Acute inflammation alters bicarbonate transport in mouse ileum

J Physiol 581.3 (2007) pp 1221–1233 1221

Acute inflammation alters bicarbonate transportin mouse ileum

Hui Zhang1,2, Nadia Ameen4, James E. Melvin2,3 and Sadasivan Vidyasagar1

1Digestive Diseases, Department of Medicine and 2Department of Pharmacology and Physiology, 3Center for Oral biology University of Rochester School

of Medicine, Rochester, NY, USA4Department of Pediatrics and Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

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 (I sc) (0.58 ± 0.07 μEq h−1 cm2 versus

1.63 ± 0.14 μEq h−1 cm2). The cAMP-stimulated I sc 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 I sc 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.

(Received 26 January 2007; accepted after revision 23 March 2007; first published online 29 March 2007)

Corresponding author S. Vidyasagar: University of Rochester School of Medicine, 601 Elmwood Ave, Box 646, Rochester,

NY 14642, USA. Email: sadasivan [email protected]

Crohn’s disease (CD) and ulcerative colitis, the two mostcommon forms of inflammatory bowel disease (IBD),are characterized by chronic, recurrent inflammation ofthe intestinal tract. Ulcerative colitis occurs mainly inthe colon, while Crohn’s disease can affect any part ofthe digestive tract from the mouth to the rectum, butprimarily occurs in the lower part of the small intestine(ileitis or enteritis). Although the exact aetiology ofIBD is unknown, it is widely accepted that the diseaseis characterized by an abnormal cell-mediated immunereaction – primarily by CD4+ T-cells – to the antigensand adjuvant of the enteric bacteria in genetically

This paper has online supplemental material.

susceptible hosts (Jump & Levine, 2004). CD tends to bea chronic, recurrent condition with periods of remissionand exacerbation. Complex and active interactions existbetween the bacterial flora, epithelium, and immune cellsin the intestine, and perturbation of these interactionscan result in intestinal inflammation. The pathologicalfeatures of CD include villus atrophy and crypt hyperplasialeading to malabsorption by villus epithelia and mostlikely increased fluid secretion from the crypt epithelium.These alterations lead to fluid accumulation and thesubsequent diarrhoea, a common clinical feature duringIBD (Ciancio & Chang, 1992; Baert et al. 1999; Bell &Kamm, 2000). Despite the abundance of data regardingintestinal inflammation-associated diarrhoea, specific ilealtransport alterations have not been clearly identified

C© 2007 The Authors. Journal compilation C© 2007 The Physiological Society DOI: 10.1113/jphysiol.2007.129262

1222 Hui Zhang and others J Physiol 581.3

(Ciancio & Chang, 1992; Radojevic et al. 1999; Musch et al.2002).

The main physiological function of the small intestine isabsorption of nutrients, electrolytes and water. However,low rates of fluid secretion in the small intestine arealso necessary to maintain luminal ionic composition,pH and motility. Consequently, there is a fine balancebetween absorption and secretion, such that electro-neutral Na+–H+ exchange coupled with Cl−–HCO3

−

exchange-stimulated fluid absorption predominates overelectrogenic anion secretion and thus fluid secretion. Anydecrease in electroneutral Na+ and Cl− absorption and/orincreased electrogenic anion secretion may result in fluidaccumulation and diarrhoea. Amongst the anions, Cl−

secretion is considered the major driving force for fluidsecretion in the small intestine. It is widely believed thatthe major route for stimulated Cl− secretion in the smallintestine occurs via the cystic fibrosis transmembraneconductance 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

− (Furukawaet al. 2005). The exchange of Cl− for HCO3

− has beenidentified in all three regions of the small intestine (i.e.duodenum, jejunum and ileum). In most regions of themouse small intestine, electroneutral HCO3

− secretion ismediated by the SLC4 family of Cl− -HCO3

− exchangers(anion exchanger (AE)) coupled to Na+–H+ exchange. Allfour AE isoforms (AE1, AE2 and AE3, AE4) of the SLC4family have been reported in the small intestine (Alperet al. 1999; Alrefai et al. 2001; Alper et al. 2002; Charneyet al. 2004).

