This paper has online supplemental material.
Acute inflammation alters bicarbonate transport in mouse ileum
Article first published online: 6 JUN 2007
The Journal of Physiology
Volume 581, Issue 3, pages 1221–1233, June 2007
How to Cite
Zhang, H., Ameen, N., Melvin, J. E. and Vidyasagar, S. (2007), Acute inflammation alters bicarbonate transport in mouse ileum. The Journal of Physiology, 581: 1221–1233. doi: 10.1113/jphysiol.2007.129262
- Issue published online: 6 JUN 2007
- Article first published online: 6 JUN 2007
- (Received 26 January 2007; accepted after revision 23 March 2007; first published online 29 March 2007)
T-cell mediated acute inflammation of the ileum may occur during Crohn's disease exacerbations. During ileal inflammation, absorption of nutrients and electrolytes by villus cells is decreased with a concomitant increase in crypt and/or villus fluid secretion. These alterations lead to fluid accumulation and the subsequent diarrhoea. Net intestinal fluid secretion consists of HCO3−-rich plasma-like fluid. However, the regulation and mechanisms of HCO3− secretion in normal and acutely inflamed ileum are not clearly understood. To study this phenomenon, anti-CD3 monoclonal antibody (mAb)- induced in vivo ileal inflammatory mouse models was used for in vitro functional studies with Ussing chamber and pH stat techniques. Three hours after anti-CD3 mAb injection, ileal mucosa stripped of muscular and serosal layers showed a significant increase in short circuit current (Isc) (0.58 ± 0.07 μEq h−1 cm2versus 1.63 ± 0.14 μEq h−1 cm2). The cAMP-stimulated Isc component was sensitive to glibenclamide but not to DIDS, suggesting that a cystic fibrosis transmembrane conductance regulator (Cftr)-mediated anion conductance was responsible. Basal Cl−-dependent HCO3− secretion, measured using a pH stat technique, was decreased significantly in anti-CD3-injected mice, with a simultaneous increase in Cl−-independent HCO3− secretion that was also inhibited by glibenclamide. Experiments using Cftr−/− mice showed neither an increase in Isc nor an increase in HCO3− secretion, confirming the role for Cftr protein in stimulating anion secretion following anti-CD3 treatment. Western blot analysis indicated that Cftr protein levels were unaltered by anti-CD3 treatment, at least acutely. Finally, an immunoassay for cAMP showed significant increases in intracellular cAMP in villus cells, but not in crypt cells. These studies therefore suggest a shift from a predominantly electroneutral Cl−HCO3− exchange in normal mice, to a predominantly electrogenic anion secretion including HCO3− that occurs via functional Cftr during anti-CD3-mediated acute inflammation.
Crohn's disease (CD) and ulcerative colitis, the two most common forms of inflammatory bowel disease (IBD), are characterized by chronic, recurrent inflammation of the intestinal tract. Ulcerative colitis occurs mainly in the colon, while Crohn's disease can affect any part of the digestive tract from the mouth to the rectum, but primarily occurs in the lower part of the small intestine (ileitis or enteritis). Although the exact aetiology of IBD is unknown, it is widely accepted that the disease is characterized by an abnormal cell-mediated immune reaction – primarily by CD4+ T-cells – to the antigens and adjuvant of the enteric bacteria in genetically susceptible hosts (Jump & Levine, 2004). CD tends to be a chronic, recurrent condition with periods of remission and exacerbation. Complex and active interactions exist between the bacterial flora, epithelium, and immune cells in the intestine, and perturbation of these interactions can result in intestinal inflammation. The pathological features of CD include villus atrophy and crypt hyperplasia leading to malabsorption by villus epithelia and most likely increased fluid secretion from the crypt epithelium. These alterations lead to fluid accumulation and the subsequent diarrhoea, a common clinical feature during IBD (Ciancio & Chang, 1992; Baert et al. 1999; Bell & Kamm, 2000). Despite the abundance of data regarding intestinal inflammation-associated diarrhoea, specific ileal transport alterations have not been clearly identified (Ciancio & Chang, 1992; Radojevic et al. 1999; Musch et al. 2002).
The main physiological function of the small intestine is absorption of nutrients, electrolytes and water. However, low rates of fluid secretion in the small intestine are also necessary to maintain luminal ionic composition, pH and motility. Consequently, there is a fine balance between absorption and secretion, such that electroneutral Na+–H+ exchange coupled with Cl−–HCO3− exchange-stimulated fluid absorption predominates over electrogenic anion secretion and thus fluid secretion. Any decrease in electroneutral Na+ and Cl− absorption and/or increased electrogenic anion secretion may result in fluid accumulation and diarrhoea. Amongst the anions, Cl− secretion is considered the major driving force for fluid secretion in the small intestine. It is widely believed that the major route for stimulated Cl− secretion in the small intestine occurs via the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP–protein kinase A (PKA)-dependent Cl− channel (Berschneider et al. 1988; Anderson & Welsh, 1991; Barrett & Keely, 2000).
