marker enzyme enrichment (e.g., alkaline phosphatase).
2.5. Uptake studies in villus cell BBMV BBMV uptake studies were performed by the rapid-filtration technique as previously described [1,23]. Cl −/HCO3− exchange experiments were performed by resuspending BBMV in vesicle medium containing 105 mM N-methyl-D-glucamine (NMG) gluconate, 50 mM HEPES-Tris pH7.5 and either 50 mM KHCO3− gassed with 5%CO2-95%N2 or 50 mM potassium gluconate gassed with 100% N2. The reaction was started by adding 5 μl of vesicle to 95 μl reaction mix containing 5 mM NMG 36Cl −, 149.7 mM potassium gluconate, 50 mM MES-Tris pH 5.5 and either 0.3 mM KHCO3− gassed with 5% CO2-95% N2 or 150 mM potassium gluconate gasses with 100% N2. 1 mM 4,4-Diisothiocyanatostilbene-2,2-disulfonic acid disodium salt (DIDS), a potent anion exchange inhibitor, was added to the reaction mix when relevant. The uptake was stopped at desired time with ice cold stop solution containing 50 mM HEPES-Tris buffer (pH7.5), 0.10 mM MgSO4, 50 mM potassium gluconate and 100 mM NMG gluconate. The mixture was filtered on 0.45 μm Millipore (HAWP) filters and washed twice with 5 ml ice-cold stop solution. Filters with BBMV were dissolved in 5 ml scintillation fluid (Ecoscint, National Diagnostics), and radioactivity was determined in a Beckman 6500 Beta Scintillation Counter. Results were calculated as the HCO3 dependent DIDS sensitive Cl− uptake. 2.6. Immunohistochemistry for DRA Normal and chronically inflamed rabbit intestinal tissue was fixed in 10% (vol/vol) neutral-buffered formalin (Richard-Allan Scientific, Kalamazoo, MI) and embedded in paraffin. Sections (4 μm) were obtained by a microtome and were mounted on glass slides. Paraffin was removed from the sections by incubating the slides with xylene, and sections were hydrated gradually by incubating with graded ethanol. Antigen retrieval was performed by incubating the sections with 10 mM sodium citrate buffer, pH 6, at 95 °C for 5 min. Nonspecific binding sites in the tissue sections were blocked by incubation with goat serum for 1 hour at room temperature. The tissue sections were then incubated overnight with DRA primary antibody. Excess antibody was removed by washing with phosphate buffered saline (PBS) and incubated with goat anti-chicken IgG secondary antibody coupled to FITC (Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 1 hour. Excess secondary antibody was removed by washing with PBS and the tissue sections were mounted by use of ProLong Gold Antifade Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). Finally, the signal generated by FITC was observed under Zeiss LSM510 confocal microscope and photographed. 2.7. Real time PCR (RTQ-PCR) for DRA Total RNA was isolated from normal, inflamed and treated rabbit intestinal villus cells using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and RTQ-PCR was performed by a two step method. First–strand cDNA synthesis from total RNA was performed using Superscript™ III from Invitrogen Life Technologies using an equal mixture of oligo (dT) primer and random hexamer. The cDNAs generated were used as templates for RTQ- PCR. The reactions were performed using TaqMan universal PCR master mix from Applied Biosystems on an ABI 7300 Real Time PCR system according to the manufacturer- recommended protocol. The primer and probe sequences used for DRA RTQ-PCR are given below. Forward primer — 5′-TGGCACTTCTAAACACATCTCTG-3′ Reverse primer — 5′-TCGTCTGGGGCTAATCTCAC-3′ TaqMan probe — 5′ FAM-TCCATTTCCGGTTCTGAGCATGATGGTG‐ TAMRA 3′ RTQ-PCR for β-actin, using specific primers for rabbit gene, was run along with the DRA-specific RTQ-PCR as an endogenous control under
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similar conditions. The expression of β-actin was used to normalize the expression levels of DRA between the individual samples. The sequences of the rabbit β-actin primers and probes are given below: Forward primer — 5′ GCTATTTGGCGCTGGACTT 3′ Reverse primer — 5′ GCGGCTCGTAGCTCTTCTC 3′ TaqMan Probe — 5′ FAM-AAGAGATGGCCACGGCCGCGAAC-TAMRA 3′ Final concentrations of primer and probe for both DRA and β-actin were 500 nM and 100 nM respectively. The parameters of Real-time PCR were 95 °C for 15 seconds and 62 °C for 1 minute. All experiments were performed in triplicate and repeated at least thrice. 