The SLC26 family of anion exchangers, like the SLC4family of Cl−–HCO3

− exchangers, is known to transporta variety of anions and in some cases, to participate inelectrogenic Cl−–HCO3

− exchange (Mount & Romero,2004). In particular, mutations in the SLC26A3 gene, alsoknown as Down Regulated in Adenoma (DRA), lead tocongenital chloride diarrhoea (CLD) (Schweinfest et al.1993; Hoglund et al. 1996). SLC26A6, the putative aniontransporter (PAT1) has also been identified in the gastro-intestinal tract (Lohi et al. 2000; Waldegger et al. 2001). Inthe intestine, DRA is mainly expressed in the colon andduodenum, 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 allregions of the small intestine but low in the large intestine(Boll et al. 2002; Wang et al. 2002). Recent studies by Wanget al. 2005 demonstrated that PAT1 plays a major rolein Cl−–HCO3

− exchange in the duodenum as the basalHCO3

− transport in Slc26A6−/− mice was reduced by∼30%. Although, the mechanisms of HCO3

− transporthave been studied in detail in the duodenum and the

colon, the exact AE and Slc26A isoforms mediatingHCO3

− transport in the ileum and how they arealtered during inflammatory states remains to beexamined.

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 controversialas HCO3

− secretion was not enhanced by increasesin cAMP in recombinant wild-type CFTR-expressingcells, suggesting that CFTR does not conduct HCO3

−

(Shumaker et al. 1999; Soleimani & Ulrich, 2000).However, functional data regarding the types of anionchannels and transporters modulating ileal fluid secretionusing 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-establishedimmune-mediated acute inflammatory mouse modelin which mice were injected with anti-CD3 mono-clonal antibody (mAb). Intraperitoneal injection of micewith anti-CD3 mAb induced acute inflammation andincreased proinflammatory cytokines transcripts andacute diarrhoea (Radojevic et al. 1999; Musch et al.2002; Clayburgh et al. 2005). Early human studies usinganti-CD3 antibodies to prevent renal transplant rejectionalso resulted in diarrhoea (Chatenoud & Bach, 1988).In anti-CD3-injected mice, maximal fluid accumulation,and hence diarrhoea, occurred within 2–3 h of anti-CD3mAb injection (Musch et al. 2002; Clayburgh et al.2005). Recently, Clayburgh et al. (2005) reported thatanti-CD3-injected mice exhibited signs of intestinalinflammation as evidenced by vasodilatation, oedema,erythema and increased intraepithelial lymphocytesFurthermore, in vivo treatment with anti-CD3 mAbincreased 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; Clayburghet al. 2005). During intestinal inflammation, a host ofsimilar proinflammatory cytokines are released by T-cells(Chatenoud & Bach, 1988; Ferran et al. 1991). In additionto these proinflammatory cytokines, anti-CD3 increasesinterleukin (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, inthe present studies we used in vivo anti-CD3 mAb-treatedmice to investigate whether the diarrhoea during acuteinflammation is due to alterations in ileal electroneutralanion exchange activity and/or simultaneous activation ofanion channels. Specifically, the cellular mechanisms ofHCO3

− secretion was examined in stripped ileal mucosaunder voltage-clamp conditions using a pH stat techniqueto study electroneutral Cl−–HCO3

− exchange and electro-genic HCO3

− secretion. In addition, Ussing chamberexperiments were performed to study changes in shortcircuit current (I sc) in anti-CD3-injected mice.