In addition to Cl−, HCO3− also plays a significant role in net fluid secretion. Under normal physiological states, the small intestine actively secretes net HCO3− (Furukawa et al. 2005). The exchange of Cl− for HCO3− has been identified in all three regions of the small intestine (i.e. duodenum, jejunum and ileum). In most regions of the mouse small intestine, electroneutral HCO3− secretion is mediated by the SLC4 family of Cl− -HCO3− exchangers (anion exchanger (AE)) coupled to Na+–H+ exchange. All four AE isoforms (AE1, AE2 and AE3, AE4) of the SLC4 family have been reported in the small intestine (Alper et al. 1999; Alrefai et al. 2001; Alper et al. 2002; Charney et al. 2004).
The SLC26 family of anion exchangers, like the SLC4 family of Cl−–HCO3− exchangers, is known to transport a variety of anions and in some cases, to participate in electrogenic Cl−–HCO3− exchange (Mount & Romero, 2004). In particular, mutations in the SLC26A3 gene, also known as Down Regulated in Adenoma (DRA), lead to congenital chloride diarrhoea (CLD) (Schweinfest et al. 1993; Hoglund et al. 1996). SLC26A6, the putative anion transporter (PAT1) has also been identified in the gastrointestinal tract (Lohi et al. 2000; Waldegger et al. 2001). In the intestine, DRA is mainly expressed in the colon and duodenum, with lower levels in the ileum (Silberg et al. 1995; Hoglund et al. 1996; Melvin et al. 1999; Jacob et al. 2002). In contrast, PAT1 mRNA levels are abundant in all regions of the small intestine but low in the large intestine (Boll et al. 2002; Wang et al. 2002). Recent studies by Wang et al. 2005 demonstrated that PAT1 plays a major role in Cl−–HCO3− exchange in the duodenum as the basal HCO3− transport in Slc26A6−/− mice was reduced by ∼30%. Although, the mechanisms of HCO3− transport have been studied in detail in the duodenum and the colon, the exact AE and Slc26A isoforms mediating HCO3− transport in the ileum and how they are altered during inflammatory states remains to be examined.
CFTR may also be permeable to HCO3− (Gray et al. 1989; Poulsen et al. 1994; Seidler et al. 1997; Illek et al. 1998; O'Reilly et al. 2000). However this remains controversial as HCO3− secretion was not enhanced by increases in cAMP in recombinant wild-type CFTR-expressing cells, suggesting that CFTR does not conduct HCO3− (Shumaker et al. 1999; Soleimani & Ulrich, 2000). However, functional data regarding the types of anion channels and transporters modulating ileal fluid secretion using primary epithelial tissue and cells are limited.
In order to investigate ileal HCO3− secretion and how ileal HCO3− secretion might be altered during acute inflammation, we used the well-established immune-mediated acute inflammatory mouse model in which mice were injected with anti-CD3 monoclonal antibody (mAb). Intraperitoneal injection of mice with anti-CD3 mAb induced acute inflammation and increased proinflammatory cytokines transcripts and acute diarrhoea (Radojevic et al. 1999; Musch et al. 2002; Clayburgh et al. 2005). Early human studies using anti-CD3 antibodies to prevent renal transplant rejection also resulted in diarrhoea (Chatenoud & Bach, 1988). In anti-CD3-injected mice, maximal fluid accumulation, and hence diarrhoea, occurred within 2–3 h of anti-CD3 mAb injection (Musch et al. 2002; Clayburgh et al. 2005). Recently, Clayburgh et al. (2005) reported that anti-CD3-injected mice exhibited signs of intestinal inflammation as evidenced by vasodilatation, oedema, erythema and increased intraepithelial lymphocytes Furthermore, in vivo treatment with anti-CD3 mAb increased the circulating levels of tumor necrosis factor (TNF)-α and interferon (IFN)-γ (Ferran et al. 1990; Radojevic et al. 1999; Musch et al. 2002; Clayburgh et al. 2005). During intestinal inflammation, a host of similar proinflammatory cytokines are released by T-cells (Chatenoud & Bach, 1988; Ferran et al. 1991). In addition to these proinflammatory cytokines, anti-CD3 increases interleukin (IL)-2, IL-3, IL-4 and IL-6 (Hirsch et al. 1989; Ferran et al. 1990, 1994; Bemelmans et al. 1994), similar to the increases found in human IBD. Thus, in the present studies we used in vivo anti-CD3 mAb-treated mice to investigate whether the diarrhoea during acute inflammation is due to alterations in ileal electroneutral anion exchange activity and/or simultaneous activation of anion channels. Specifically, the cellular mechanisms of HCO3− secretion was examined in stripped ileal mucosa under voltage-clamp conditions using a pH stat technique to study electroneutral Cl−–HCO3− exchange and electrogenic HCO3− secretion. In addition, Ussing chamber experiments were performed to study changes in short circuit current (Isc) in anti-CD3-injected mice.