2.8. Western blot studies for DRA Polyclonal antibody against rabbit DRA was raised in chicken by using the custom antibody services provided by Invitrogen Life Technologies. Western blotting for DRA was performed according to the standard protocols. Briefly, BBM from villus cells of normal, inflamed and treated rabbit ileum were solubilized in RIPA buffer (50 mM Tris–HCl pH 7.4, 1% Igepal, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF) containing protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Equal volume of 2× SDS/sample buffer (100 mM Tris, 25% glycerol, 2%SDS, 0.01% bromophenol blue, 10% 2- mercapto ethanol, pH 6.8) was added and the proteins were separated on a 4–20% Ready Gel (Bio-Rad Laboratories, Hercules, CA, USA). The separated proteins were transferred on to polyvinylidenedifluoride membrane and probed with the primary antibody (Ab) against rabbit DRA Ab at a dilution of 1:2000 and incubated overnight at 4 °C. Rabbit anti-chicken secondary antibody coupled to horseradish peroxidase was used to monitor the binding of the primary antibody. ECL western blotting detection reagent (GE Healthcare Biosciences, Piscataway, NJ, USA) was used to detect the DRA specific signal. The chemiluminescent signal was quantitated by using a Molecular Dynamics (Sunnyvale, CA, USA) densitometric scanner. All experiments were performed in triplicate. 2.9. Data presentation Results presented represent means ± SE of experiments performed and calculated using GraphPadInstat program. All uptakes were done in triplicate. Student t-test was performed for statistical analysis and is significant if the p value is b 0.05. The n number in each figure represents the number of animals with respective conditions that were studied.
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3. Results 3.1. Effect of inhibition of AA release on Cl −/HCO3− exchange Cl −/HCO3− exchange, defined as HCO3−-dependent and DIDSsensitive Cl uptake, is diminished in villus cell BBMV from the chronically inflamed rabbit intestine. In order to determine whether arachidonic acid metabolites are responsible for the decrease in Cl −/HCO3− activity during chronic inflammation, rabbits were treated in vivo with ATMK. ATMK treatment reversed Cl −/HCO3− exchange inhibition in villus cell BBMV (756 ± 55 pmol/mg protein/min in control, 159 ± 22 in inflamed, and 678 ± 47 in inflamed treated with ATMK, p b 0.01, n = 5; Fig. 1A). Interestingly, ATMK treatment stimulated Cl−/HCO3− exchange in villus cell BBMV from normal rabbit intestine (1438 ± 114 pmol/mg protein/min in ATMK treated, p b 0.05, n = 5; and Fig. 1B). These data indicate that blocking the release of AA reverses the inhibition of Cl −/HCO3− activity during chronic inflammation. 3.2. Kinetic studies for Cl −/HCO3− exchange during inhibition of AA release Previous studies from our lab have shown that HCO3−-dependent and DIDS-sensitive 36Cl uptake in BBMV was linear for at least 10 seconds, hence uptake studies for various concentrations were carried out at 3 seconds. Fig. 2A shows 36Cl uptake in BBMV as a function of varying concentrations of extra-vesicular Cl. As the concentration of extra-vesicular Cl was increased, 36Cl uptake was stimulated and subsequently became saturated in all conditions. Kinetic parameters demonstrated that in the chronically inflamed intestine inhibition of Cl −/HCO3− exchange activity occurs as a result of an increase in the Km, and in vivo ATMK treatment revered this change (Km for Cl − uptake in villus cell BBMV was 20.6 ± 1.0 mM in normal, 51.3 ± 1.4 in inflamed, 21.7 ± 1.2* inflamed + ATMK, n = 3, p b 0.05). The maximal rate of uptake (Vmax) was unaffected in all conditions (Vmax was 43.5 ± 1.5 nmol/mg protein min in normal, 45.4 ± 1.3 inflamed, 46.2 ± 1.4 inflamed + ATMK; n = 3). In the normal intestine, kinetic parameters demonstrated that ATMK treatment increased the maximal rate of uptake of Cl (Vmax for Cl− - uptake in BBMV was 98.6 ± 2.7 nmol/mg protein min in normal + ATMK, n = 3, p = 0.01 (Fig. 2B), without a change in the affinity of the transporter for Cl. These data indicate that the mechanism of reversal of inhibition of Cl −/HCO3− exchange by ATMK during chronic intestinal inflammation was secondary to a restoration in the affinity for Cl − rather than an alteration in the number of BBM of Cl −/HCO3− exchangers.