C© 2007 The Authors. Journal compilation C© 2007 The Physiological Society

J Physiol 581.3 Inflammation alters ileal transport 1223

Table 1. Composition of experimental solutions

Bath Lumen

Constituent (mM) Ringer HCO3−-free Ringer Cl−-containing Cl−-free

Na+ 140 140 140 140Cl− 119.8 119.8 119.8 —HCO3

− 25 — — —HPO4

− 2.4 2.4 — —H2PO4

− 0.4 0.4 — —SO4

2− — — 2.4 2.4Cyclamide — — 0.4 5.2Isethionate — 25 25 140

pH of all Cl-containing solutions was adjusted to 7.4 with HCl while that of Cl−-freesolutions was adjusted with H2SO4. HCO3

−-containing solutions were gassed with 95%O2–5%CO2; HCO3

−-free solutions were gassed with 100% O2. Both luminal and bathsolutions also contained 10 mM glutamine, 5.2 mM K+, 1.2 mM Ca2+, and 1.2 mM Mg2+. Allluminal solutions were HCO3

−-free and contained 0.1 mM Hepes (pH 7.4).

Methods

Animal model

Non-fasting male C57BL/6 mice, 6–8 weeks old-were usedin all experiments except experiments in which Cftr−/−

(Snouwaert et al. 1992) and Cftr+/+ mice (age- andsex-matched littermates as controls) were used. Micewith a truncation mutation beginning at position S489were used to compare the effect of anti-CD3 mAb onI sc and HCO3

− movement. Following exsanguinations,ileal mucosa was obtained from the segment close tothe caecum. HCO3

− transport studies were performedin mucosa that was stripped through the submucosallayers to remove the serosal and muscular layers under adissection microscope. Mucosa was mounted between thetwo halves of a Ussing-type Lucite chamber, as previouslydescribed (Vidyasagar & Ramakrishna, 2002; Vidyasagaret al. 2004). In experiments in which the effects ofanti-CD3 were studied, 0.2 mg/0.2 ml of anti-CD3 mAbwas injected i.p. and animals were killed 3 h later toremove the ileum. Musch et al. (2002) demonstrated thatmaximal intraluminal fluid accumulation occurs at 3 hof in vivo anti-CD3 mAb treatment. Control animalswere sham-injected with 0.2 ml PBS. Mice were killed bysodium pentobarbital injection (50 mg kg−1, i.p.) followedby cervical dislocation 3 h after i.p. injection. Tissues wereharvested only after apnoea had occurred. All experimentswere approved by the University of Rochester InstitutionalAnimal Care and Use Committee.

Bioelectric measurements

The ileal sheets were stripped through the submucosallayer such that almost all of the muscular layers wereremoved. Stripped ileal sheets showed significantly lower

I sc compared to unstripped ileal sheets (Fig. 1). Thus, forall Ussing chamber experiments, stripped ileal sheets weremounted in Lucite chambers (0.3 cm2 exposed surfacearea). Intestinal preparations were bathed bilaterally inregular Ringer’s solution (Table 1) containing 10 mm

glutamine and gassed with a mixture of 95% O2 and5% CO2. Glutamine was substituted for glucose to

Figure 1. Stripping reduces Isc

A, left panel shows ileal tissue before stripping of all serosal andmuscular layers. After stripping only epithelial cells are present (rightpanel). B, the basal Isc was significantly reduced after stripping (n = 4).∗P < 0.01. Error bars represent S.E.M.



avoid inward current resulting from Na+-coupled glucosecotransport.

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 witha hysteresis of 0.05 and thus titrating between 7.35 and7.45 (physiological limits of pH range for body fluids), aspreviously described (Vidyasagar et al. 2004, 2005). Briefly,luminal solution pH was continuously maintained at aconstant (or stat) pH by the addition of 0.025 m H2SO4.Pumps were programmed to operate in real time to thechanges in luminal pH delivering a minimum of 0.01 μlat a given time. Standard pH calibration to stat pH wasestablished by adding a known quantity of H2SO4 to a lowbuffering solution containing increasing concentration ofHCO3