Non-fasting male C57BL/6 mice, 6–8 weeks old-were used in all experiments except experiments in which Cftr−/− (Snouwaert et al. 1992) and Cftr+/+ mice (age- and sex-matched littermates as controls) were used. Mice with a truncation mutation beginning at position S489 were used to compare the effect of anti-CD3 mAb on Isc and HCO3− movement. Following exsanguinations, ileal mucosa was obtained from the segment close to the caecum. HCO3− transport studies were performed in mucosa that was stripped through the submucosal layers to remove the serosal and muscular layers under a dissection microscope. Mucosa was mounted between the two halves of a Ussing-type Lucite chamber, as previously described (Vidyasagar & Ramakrishna, 2002; Vidyasagar et al. 2004). In experiments in which the effects of anti-CD3 were studied, 0.2 mg/0.2 ml of anti-CD3 mAb was injected i.p. and animals were killed 3 h later to remove the ileum. Musch et al. (2002) demonstrated that maximal intraluminal fluid accumulation occurs at 3 h of in vivo anti-CD3 mAb treatment. Control animals were sham-injected with 0.2 ml PBS. Mice were killed by sodium pentobarbital injection (50 mg kg−1, i.p.) followed by cervical dislocation 3 h after i.p. injection. Tissues were harvested only after apnoea had occurred. All experiments were approved by the University of Rochester Institutional Animal Care and Use Committee.
The ileal sheets were stripped through the submucosal layer such that almost all of the muscular layers were removed. Stripped ileal sheets showed significantly lower Isc compared to unstripped ileal sheets (Fig. 1). Thus, for all Ussing chamber experiments, stripped ileal sheets were mounted in Lucite chambers (0.3 cm2 exposed surface area). Intestinal preparations were bathed bilaterally in regular Ringer's solution (Table 1) containing 10 mm glutamine and gassed with a mixture of 95% O2 and 5% CO2. Glutamine was substituted for glucose to avoid inward current resulting from Na+-coupled glucose cotransport.
pH stat recordings
HCO3− secretion was quantified using Bi-burette TIM 856 (Radiometer Analytical S.A., Villeurbanne, France) that titrates both above and below a stat pH 7.4 with a hysteresis of 0.05 and thus titrating between 7.35 and 7.45 (physiological limits of pH range for body fluids), as previously described (Vidyasagar et al. 2004, 2005). Briefly, luminal solution pH was continuously maintained at a constant (or stat) pH by the addition of 0.025 m H2SO4. Pumps were programmed to operate in real time to the changes in luminal pH delivering a minimum of 0.01 μl at a given time. Standard pH calibration to stat pH was established by adding a known quantity of H2SO4 to a low buffering solution containing increasing concentration of HCO3− to get a linear curve. The amount of HCO3− measured in the luminal solution was always within the linear range of this curve. The acid used for the titration was diluted in the same ionic solution as used in that particular experiment to give a final concentration of 0.025 m. Ileal tissues were always exposed to a buffered solution on the bath side, while the luminal side was exposed to a low buffered solution (0.1 mm Hepes buffer, pH 7.4). HCO3− secretion is equivalent to the amount of acid required to maintain pH at 7.4. All experiments were performed under voltage-clamp conditions and HCO3−-free solutions were gassed with 100% O2, while HCO3−-containing solutions were gassed with 95% O2 and 5% CO2. HCO3− secretion was expressed as μEq h−1 cm2.
Initial studies demonstrated that immediately after the tissue was mounted, HCO3− secretion was present in the absence of bath HCO3−, but rapidly fell towards zero within 20–30 min. If bath HCO3− was not added, HCO3− secretion remained close to zero. Addition of HCO3− to the bath solution resulted in a rapid increase in HCO3− secretion that remained constant for at least 60 min. These experimental methods were similar to those of earlier published work (Vidyasagar et al. 2004). All experiments were performed during this 1 h steady-state period.
In experiments where inhibitors were added to the mucosal solution, the specific drug was added during the initial steady-state period and pH adjusted and allowed to equilibrate for 30 min until a steady rate of HCO3− secretion was again observed. In experiments where the inhibitor was added to the serosal side, the tissue was also equilibrated for 30 min to achieve a steady state of HCO3− secretion. The composition of the several solutions used in these experiments is presented in Table 1. In brief, in Cl−-free experiments, isethionate was used as a substitute for Cl−; in Na+-free experiments NMDG was used as a Na+ substitute; 10 mm glutamine was added to all solutions. Only one tissue from each animal was used for a specific experiment, and only one experimental condition was used with each tissue. All experiments were repeated at least four times.
Intestinal lysates were prepared from mucosal scrapings from anti-CD3 mAb-treated and control mouse ileum and analysed for Cftr protein by Western blots. Mucosal scrapings were lysed in TGH buffer containing 25 mm Hepes, 10% glycerol, 1% Triton X-100, containing a protease inhibitor mixture (10 mm iodoactamide, 1 mm phenylmethylsulphonyl fluoride, and 2 μg ml−1 leupeptin) pH 7.4, and protein concentration was determined in samples using the Bradford assay. Equivalent loads of protein from anti-CD3-treated and control samples were analysed by SDS PAGE. Proteins were transferred onto PVDF membranes and Cftr detected using the affinity-purified polyclonal anti-Cftr antibody, AME 4991 as described before (Golin-Bisello et al. 2005).
Colorimetric cAMP immunoassay
Ileal lysates from anti-CD3 mAb-treated and sham-injected mice were used for cAMP assay using cAMP direct immunoassay kit (Calbiochem, USA). Cells were treated with 0.1 m HCl to stop endogenous phosphodiesterase activity. Competitive immunoassay for the quantitative assay of cAMP used a polyclonal antibody to cAMP that binds to cAMP in samples in a competitive manner. After a simultaneous incubation at room temperature, the excess reagents were washed away and substrate added. After a short incubation time, the reaction is stopped and the yellow colour generated is read at 405 nm. The intensity of the colour is inversely proportional to the concentration of cAMP in standards and samples. Forskolin-treated cells were incubated for 45 min after its addition and before the cAMP immunoassay. cAMP levels were standardized to protein levels from respective fractions and expressed in pmol (mg protein)−1. All the assays were done in triplicate and repeated to get n= 4 different mice.