Fig. 1. Effect of ATMK treatment on Cl−/HCO3− exchange in villus cell BBMV from the normal and chronically inflamed rabbit ileum. Cl−/HCO3− exchange was significantly inhibited during chronic intestinal inflammation. A: Inhibited Cl−/HCO3− exchange in chronically inflamed rabbit BBMV was reversed back to normal levels by ATMK treatment, n = 5, *p b 0.05. B: ATMK treatment significantly stimulated Cl−/HCO3− exchange in normal rabbit BBMV, n = 5, *p b 0.05.
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Fig. 3. Effect of piroxicam treatment on Cl−/HCO3− exchange in villus cell BBMV from the normal and chronically inflamed rabbit ileum. Cl−/HCO3− exchange that is inhibited during chronic intestinal inflammation was reversed back to normal levels by piroxicam treatment (1147±73 pmol/mg protein min in normal; 324±16* in inflamed and 1100±34* in inflamed+piroxicam, n=3, *pb 0.001). Piroxicam treatment did not have any effect on the normal rabbit BBMV (1037±33 pmol/mg protein min, n=3).
3.5. Kinetic studies for Cl −/HCO3− exchange during inhibition of COX pathway
Fig. 2. Kinetics of Cl−/HCO3− exchange in villus cell BBMV from normal, inflamed and ATMK treated rabbit ileum. HCO3−-dependent and DIDS-sensitive 36Cl uptake is shown as a function of varying concentrations of extra vesicular Cl− at 3 secs. As the concentration of extra-vesicular Cl− was increased, 36Cl uptake was stimulated and subsequently became saturated in all conditions. Analysis of the data yielded kinetic parameters. A: In chronically inflamed BBMV the Cl−/HCO3− exchange was inhibited by altering the affinity of the transporter for Cl− and ATMK treatment significantly reversed the inhibited Cl−/HCO3− exchange in chronically inflamed villus cell BBMV by restoring the affinity of the transporter (Km) without an alteration in the maximal rate of uptake (Vmax) of Cl, n = 3. B: In normal rabbit villus cell BBMV ATMK treatment stimulated Cl−/HCO3− exchange by altering the number of transporters (Vmax), n = 3.