− to get a linear curve. The amount of HCO3−

measured in the luminal solution was always within thelinear range of this curve. The acid used for the titration wasdiluted in the same ionic solution as used in that particularexperiment to give a final concentration of 0.025 m. Ilealtissues were always exposed to a buffered solution on thebath side, while the luminal side was exposed to a lowbuffered solution (0.1 mm Hepes buffer, pH 7.4). HCO3

−

secretion is equivalent to the amount of acid required tomaintain pH at 7.4. All experiments were performed undervoltage-clamp conditions and HCO3

−-free solutions weregassed with 100% O2, while HCO3

−-containing solutionswere gassed with 95% O2 and 5% CO2. HCO3

− secretionwas expressed as μEq h−1 cm2.

Initial studies demonstrated that immediately after thetissue was mounted, HCO3

− secretion was present in theabsence of bath HCO3

−, but rapidly fell towards zerowithin 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. Theseexperimental methods were similar to those of earlierpublished work (Vidyasagar et al. 2004). All experimentswere performed during this 1 h steady-state period.

In experiments where inhibitors were added to themucosal solution, the specific drug was added during theinitial steady-state period and pH adjusted and allowedto equilibrate for 30 min until a steady rate of HCO3

−

secretion was again observed. In experiments where theinhibitor was added to the serosal side, the tissue wasalso equilibrated for 30 min to achieve a steady state ofHCO3

− secretion. The composition of the several solutionsused in these experiments is presented in Table 1. Inbrief, in Cl−-free experiments, isethionate was used as asubstitute for Cl−; in Na+-free experiments NMDG wasused as a Na+ substitute; 10 mm glutamine was added to all

solutions. Only one tissue from each animal was used for aspecific experiment, and only one experimental conditionwas used with each tissue. All experiments were repeatedat least four times.

Western blot

Intestinal lysates were prepared from mucosal scrapingsfrom anti-CD3 mAb-treated and control mouse ileumand analysed for Cftr protein by Western blots. Mucosalscrapings were lysed in TGH buffer containing 25 mm

Hepes, 10% glycerol, 1% Triton X-100, containinga protease inhibitor mixture (10 mm iodoactamide,1 mm phenylmethylsulphonyl fluoride, and 2 μg ml−1

leupeptin) pH 7.4, and protein concentration wasdetermined in samples using the Bradford assay.Equivalent loads of protein from anti-CD3-treated andcontrol samples were analysed by SDS PAGE. Proteinswere transferred onto PVDF membranes and Cftr detectedusing 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 andsham-injected mice were used for cAMP assay usingcAMP direct immunoassay kit (Calbiochem, USA).Cells were treated with 0.1 m HCl to stop endogenousphosphodiesterase activity. Competitive immunoassayfor the quantitative assay of cAMP used a polyclonalantibody to cAMP that binds to cAMP in samples in acompetitive manner. After a simultaneous incubationat room temperature, the excess reagents were washedaway and substrate added. After a short incubationtime, the reaction is stopped and the yellow colourgenerated is read at 405 nm. The intensity of the colouris inversely proportional to the concentration of cAMPin standards and samples. Forskolin-treated cells wereincubated for 45 min after its addition and before thecAMP immunoassay. cAMP levels were standardized toprotein levels from respective fractions and expressed inpmol (mg protein)−1. All the assays were done in triplicateand repeated to get n = 4 different mice.

Statistics

Results are presented as mean ± s.e.m. Statistical analyseswere performed using paired and unpaired t test andBonferroni’s one-way ANOVA post hoc test. P < 0.05 wasconsidered significant.



Table 2. Change in Isc in anti-CD3-injected mice

Sham-injected Anti-CD3 mAb-injected

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 ± 0.18NPPB 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∗∗

Experiments were done in the presence of luminal Cl− and HCO3−-containing Ringer solution. Isc (μEq h−1 cm2) after addition of 10 μM

forskolin are taken from the highest values. Values represent mean ± S.E.M. n = 12 in the basal group and n = 6 in which blockerswere added. ∗P < 0.001, comparison between presence and absence (basal) of NPPB. ∗∗P < 0.001, comparison between presence andabsence (basal) of glibenclamide. ns = no significant difference between the groups.