Results are presented as mean ±s.e.m. Statistical analyses were performed using paired and unpaired t test and Bonferroni's one-way ANOVA post hoc test. P < 0.05 was considered significant.
Chemicals and solutions
Forskolin; 4,4′-diisothiocyanato-stilbene-2,2′-disulphonic acid (DIDS); dimethyl sulfoxide (DMSO) grade I; glibenclamide; N-[2-hydroxyethyl]piperazine-N-[2-ethanesulphonic acid] (Hepes); isethionic acid; N-methyl-d-glucamine (NMDG); 5-nitro-3-(3-phenylpropyl-amino)benzoic acid (NPPB); potassium gluconate and sodium gluconate were obtained from Sigma. Anti-CD3 mAb was a generous gift from Terrance Barrett, North-western University Feinberg School of Medicine, Chicago, USA.
Anti-CD3-induced change in Isc
Ussing chamber experiments were performed 3 h after anti-CD3 mAb injection to determine the effects of T-cell activation on transepithelial Isc and conductance (G). Anti-CD3 injection did not result in a significant increase in conductance in totally stripped ileal tissue (Table 2). In contrast, basal Isc increased by ∼260% as compared to sham-injected mice (Table 2). To assess whether the observed increase in Isc in anti-CD3 mAb-injected mice was associated with an apical membrane anion channel, 100 μm NPPB (non-specific Cl− channel blocker; Tilmann et al. 1991; Fuller & Benos, 1992) was added to the lumen solution. NPPB decreased the anti-CD3-stimulated Isc to levels similar to that of sham-injected (control) basal Isc. In control mice, NPPB addition resulted in a marginal but significant decrease in Isc (Table 2). This suggests that anti-CD3 treatment enhances electrogenic anion secretion via an anion channel. As Cftr is postulated to contribute the bulk of anion conductance in the small intestine, an inhibitor study was performed with 300 μm glibenclamide, a concentration at which glibenclamide is a relatively specific inhibitor of Cftr. Exposure to glibenclamide decreased significantly the basal Isc in anti-CD3 injected mice, suggesting increased electrogenic anion secretion via Cftr during acute inflammation (Table 2).
|G (mS cm−2)||Basal Isc||cAMP-stimulated Isc||G (mS cm−2)||Basal Isc||cAMP-stimulated Isc|
|Basal||11.5 ± 1.1||0.6 ± 2.1||3.0 ± 0.16||13.3 ± 1.4||2.1 ± 0.14||2.5 ± 0.18|
|NPPB||12.6 ± 1.1ns||0.3 ± 0.9*||0.3 ± 0.04*||13.5 ± 1.1ns||0.4 ± 0.05*||0.4 ± 0.05*|
|Glibenclamide||12.1 ± 1.2ns||0.4 ± 1.9**||0.3 ± 0.06**||14.1 ± 1.1ns||0.3 ± 0.05**||0.24 ± 0.06**|
cAMP-stimulated changes on anti-CD3-induced Isc
Cftr conductance is activated by elevations in intracellular cAMP. Whether cAMP also modifies anion secretion was examined in both normal and anti-CD3 mAb-injected mice. Exposure of ileal tissue to 10 μm forskolin significantly increased Isc in both sham-injected and anti-CD3 mAb-injected mice. Maximal increase in Isc was seen in most tissues in less than 5 min. However, the cAMP-mediated increase in Isc was significantly lower in anti-CD3 mAb-injected mice when compared to that of the control. The forskolin response could be attenuated either as a result of a general reduction in the response to secretagogues or, alternatively, as the basal Isc was already elevated in anti-CD3 mAb-injected mice. Luminal addition of NPPB inhibited cAMP-stimulated increase in Isc in both sham-injected (Supplemental Fig. 1) and anti-CD3 mAb-injected mice. The subsequent addition of 300 μm glibenclamide also abolished the cAMP-stimulated increase in Isc in both control (Supplemental Fig. 2) and anti-CD3 mAb-injected mice. In anti-CD3 mAb-injected mice, glibenclamide decreased the basal Isc to the levels of control mice (Table 2).
Dependence of luminal Cl− on HCO3− secretion
The endogenous HCO3− secretion contributing to total HCO3− secretion measured using the pH stat technique was ruled out by using nominally HCO3−-free solution in both luminal and basolateral bathing solutions. Under conditions in which the ileal mucosa was bathed with nominally HCO3−-free, Cl−containing unbuffered solution in both the luminal and basolateral surfaces, HCO3− secretion rate was minimal (0.2 ± 0.03 μEq h−1 cm2, n= 6). The subsequent addition of HCO3− and simultaneous bubbling with CO2 on the basolateral solution significantly increased luminal HCO3− secretion rate to 4.2 ± 0.2 μEq h−1 cm2 (n= 6), indicating that basolateral HCO3− and bubbling with CO2 are required for basal HCO3− secretion (Fig. 2).