To determine the mechanism of piroxicam-mediated reversal of inhibition of Cl−/HCO3− exchange in the chronically inflamed intestine, kinetics studies were performed. These data demonstrated that, piroxicam did not alter the Vmax for Cl− uptake (Vmax for Cl− uptake in BBMV was 47.5 ± 2.2 nmol/mg protein. Min in normal, 42.8 ±2.8 in normal + piroxicam, 42.4 ±1.0 in inflamed and 45.1 ± 1.8 in inflamed + piroxicam; n = 3). However, the affinity for Cl− uptake, which was reduced in the chronically inflamed rabbit intestine, was restored by piroxicam treatment (Km for Cl− uptake in BBMV was 19.0 ± 1.0 mM in normal, 43.3 ±1.8*inflamed, 20.5 ± 2.5 normal+ piroxicam, and 23.1 ± 1.2*inflamed+ piroxicam; n = 3, *p b 0.05). Thus, these data indicated that the mechanism of reversal of inhibition of Cl −/HCO3− exchange by piroxicam during chronic intestinal inflammation was secondary to a restoration of the affinity for Cl− rather than an increase in transporter numbers. 3.6. Identification of the Cl −/HCO3− exchanger in rabbit ileum BBMV–DRA Different members of SLC26A protein family may mediate Cl−/HCO3− exchange depending upon their tissue distribution. In order to identify
3.3. Effect of inhibition of COX pathway on Cl −/HCO3− exchange Piroxicam, was utilized to inhibit cyclooxygenase (COX1 and COX2) pathway in vivo in the chronically inflamed intestine. Piroxicam reversed the inhibition of Cl−/HCO3− exchange in villus cell BBMV (1147 ± 73 pmol/mg protein min in normal; 324 ±16 in inflamed; 1037± 33 in normal + piroxicam and 1100 ±34* in inflamed+ piroxicam, n = 3, *p b 0.01; Fig. 3). These data indicate that the COX pathway likely mediates the inhibition of Cl −/HCO3− exchange during chronic intestinal inflammation.
3.4. Effect of inhibition of LOX pathway on Cl −/HCO3− exchange Since AA can be metabolized by the LOX pathway as well, MK-886 was utilized to inhibit this pathway. In vivo treatment of rabbits with chronic intestinal inflammation with MK-886 did not reverse the diminished Cl −/HCO3−exchange in villus cell BBMV (Fig. 4). Thus, these data indicate that inhibition of Cl −/HCO3− exchange during chronic intestinal inflammation is not mediated by the LOX pathway.
Fig. 4. Effect of MK-886 treatment on Cl−/HCO3− exchange in villus cell BBMV from chronically inflamed rabbit ileum. Cl−/HCO3− exchange that was inhibited during chronic intestinal inflammation was not reversed by MK-886 treatment (324 ± 16 in inflamed and 411 ± 46 in inflamed + MK-886, n = 3, p = not significant).
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with kinetic studies that Cl−/HCO3− exchange inhibition during chronic inflammation is not secondary to a decrease in protein expression. 3.8. RTQ-PCR studies for DRA Next to determine the molecular mechanism of COX mediated inhibition of DRA, we determined its mRNA abundance in villus cells by RTQ-PCR. DRA specific mRNA abundance was not significantly altered in any condition (Fig. 7). 3.9. Western blot studies for DRA
Fig. 5. Relative abundance of anion exchangers in normal rabbit ileum by RTQ-PCR. mRNA abundance is expressed relative to PAT1. Expression of mRNA levels of DRA were 10 fold higher compared to PAT1 and Pendrin, n = 3, *p b 0.05.
the major anion exchanger responsible for Cl−/HCO3− in the rabbit ileal villus cells, RTQ-PCR for DRA (SLC26A3), PAT1 (SLC26A) and Pendrin (SLC26A4) were performed. The results show that expression of DRA was 10 fold higher than PAT1 or Pendrin in rabbit villus cells (Fig. 5).
Since mRNA level may not necessarily correlate with immunoreactive protein in the BBM, we quantitated DRA specific immunoreactive protein. There was no significant difference in the immunoreactive levels of DRA in any of the conditions tested (Fig. 8A). Densitometric quantitation confirmed these observations (Fig. 8B). These results, in conjunction with kinetic parameters and RTQ-PCR data demonstrate that the mechanism of inhibition of DRA by the COX pathway during chronic intestinal inflammation was secondary to a decrease in the affinity of the transporter for Cl− without a change in the transporter numbers.