Chemicals and solutions

Forskolin; 4,4′-diisothiocyanato-stilbene-2,2′-disulphonicacid (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 gluconateand 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.

Results

Anti-CD3-induced change in Isc

Ussing chamber experiments were performed 3 h afteranti-CD3 mAb injection to determine the effects of T-cellactivation on transepithelial I sc and conductance (G).Anti-CD3 injection did not result in a significant increasein conductance in totally stripped ileal tissue (Table 2).In contrast, basal I sc increased by ∼260% as comparedto sham-injected mice (Table 2). To assess whether theobserved increase in I sc in anti-CD3 mAb-injected micewas associated with an apical membrane anion channel,100 μm NPPB (non-specific Cl− channel blocker; Tilmannet al. 1991; Fuller & Benos, 1992) was added to thelumen solution. NPPB decreased the anti-CD3-stimulatedI sc to levels similar to that of sham-injected (control)basal I sc. In control mice, NPPB addition resulted ina marginal but significant decrease in I sc (Table 2).This suggests that anti-CD3 treatment enhances electro-genic anion secretion via an anion channel. As Cftr ispostulated to contribute the bulk of anion conductancein the small intestine, an inhibitor study was performedwith 300 μm glibenclamide, a concentration at whichglibenclamide is a relatively specific inhibitor of Cftr.Exposure to glibenclamide decreased significantly the basalI sc in anti-CD3 injected mice, suggesting increased electro-genic anion secretion via Cftr during acute inflammation(Table 2).

cAMP-stimulated changes on anti-CD3-induced Isc

Cftr conductance is activated by elevations in intra-cellular cAMP. Whether cAMP also modifies anionsecretion was examined in both normal and anti-CD3mAb-injected mice. Exposure of ileal tissue to 10 μm

forskolin significantly increased I sc in both sham-injectedand anti-CD3 mAb-injected mice. Maximal increase inI sc was seen in most tissues in less than 5 min. However,the cAMP-mediated increase in I sc was significantlylower in anti-CD3 mAb-injected mice when comparedto that of the control. The forskolin response could beattenuated either as a result of a general reduction in theresponse to secretagogues or, alternatively, as the basalI sc was already elevated in anti-CD3 mAb-injected mice.Luminal addition of NPPB inhibited cAMP-stimulatedincrease in I sc in both sham-injected (SupplementalFig. 1) and anti-CD3 mAb-injected mice. The subsequentaddition of 300 μm glibenclamide also abolished thecAMP-stimulated increase in I sc in both control(Supplemental Fig. 2) and anti-CD3 mAb-injected mice.In anti-CD3 mAb-injected mice, glibenclamide decreasedthe basal I sc 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 bathingsolutions. Under conditions in which the ileal mucosawas bathed with nominally HCO3

−-free, Cl−containingunbuffered solution in both the luminal and baso-lateral surfaces, HCO3

− secretion rate was minimal(0.2 ± 0.03 μEq h−1 cm2, n = 6). The subsequent additionof HCO3

− and simultaneous bubbling with CO2 onthe basolateral solution significantly increased luminalHCO3

− 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).



Table 3. HCO3− secretion (μEq h cm2) in sham and anti-CD3 mAb-injected mice

Sham injected Anti-CD3mAb injected

Basal cAMP Basal cAMP

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‡

Experiments were done in the presence and absence of luminal Cl− and HCO3−.

Values represent mean ± S.E.M. n = 6. DIDS (100 μM) was added to the mucosal side inluminal Cl−-containing experiments and HCO3

− secretion compared to luminal Cl−-freeexperiments. ∗P < 0.001, comparison between presence and absence of luminal Cl− onHCO3

− secretion. †P < 0.001, comparison between luminal Cl−-free and luminal additionof NPPB on Cl−-independent HCO3

− secretion. ‡ P < 0.001, comparison between luminalCl−-free and luminal addition of glibencalmide on Cl−-independent HCO3

− secretion.§P < 0.01, comparison between anti-CD3 and normal mice. ns = no significant differencebetween the groups.