As shown in Table 3, absence of luminal Cl− resulted in almost complete elimination of basal HCO3− secretion (0.5 ± 0.04 μEq h−1 cm2, n= 6). Consequently, basal HCO3− secretion was completely dependent on luminal Cl− and thus, will now be referred to as Cl−-dependent HCO3− secretion. To establish whether Cl−-dependent HCO3− secretion involves an apical membrane Cl−–HCO3− exchange, experiments were performed in the presence of a non-specific anion exchange inhibitor, DIDS. Luminal addition of 100 μm DIDS (a concentration at which it is known to inhibits Cl− anion exchanges; Tilmann et al. 1991; Vidyasagar et al. 2004; Vidyasagar et al. 2005), completely inhibited HCO3− secretion (0.5 ± 0.05 μEq h−1 cm2versus 4.2 ± 0.2 μEq h−1 cm2n= 6), which was consistent with apical membrane Cl−–HCO3− exchange (Fig. 2).
|Sham injected||Anti-CD3mAb injected|
|Luminal Cl−||4.2 ± 0.2||7.9 ± 0.1||3.1 ± 0.1§||6.2 ± 0.1§|
|Luminal Cl−-free||0.5 ± 0.04*||7.2 ± 0.1ns||1.9 ± 0.1*||6.3 ± 0.1ns|
|DIDS||0.3 ± 0.05ns||7.1 ± 0.2ns||2.1 ± 0.1ns||6.3 ± 0.2ns|
|NPPB||0.1 ± 0.01ns||0.2 ± 0.05†||0.2 ± 0.05‡||0.2 ± 0.04†|
|Glibenclamide||0.1 ± 0.01ns||0.2 ± 0.04‡||0.3 ± 0.1‡||0.3 ± 0.1‡|
Anti-CD3 mAb-induced alterations in Cl−–HCO3− exchange
Previous studies using the pH stat titration technique on rat distal colon have shown that cAMP stimulates anion secretion and simultaneously inhibits Cl−–HCO3−exchange (Vidyasagar et al. 2004, 2005). We thus performed studies to determine if the increased basal Isc in anti-CD3-treated mice alters Cl−–HCO3− exchange. As shown in Table 3, ileal HCO3− secretion was significantly lower in anti-CD3-treated mice as compared to sham-injected mice. The subsequent removal of luminal Cl− significantly decreased basal HCO3− secretion in anti-CD3 mice. However, in contrast to control mice, HCO3− secretion was not completely abolished by luminal Cl− removal. Furthermore, HCO3− secretion in the absence of luminal Cl− was not inhibited by the apical addition of 100 μm DIDS (data not shown). This suggests that during acute inflammation, HCO3− secretion occurred via a DIDS-insensitive mechanism and was not totally dependent upon apical Cl−–HCO3−exchange (Table 3).
To test this directly, 100 μm NPPB was added to the luminal chamber depleted of Cl−. This addition abolished the Cl−-independent HCO3− secretion, and suggests that during acute inflammation an anion channel-mediated HCO3− secretion is upregulated. In the presence of 300 μm glibenclamide, the anion channel-mediated HCO3− secretion in anti-CD3-treated mice was abolished. These observations suggest that during acute inflammation, Cl−–HCO3− exchange was inhibited with a simultaneous activation of HCO3− secretion via Cftr channel, in addition to the enhanced Cl− secretion previously demonstrated above.
cAMP-induced alterations in HCO3− secretion in anti-CD3-injected mice
Electrogenic ileal anion secretion was enhanced by cAMP, and the forskolin-induced increase in Isc was sensitive to both NPPB and glibenclamide. We therefore examined the effects of cAMP on ileal HCO3− secretion in control and anti-CD3 mAb-injected mice. As shown in Table 3, in the absence of luminal Cl−, HCO3− secretion is minimal in normal mice. Addition of forskolin increased Cl−-independent ileal HCO3− secretion in control and anti-CD3 mAb-injected mice. However, the cAMP-induced HCO3− secretion was slightly lower in anti-CD3-injected mice as compared to control mice (Table 3) (P < 0.01). To assess whether cAMP-stimulated HCO3− secretion occurs via an apical anion channel, the effect of NPPB was first examined in both control and anti-CD3-injected mice. Table 3 demonstrates that cAMP-stimulated HCO3− secretion is significantly inhibited by luminal 100 μm NPPB. In subsequent studies, luminal addition of 300 μm glibenclamide completely inhibited the cAMP-stimulated HCO3− (Table 3), while 100 μm DIDS did not have significant effects (Table 3) on cAMP-stimulated HCO3− secretion. This suggests a role for Cftr in cAMP-stimulated HCO3− secretion in both control and anti-CD3 injected mice.