3.7. Immunocytochemistry studies of DRA
4. Discussion
In order to localize DRA in the rabbit ileum and to investigate if it is affected by chronic intestinal inflammation, immunocytochemistry was performed. Fig. 6 demonstrates that DRA is present in the BBM of rabbit small intestinal villus cells. Further, DRA protein expression was unaltered during chronic intestinal inflammation. This is consistent
This study demonstrated that in a mammalian animal model of IBD, inhibition of coupled NaCl absorption occurs secondary to the inhibition of Cl−/HCO3− but not, Na/H exchange in the BBM of absorptive villus cells. This rabbit ileal villus cell BBM Cl−/HCO3− exchanger was identified to be DRA. The inhibition of DRA in the chronically inflamed
Fig. 6. Immunofluorescence localization of DRA in rabbit ileum. A & B: Immunofluorescent detection of DRA in rabbit ileum was performed using anti-DRA antibody following a pre-incubation of the antibody with antigenic peptide. C & D: Immunofluorescence of DRA expression compared between normal and chronically inflamed rabbit ileum.
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Fig. 7. RTQ-PCR analysis for DRA in rabbit ileum. DRA specific mRNA abundance was not changed between normal, inflamed and piroxicam treated rabbit ileum, n=3, p= insignificant.
intestine was mediated by arachidonic acid metabolites, specifically by the metabolites of the cyclooxygenase, but not the lipoxygenase pathway. The most common and disabling morbidity of IBD is diarrhea which is caused by malabsorption and secretion of electrolytes and water These electrolyte transport abnormalities are likely mediated by immune-inflammatory mediators known to be elevated in the mucosa of the chronically inflamed intestine [26–28]. Given that treatments for IBD are few and fraught with numerous side effects, it is necessary to more clearly define which immune inflammatory mediators affect electrolyte absorption so a more precise treatment can be developed with fewer side effects. Therefore, this study was designed to begin to determine which immune inflammatory mediators regulate coupled NaCl absorption, specifically Cl −/HCO3−exchange, in the chronically inflamed intestine. In previous studies we had demonstrated that Cl −/HCO3−exchange was inhibited in the BBM of villus cells from the chronically inflamed rabbit intestine. The mechanism of inhibition was secondary to a reduction in the affinity of the transporter for Cl without a change in the number of transporters in the BBM. However, the molecular identity of this Cl −/HCO3−exchanger in the rabbit intestine was unknown. This study demonstrated that DRA mediates Cl −/HCO3−
Fig. 8. Western blot analysis for DRA in rabbit ileum. A: DRA specific protein expression was not altered between normal, inflamed and piroxicam treated rabbit ileum. B: Densitometric analysis shows the relative expression of DRA protein levels in all the different experimental condition with reference to its level in villus cell BBM from normal intestine, n = 3, p = insignificant.
exchange in rabbit ileal villus cells. This observation is different from that in mouse where PAT1 is the major Cl −/HCO3− exchanger expressed in BBM of the small intestine [3,37], while DRA is abundantly expressed in apical membranes of colonocytes [3,38]. Despite the low expression of DRA in the small intestine, functional studies have shown that DRA is the major Cl −/HCO3− exchanger coupled with NHE3 for electro neutral NaCl absorption in murine small intestine [39]. In contrast to mouse, RTQ-PCR studies showed that expression of mRNA of DRA was 10 fold higher compared to PAT1 and Pendrin in the rabbit small intestine (Fig. 5). Consistent with RTQ-PCR data, immunocytochemistry and Western blot studies showed that DRA was indeed the predominant anion exchanger in the BBM of villus cells in the rabbit ileum (Fig. 6). Thus, these data clearly indicate that DRA mediates Cl −/HCO3− exchange in villus cells in the rabbit small intestine. Other studies have demonstrated the role of DRA in the causation of diarrhea. Mutation in DRA causes