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 onluminal Cl− and thus, will now be referred to asCl−-dependent HCO3

− secretion. To establish whetherCl−-dependent HCO3

− secretion involves an apicalmembrane Cl−–HCO3

− exchange, experiments wereperformed in the presence of a non-specific anionexchange inhibitor, DIDS. Luminal addition of 100 μm

DIDS (a concentration at which it is known to

0

1

2

3

4

5

(4)

(6)

+ - +

HC

O3 S

ecre

tio

n (

meq

/h.c

m2)

Bath HCO3-

(4)

DIDS

Figure 2. Basal Cl−-dependent HCO3− secretion is bath HCO3

−

dependent and DIDS sensitiveExperiments were done in the presence of 119.8 mM luminal Cl−. InHCO3

−-free experiments, 25 mM HCO3− was replaced by 25 mM

isethionate. Exposure to 100 μM DIDS reduced HCO3− secretion to

basal levels. Numbers in parentheses indicate number of tissuesstudied in each group. ∗P < 0.001 compared with group without bathHCO3

−. Error bars represent S.E.M.

inhibits Cl− anion exchanges; Tilmann et al. 1991;Vidyasagar et al. 2004; Vidyasagar et al. 2005), completelyinhibited HCO3

− secretion (0.5 ± 0.05 μEq h−1 cm2

versus 4.2 ± 0.2 μEq h−1 cm2 n = 6), which was consistentwith apical membrane Cl−–HCO3

− exchange (Fig. 2).

Anti-CD3 mAb-induced alterations in Cl−–HCO3−

exchange

Previous studies using the pH stat titrationtechnique on rat distal colon have shown that cAMPstimulates anion secretion and simultaneously inhibitsCl−–HCO3

−exchange (Vidyasagar et al. 2004, 2005). Wethus performed studies to determine if the increasedbasal I sc 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 comparedto sham-injected mice. The subsequent removal of luminalCl− significantly decreased basal HCO3

− secretion inanti-CD3 mice. However, in contrast to control mice,HCO3

− secretion was not completely abolished byluminal Cl− removal. Furthermore, HCO3

− secretion inthe absence of luminal Cl− was not inhibited by the apicaladdition of 100 μm DIDS (data not shown). This suggeststhat during acute inflammation, HCO3

− secretionoccurred via a DIDS-insensitive mechanism and wasnot totally dependent upon apical Cl−–HCO3

−exchange(Table 3).

To test this directly, 100 μm NPPB was added to theluminal chamber depleted of Cl−. This addition abolishedthe Cl−-independent HCO3

− secretion, and suggests thatduring acute inflammation an anion channel-mediatedHCO3

− 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 simultaneousactivation of HCO3

− secretion via Cftr channel, in additionto the enhanced Cl− secretion previously demonstratedabove.

cAMP-induced alterations in HCO3− secretion

in anti-CD3-injected mice

Electrogenic ileal anion secretion was enhanced bycAMP, and the forskolin-induced increase in I sc wassensitive to both NPPB and glibenclamide. We thereforeexamined the effects of cAMP on ileal HCO3

− secretionin control and anti-CD3 mAb-injected mice. As shownin Table 3, in the absence of luminal Cl−, HCO3

−

secretion is minimal in normal mice. Addition of forskolinincreased Cl−-independent ileal HCO3

− secretion incontrol and anti-CD3 mAb-injected mice. However, thecAMP-induced HCO3

− secretion was slightly lower inanti-CD3-injected mice as compared to control mice(Table 3) (P < 0.01). To assess whether cAMP-stimulatedHCO3

− secretion occurs via an apical anion channel,the effect of NPPB was first examined in both controland anti-CD3-injected mice. Table 3 demonstratesthat cAMP-stimulated HCO3

− secretion is significantlyinhibited by luminal 100 μm NPPB. In subsequent studies,luminal addition of 300 μm glibenclamide completelyinhibited the cAMP-stimulated HCO3

− (Table 3), while100 μm DIDS did not have significant effects (Table 3)on cAMP-stimulated HCO3

− secretion. This suggests arole for Cftr in cAMP-stimulated HCO3

− secretion in bothcontrol and anti-CD3 injected mice.