Anti-CD3-induced and cAMP-stimulated anion secretion occurs via Cftr
To characterize further the role of Cftr in anti-CD3-injected and cAMP-stimulated increase in Isc, Cftr−/− mice were used. Paired littermates were used as controls. Basal Isc was significantly lower in sham-injected Cftr−/− mice (0.1 ± 0.01 μEq h−1 cm2) when the values were compared to that of Cftr+/+ mice (0.6 ± 0.07 μeq h−1 cm2) (P < 0.001, n= 3). Anti-CD3-injected Cftr−/− mice did not show increased Isc as seen with anti-CD3-injected Cftr+/+ mice (0.3 ± 0.01 versus 2.5 ± 0.16 μeq h−1 cm2) (Fig. 3). However there was a small but significant increase in Isc in anti-CD3-injected Cftr−/− mice when compared to that of the sham-injected Cftr−/− mice (0.3 ± 0.01 versus 0.1 ± 0.01 μeq h−1cm2). Addition of forskolin resulted in an increase in Isc in Cftr+/+ mice and not in Cftr−/− mice (3.5 ± 0.2 versus 0.3 ± 0.01 μeq h cm2) (Fig. 3). These studies clearly indicate that increases in Isc in anti-CD3-injected mice were due to increased electrogenic anion secretion via Cftr.
Isc was drastically reduced with Cftr−/− mice under all conditions in both control and anti-CD3-injected mice and was unresponsive to cAMP. Positive control experiments were therefore undertaken by replacing glutamine with glucose at the end of each experiment for ascertaining the viability of the tissue. Addition of d-glucose (10 mm) resulted in a robust increase in Isc (0.4 ± 0.03 versus 1.1 ± 0.05; n= 5), indicating a functionally active and viable tissue activating electrogenic sodium-coupled glucose transport (SGLT1) (Supplemental Fig. 3).
Studies were then done using a pH stat technique to determine the role of Cftr in mediating luminal, Cl−-independent HCO3− secretion in anti-CD3-injected mice. In control Cftr−/− mice, the presence of Cl− in the luminal solution resulted in a significant HCO3− secretion (Table 4). Addition of forskolin to the serosal solution abolished HCO3− secretion, indicating inhibition of luminal Cl--dependent HCO3− secretion. This was further confirmed in pH stat experiments performed in the absence of luminal Cl−. Removal of Cl− from the luminal solution abolished basal HCO3− secretion in Cftr−/− mice. Addition of 10 μm forskolin to the serosal side failed to stimulate HCO3− secretion in the absence of luminal Cl− in Cftr−/− mice (Table 4). This indicates functional Cftr channels are essential for cAMP-stimulated HCO3− secretion.
|Sham-injected Cftr−/−||Anti-CD3-injected Cftr−/−|
|Luminal Cl−||6.8 ± 0.1||0.5 ± 0.2†||0.6 ± 0.2||0.7 ± 0.3†|
|Luminal Cl−-free||0.4 ± 0.1*||0.4 ± 0.1ns||0.5 ± 0.1*||0.4 ± 0.2ns|
|DIDS||0.5 ± 0.1ns||—||0.5 ± 0.1ns||—|
Anti-CD3 injected Cftr−/− mice showed significantly lower HCO3− secretion in the presence of luminal Cl− compared to the sham-injected Cftr−/− mice (Table 4). This indicates that anti-CD3 abolished luminal Cl−-dependent HCO3− secretion. Lack of HCO3− secretion in the absence of luminal Cl− in anti-CD3-injected Cftr−/− mice indicates a failure to stimulate a luminal Cl−-independent HCO3− secretion, which was thought to occur via functional Cftr. This was further confirmed by addition of forskolin to the bath solution in the absence of, luminal Cl− in anti-CD3-injected mice. No significant increase in HCO3− secretion was seen (Table 4 and Supplemental Fig. 4). Together from these experiments it was seen that anti-CD3 injection inhibits electroneutral luminal Cl−-dependent HCO3− secretion, and at the same time stimulates a HCO3− secretion that was luminal Cl−-independent and mediated via a functional Cftr. Thus functional Cftr channels are essential for electrogenic HCO3− secretion in both anti-CD3-injected and cAMP-stimulated HCO3− secretion.
Western blot for Cftr protein in anti-CD3 mAb-injected and normal mice
Increased Isc in anti-CD3-injected mice was inhibited by glibenclamide and was absent in Cftr−/− mice, indicating that functional Cftr channels are essential for increased electrogenic anion secretion. However, Western blot analysis for Cftr protein did not show an increase but instead showed a non-significant decrease (Fig. 4) in anti-CD3-injected mice, indicating that the increase in Isc was not due to increased Cftr protein expression.
Colorimetric immunoassay for intracellular cAMP
Anti-CD3-injected mice showed a significant increase in basal Isc and luminal Cl−-independent HCO3− secretion that was inhibited by NPPB and glibenclamide. This indicates that the anion channel mediating electrogenic anion secretion is probably Cftr. However, increased electrogenic activity did not reflect increased Cftr protein expression. Since Cftr channels are activated by cAMP, we asked if the increase in Isc and luminal Cl−-independent HCO3− secretion was because of an increase in intracellular cAMP levels. As a control, it was first established that forskolin treatment significantly increased intracellular cAMP levels in crypt cells, villus cells and unfractionated cells (Fig. 5A–C). Anti-CD3-treated crypt cells did not show an increase in cAMP levels (Fig. 5A). By contrast, villus cells showed a significant increase in cAMP in anti-CD3-treated mice (Fig. 5B). These changes were also reflected in unfractionated cells (Fig. 5C). Forskolin did not result in a significant increase in intracellular cAMP in either the anti-CD3-treated villus cells or unfractionated cells. However, crypt cells showed a significant increase in intracellular cAMP in forskolin-treated anti-CD3 mice. These studies therefore indicate that anti-CD3-induced changes in Isc may be mediated by an increase in intracellular cAMP resulting in a secondary activation of Cftr channels in villus cells. One implication of these results is that villus cells can contribute to anion secretion in inflamed tissue.