congenital chloride diarrhea both in patients and experimental animal models [40,41] and loss of transporter function of DRA plays a crucial role in the pathogenesis of diarrhea in intestinal inflammation [42]. The inhibition of DRA in the chronically inflamed rabbit intestine is likely mediated by immune-inflammatory mediators known to be elevated in the mucosa. We previously reported that a variety of immune-inflammatory mediators including prostaglandins and leukotrienes are markedly increased in the mucosa of the chronically inflamed rabbit intestine [35]. Further, previous studies have demonstrated that these agents likely mediate multiple unique alterations in electrolyte, Na-nutrient and Na-bile acid transporters during chronic intestinal inflammation [1,15,16,29,30]. For example Na-glucose co-transporter was inhibited by a decrease in the number of cotransporter [16] while Na-alanine co-transporter was inhibited by a reduction in the affinity for amino acid [15]. Our in vitro studies have demonstrated that prostaglandin E2 (PGE2), a cyclooxygenase pathway metabolite, inhibits Na-glucose co-transport while leukotriene D4 (LTD4), a 5-lipoxygenase pathway metabolite, was responsible for inhibition Na-alanine co-transport in IEC-18 cells [36]. That DRA inhibition in the chronically inflamed rabbit intestine is likely mediated by immune inflammatory mediators as well is supported by our previous observation that broad spectrum immune modulation alleviated this inhibition. Glucocorticoid ethylprednisolone is a common treatment for IBD and as a nonspecific immune modulator known to block multiple pathways of the immune system including the arachidonic acid metabolite cascade. Further, as a broad spectrum immune modulator, glucocorticoid treatment of IBD has been shown to alleviate the inhibition of Cl−/HCO3−exchange as well as other transporters [31–34]. Nevertheless, more specific immune mechanism of regulation of Cl −/HCO3−exchange in the chronically inflamed intestine was not well understood. In chronically inflamed rabbit intestine, specific inhibition of the formation arachidonic acid reversed the inhibition of Cl −/HCO3− exchange activity, Kinetic studies showed that the mechanism of reversal of Cl −/HCO3− exchange by ATMK treatment was secondary to restoration in the affinity of the transporter for Cl − without affecting transporter number (Vmax) in chronically inflamed rabbit ileum. These results indicate that AAM mediate the inhibition of Cl −/HCO3− exchange during chronic enteritis Interestingly, ATMK treatment stimulated Cl −/HCO3− exchange in the normal intestine and the mechanism of stimulation was secondary to an increase in exchanger numbers in the BBM. This mechanism of regulation of Cl −/HCO3− exchange appears to be lost in the chronically inflamed intestine. It may be that AAM produced in the normal intestine have a baseline inhibitory effect on this exchanger which is alleviated by ATMK. Future studies will need to further elucidate this finding. Since AAM produced by both the COX and/or LOX pathways are known to be elevated in the chronically inflamed intestine, each pathway was selectively inhibited to determine which specifically mediated
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the inhibition of Cl−/HCO3− exchange. It was demonstrated that the cyclooxygenase inhibitor piroxicam reversed the Cl−/HCO3− exchange while 5-lipoxygenase inhibitor MK-886 did not have an effect on Cl−/HCO3− exchange during chronic intestinal inflammation. This indicated that during chronic intestinal inflammation, Cl−/HCO3− exchange is regulated by immune inflammatory mediators produced by the cyclooxygenase but not, the 5-lipoxygenase pathway. Other investigators have also shown the importance of DRA regulation by immune inflammatory mediators. For example, Saksena et al., have demonstrated that IFN-γ significantly inhibited DRA gene expression and promoter activity in Caco-2 cells. The inhibition of DRA was mediated by signal transducer and activator of transcription factor 1 (STAT1) [43,44]. Therefore, delineating the pathway responsible