Anti-CD3-induced and cAMP-stimulated anionsecretion occurs via Cftr

To characterize further the role of Cftr inanti-CD3-injected and cAMP-stimulated increase inI sc, Cftr−/− mice were used. Paired littermates wereused as controls. Basal I sc was significantly lower insham-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 increasedI sc 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 inI sc in anti-CD3-injected Cftr−/− mice when comparedto that of the sham-injected Cftr−/− mice (0.3 ± 0.01versus 0.1 ± 0.01 μeq h−1cm2). Addition of forskolinresulted in an increase in I sc in Cftr+/+ mice and notin Cftr−/− mice (3.5 ± 0.2 versus 0.3 ± 0.01 μeq h cm2)(Fig. 3). These studies clearly indicate that increases

in I sc in anti-CD3-injected mice were due to increasedelectrogenic anion secretion via Cftr.

I sc was drastically reduced with Cftr−/− mice underall conditions in both control and anti-CD3-injectedmice and was unresponsive to cAMP. Positive controlexperiments were therefore undertaken by replacingglutamine with glucose at the end of each experimentfor ascertaining the viability of the tissue. Additionof d-glucose (10 mm) resulted in a robust increasein I sc (0.4 ± 0.03 versus 1.1 ± 0.05; n = 5), indicatinga functionally active and viable tissue activatingelectrogenic sodium-coupled glucose transport (SGLT1)(Supplemental Fig. 3).

Studies were then done using a pH stat techniqueto determine the role of Cftr in mediating luminal,Cl−-independent HCO3

− secretion in anti-CD3-injectedmice. In control Cftr−/− mice, the presence of Cl− inthe luminal solution resulted in a significant HCO3

−

secretion (Table 4). Addition of forskolin to the serosalsolution abolished HCO3

− secretion, indicating inhibitionof luminal Cl−-dependent HCO3

− secretion. This wasfurther confirmed in pH stat experiments performed in theabsence of luminal Cl−. Removal of Cl− from the luminalsolution abolished basal HCO3

− secretion in Cftr−/− mice.Addition of 10 μm forskolin to the serosal side failedto stimulate HCO3

− secretion in the absence of luminalCl− in Cftr−/− mice (Table 4). This indicates functionalCftr channels are essential for cAMP-stimulated HCO3

−

secretion.

0 20 40 60 80 100 1200.00

0.85

1.70

2.55

3.40

4.25

Isc(μ

eq

/h.c

m2)

Time (minutes)

Sham Cftr+/+

Sham Cftr-/-

AntiCD3 Cftr-/-

AntiCD3 Cftr+/+

Figure 3. A functional Cftr channel is necessary foranti-CD3-induced increase in Isc

Representative trace showing increased basal Isc in anti-CD3-injectedCftr+/+ mice compared to sham-injected Cftr+/+ mice. Addition offorskolin (black arrow) increased Isc in both sham-injected andanti-CD3 Cftr+/+ mice. Anti-CD3 injection and subsequent addition offorskolin failed to increase Isc in Cftr−/− mice. Experiments were doneon identical HCO3

−-containing buffered solution on both sides of thetissue.



Table 4. HCO3− secretion (μEq h−1 cm2) in sham and anti-CD3 mAb-injected Cftr−/−

mice

Sham-injected Cftr−/− Anti-CD3-injected Cftr−/−

Basal cAMP Basal cAMP

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 —

Experiments were done in the presence and absence of luminal Cl− and HCO3−.