During jejunal and ileal inflammation, it is widely accepted that absorption of nutrients, electrolytes and water by villus cells is significantly decreased (Sundaram & West, 1997b). In addition, fluid secretion by crypts may also be enhanced. Thus, the fine balance between absorption and secretion is altered, resulting in fluid accumulation and the subsequent diarrhoea. Our present findings of increased Isc after anti-CD3 treatment suggests that fluid secretion may indeed be increased during acute ileal inflammation. Increased Isc in anti-CD3 treated mice was inhibited by 100 μm NPPB, a non-specific anion channel blocker, and 300 μm glibenclamide, a relatively specific CFTR blocker. To exclude an effect of glibenclamide on apical membrane K+ channels, 5 mm barium was added to the luminal solution as shown previously (Vidyasagar et al. 2004). 8-Br-cAMP-stimulated HCO3− secretion measured in the luminal Cl−-free solution, failed to show significant difference in the presence 5 mm barium (4.7 ± 0.06 versus 4.4 ± 0.08 μEq h−1 cm2, n= 4). These studies indicate that anti-CD3 treatment results in an increased anion conductance in mice that probably arises via Cftr channels. However, the cAMP-stimulated Isc in anti-CD3 mice was lower when compared with normal mice (Table 2). Since the basal Isc was significantly higher in treated mice, Cftr channels may already be in a state of maximal conductance, resulting in an attenuated response to further stimulation by cAMP.
In contrast to our studies showing increased Isc with anti-CD3 treatment, Musch et al. (2002) did not observe significant alterations in jejunum Isc after a similar anti-CD3 mAb treatment. Similarly, Musch et al. (2002) using the same model had shown a possible defect in the epithelial barrier function. However, the transepithelial measurement of conductance in Ussing chamber studies did not show an increase in conductance in anti-CD3-injected mice. These differing observations may reflect segmental heterogeneity and/or differences in tissue-stripping techniques. In particular, our ileal tissue was completely stripped of all muscle layers (Fig. 1A), thus ensuring that any observed changes in Isc were mostly due to responses from epithelial cells (Fig. 1B). Furthermore, compared with the ileum, fluid and electrolyte absorption is significantly higher in the jejunum.
Also in the pH stat studies with a large serosal-to-mucosal gradient for HCO3−, no bicarbonate secretion was detected in the absence of luminal Cl−. This rules out the possibility of an increased HCO3− secretion that could occur via the paracellular route due to the large transepithelial HCO3− gradient. If HCO3− secretion occurred via a paracellular route, a luminal Cl--independent HCO3− secretion should have been detected in anti-CD3-treated mice. Our studies therefore show no evidence for increased barrier permeability in anti-CD3-injected mice.
As Cl− secretion is the driving force for fluid secretion in the small intestine, the increased Cl− secretion during acute inflammation may be an adaptive measure to flush the toxins and/or the elevated proinflammatory cytokines from the lumen. In addition to Cl− secretion, net fluid secretion, which is associated with most acute and chronic clinical diarrhoeas and those induced experimentally in vivo, also contains HCO3−-rich, plasma-like solution (Fordtran, 1967). Under normal physiological states, the small intestine actively secretes net HCO3− (Dietz & Field, 1973; Sheerin & Field, 1975; Charney & Haskell, 1983; Sundaram et al. 1991a, b; Minhas et al. 1993; MacLeod et al. 1996; Alper et al. 1999) for the proper maintenance of many biological functions. Pathophysiological conditions such as acute severe diarrhoea and chronic persistent diarrhoea, result in significant HCO3− loss and often lead to metabolic acidosis.
Our present findings in the normal mice ileum demonstrates that HCO3− secretion is luminal Cl− dependent and DIDS sensitive, indicating that ileal Cl−–HCO3− activity is mediated by AE and is electroneutral. However, during acute inflammation, HCO3− secretion was significantly decreased. The subsequent removal of luminal Cl− did not completely abolish HCO3− secretion in anti-CD3-treated mice. Furthermore, the Cl−-independent HCO3− secretion was blocked by glibenclamide but insensitive to DIDS (Table 3). Thus, during acute inflammation an electrogenic HCO3− component is upregulated, and this is probably mediated by Cftr channels. Similar experiments done using anti-CD3-injected Cftr−/− mice showed minimal HCO3− secretion when compared to that in sham-injected Cftr+/+ mice, indicating the role of Cftr channels in mediating Cl−-independent HCO3− secretion (Table 4). Indeed, CFTR has been shown to be permeable to HCO3− in a variety of cell types (Gray et al. 1989; Poulsen et al. 1994; Seidler et al. 1997; Illek et al. 1998; O'Reilly et al. 2000). In contrast, in recombinant wild-type CFTR-expressing cells, HCO3− secretion was not enhanced by increases in cAMP, suggesting that CFTR does not conduct HCO3− (Shumaker et al. 1999; Soleimani & Ulrich, 2000). In the present studies, increases in cAMP levels enhanced luminal Cl−-independent HCO3− secretion in both normal and anti-CD3-treated mice, and at the same time inhibited luminal Cl−-dependent HCO3− secretion. Similar findings with cAMP were reported in the rat distal colon (Vidyasagar et al. 2004; Vidyasagar et al. 2005). However, the cAMP-stimulated increase in total HCO3− secretion in anti-CD3 mAb-injected mice was significantly lower than that in normal mice (Table 3). The HCO3− transport studies correlated with the Isc data and support the fact that HCO3− contributes to net anion secretion seen during inflammation. Thus, one of the major transport alterations during inflammation is inhibition of electroneutral Cl−–HCO3− exchange and stimulation of electrogenic anion secretion which includes luminal Cl−-independent HCO3− secretion.