for alteration of coupled NaCl absorption by altering DRA may yield precise targeted therapies for patients with IBD. The COX pathway mediated alteration of Cl−/HCO3− exchange at the molecular level appears to be post translational. RTQ-PCR and Western blot studies showed that there was no change in DRA specific mRNA abundance and protein expression respectively in the chronically inflamed intestine(Figs. 7 and 8). Immunocytochemistry studies also showed there was no difference in expression of DRA between normal and inflamed rabbit villus cell BBM (Fig. 6C and D). Further, both ATMK and piroxicam treatment reversed the inhibited Cl−/HCO3− exchange by restoring the affinity of the transporter. Thus, together our functional and molecular data demonstrate that the mechanism of inhibition of DRA is post translational during chronic enteritis. The alteration in the affinity of the DRA for Cl− during chronic ileitis may be due to changes in glycosylation and/or phosphorylation of the transporter since they are the most common post translational modifications observed in membrane proteins. In conclusion, this study demonstrates that DRA is the primary villus cell BBM Cl−/HCO3− exchanger in the rabbit intestine. It is inhibited secondary to a reduction in the affinity of the transporter for Cl in the chronically inflamed intestine. This inhibition of DRA is mediated by arachidonic acid metabolites during chronic enteritis. Finally, cyclooxygenase but not the 5-lipoxygenase pathway metabolites mediate the inhibition of DRA during chronic intestinal inflammation. References [1] U. Sundaram, A.B. West, Effect of chronic inflammation on electrolyte transport in rabbit ileal villus and crypt cells, Am. J. Physiol. 272 (1997) G732–G741. [2] U. Sundaram, R.G. Knickelbein, J.W. Dobbins, Mechanism of intestinal secretion. Effect of serotonin on rabbit ileal crypt and villus cells, J. Clin. Invest. 87 (1991) 743–746. [3] Z. Wang, S. Petrovic, E. Mann, M. Soleimani, Identification of an apical Cl(−)/HCO3(−) exchanger in the small intestine, Am. J. Physiol. Gastrointest. Liver Physiol. 282 (2002) G573–G579. [4] D.B. Mount, M.F. Romero, The SLC26 gene family of multifunctional anion exchangers, Pflugers Arch. 447 (2004) 710–721. [5] J. Kere, H. Lohi, P. Hoglund, Genetic disorders of membrane transport III. Congenital chloride diarrhea, Am. J. Physiol. 276 (1999) G7–G13. [6] T.R. Traynor, D.R. Brown, S.M. O'Grady, Effects of inflammatory mediators on electrolyte transport across the porcine distal colon epithelium, J. Pharmacol. Exp. Ther. 264 (1993) 61–66. [7] M.W. Musch, J.F. Kachur, R.J. Miller, M. Field, J.S. Stoff, Bradykinin-stimulated electrolyte secretion in rabbit and guinea pig intestine. Involvement of arachidonic acid metabolites, J. Clin. Invest. 71 (1983) 1073–1083. [8] S.A. Hojvat, M.W. Musch, R.J. Miller, Stimulation of prostaglandin production in rabbit ileal mucosa by bradykinin, J. Pharmacol. Exp. Ther. 226 (1983) 749–755. [9] C.S. Hyun, H.J. Binder, Mechanism of leukotriene D4 stimulation of Cl- secretion in rat distal colon in vitro, Am. J. Physiol. 265 (1993) G467–G473. [10] H. Zhang, N. Ameen, J.E. Melvin, S. Vidyasagar, Acute inflammation alters bicarbonate transport in mouse ileum, J. Physiol. 581 (2007) 1221–1233. [11] M. Romero, R. Artigiani, H. Costa, C.T. Oshima, S. Miszputen, M. Franco, Evaluation of the immunoexpression of COX-1, COX-2 and p53 in Crohn's disease, Arq. Gastroenterol. 45 (2008) 295–300. [12] E. Carty, C. Nickols, R.M. Feakins, D.S. Rampton, Thromboxane synthase immunohistochemistry in inflammatory bowel disease, J. Clin. Pathol. 55 (2002) 367–370. [13] T. Nishida, H. Miwa, A. Shigematsu, M. Yamamoto, M. Iida, M. Fujishima, Increased arachidonic acid composition of phospholipids in colonic mucosa from patients with active ulcerative colitis, Gut 28 (1987) 1002–1007. [14] U. Seidler, H. Lenzen, A. Cinar, T. Tessema, A. Bleich, B. Riederer, Molecular mechanisms of disturbed electrolyte transport in intestinal inflammation, Ann. N. Y. Acad. Sci. 1072 (2006) 262–275.
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