Increased anion conductance during acute inflammation may result from increased Cftr expression, increased recruitment to the membrane, and/or an increased conductance, possibly via stimulation of intracellular cAMP levels. We used Western blot analysis of mucosal tissue from control and anti-CD3-injected mice to rule out increased Cftr protein levels (Figs 4, n= 6), though this approach does not rule out mobilization of Cftr from intracellular organelles. Cftr protein levels were not increased in anti-CD3-injected mice. Changes in protein levels might be a late response to inflammation as a 3 h anti-CD3 treatment time may be insufficient for translation of mRNA and post-translation of protein to sufficiently increase Cftr protein level in the cell and its express on the membrane. Changes with acute inflammation may not represent changes seen with chronic inflammation. This could also explain the increased CFTR expression reported in human crypts during chronic ulcerative colitis (Sundaram & West, 1997a), and may reflect an effect of long-term inflammation on modifications on protein expression. In addition, hyperproliferated mouse colonic crypts exhibited increased cAMP-stimulated Cl− secretion and Cftr expression (Umar et al. 2000).
However, an immunoassay for intracellular cAMP showed significant increases in basal cAMP levels in both villus cell and unfractionated cells isolated from anti-CD3-treated mice. Similar changes were not observed in a crypt cell fraction (Fig. 5A). Addition of forskolin to villus and unfractionated cells of anti-CD3-treated mice did not result in a further increase in intracellular cAMP level, while crypt cell fractions on the other hand showed significantly increased intracellular cAMP levels in anti-CD3-treated mice. These studies therefore indicate that increased anion secretion seen with anti-CD3 treatment may result from increased intracellular cAMP. These observations correlate well with our electrophysiology data, where forskolin did not result in further increase in Isc in anti-CD3-treated mice, as opposed to the sham-treated mice (Table 2). Although cAMP-stimulated anion secretion is associated with crypt cells under normal conditions (Barrett & Keely, 2000), the present studies show that villus cells may also contribute to anion secretion in both forskolin-stimulated and anti-CD3-treated mice. This may reflect a pre-existing pool of unutilized Cftr in ileal villus cells under normal conditions.
A family of anion exchangers (AEs) including apical AE1 and AE3 and basolateral AE2 has been described in the small intestine (Alper et al. 1999, 2002; Simpson et al. 2005; Wang et al. 2005; Tuo et al. 2006). The majority of the studies investigating AEs in the small intestine have focused on the duodenum, with relatively little information in the ileum. Furthermore, functional studies using pH stat and Ussing chamber techniques cannot accurately delineate between the different types of HCO3− secretory mechanisms. Thus, the exact identity of the ileal HCO3− transporter(s) modulating HCO3− movement remains to be resolved. The SLC26A3 gene is mutated in congenital chloride diarrhoea (CLD) (Hoglund et al. 1996). CLD is characterized by defects in intestinal HCO3− secretion and massive loss of Cl− in the stool. These defects result in acidic stool and systemic metabolic alkalosis. Cl− loss along with acidic stool increases the electrochemical gradient for protons, and secondarily impairs the intestinal absorption of Na+ resulting in loss of both sodium and chloride in the stool (Holmberg et al. 1975). SLC26A3 has been observed in villus and surface epithelium of ileum and colon, respectively, and is thought to mediate Na+-independent Cl−–OH− and Cl−–HCO3− exchange (Hoglund et al. 1996; Melvin et al. 1999; Moseley et al. 1999).
Taken together, these studies demonstrate that acute inflammation induces a host of complex changes at the cellular and molecular levels. In particular, the increased anion secretion together with a shift from electroneutral to electrogenic process may represent an adaptive mechanism to ‘flush’ harmful bacteria and/or the circulating pro-inflammatory cytokines in the ileal mucosa. Thus, our studies indicate that acute ileal inflammation downregulates electroneutral Cl−–HCO3− exchange, simultaneously inducing an electrogenic anion secretion that includes HCO3−. These alterations in epithelial electrolyte movement lead to decreased absorption of electrolytes with a concomitant increase in anion secretion resulting in diarrhoea. Further studies are, however, needed to resolve the complex interaction between inflammatory mediators and altered epithelial transport.
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This work was supported in part by the Public Health Grants R01DE09692 and R01DE08921 (J.E.M). We gratefully acknowledge the experimental assistance of Daniel G. Greener and Dr Tara Rajesh and, for helpful discussions, Dr Richard Farmer and Dr Raju Vulapalli.