Values represent mean ± S.E.M. n = 3. DIDS (100 μM) was added to the mucosal side inluminal Cl−-containing experiments and HCO3

−secretion compared to luminal Cl-freeexperiments. ∗P < 0.001, comparison between presence and absence of luminal Cl−

on HCO3− secretion. †P < 0.01, comparison between basal and cAMP-treated tissue.

ns = no significant difference between the groups.

Anti-CD3 injected Cftr−/− mice showed significantlylower HCO3

− secretion in the presence of luminalCl− compared to the sham-injected Cftr−/− mice(Table 4). This indicates that anti-CD3 abolishedluminal Cl−-dependent HCO3

− secretion. Lack ofHCO3

− secretion in the absence of luminal Cl− inanti-CD3-injected Cftr−/− mice indicates a failure tostimulate a luminal Cl−-independent HCO3

− secretion,which was thought to occur via functional Cftr. Thiswas further confirmed by addition of forskolin tothe bath solution in the absence of, luminal Cl− inanti-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-CD3injection inhibits electroneutral luminal Cl−-dependentHCO3

− secretion, and at the same time stimulates aHCO3

− secretion that was luminal Cl−-independent andmediated via a functional Cftr. Thus functional Cftrchannels are essential for electrogenic HCO3

− secretion

Figure 4. Western blot detection of Cftr in mucosal ileal lysatesfrom anti-CD3-injected and sham-injected miceEquivalent loads (15 μg) of protein from lysates prepared from CD3 orsham-treated mice were loaded and analysed by SDS PAGE.Immunoblots were probed with antibodies to Cftr and actin.Consistent with the appearance of rodent Cftr on Western blots ofmolecular mass 178–180 kDa, mature glycosylated Cftr (band C) andimmature form of Cftr (band B) are detected in both samples, withslight reduction in both mature and immature band forms in theanti-CD3-treated sample. Detection of actin in samples confirmsequivalent protein loading as shown.

in both anti-CD3-injected and cAMP-stimulated HCO3−

secretion.

Western blot for Cftr protein in anti-CD3 mAb-injected and normal mice

Increased I sc in anti-CD3-injected mice was inhibited byglibenclamide and was absent in Cftr−/− mice, indicatingthat functional Cftr channels are essential for increasedelectrogenic anion secretion. However, Western blotanalysis for Cftr protein did not show an increase butinstead showed a non-significant decrease (Fig. 4) inanti-CD3-injected mice, indicating that the increase in I sc

was not due to increased Cftr protein expression.

Colorimetric immunoassay for intracellular cAMP

Anti-CD3-injected mice showed a significant increase inbasal I sc and luminal Cl−-independent HCO3

− secretionthat was inhibited by NPPB and glibenclamide. Thisindicates that the anion channel mediating electrogenicanion secretion is probably Cftr. However, increasedelectrogenic activity did not reflect increased Cftr proteinexpression. Since Cftr channels are activated by cAMP, weasked if the increase in I sc and luminal Cl−-independentHCO3

− secretion was because of an increase inintracellular cAMP levels. As a control, it was firstestablished that forskolin treatment significantly increasedintracellular cAMP levels in crypt cells, villus cells andunfractionated cells (Fig. 5A–C). Anti-CD3-treated cryptcells did not show an increase in cAMP levels (Fig. 5A). Bycontrast, villus cells showed a significant increase in cAMPin anti-CD3-treated mice (Fig. 5B). These changes werealso reflected in unfractionated cells (Fig. 5C). Forskolindid not result in a significant increase in intracellular cAMPin either the anti-CD3-treated villus cells or unfractionatedcells. However, crypt cells showed a significant increasein intracellular cAMP in forskolin-treated anti-CD3 mice.These studies therefore indicate that anti-CD3-induced



changes in I sc may be mediated by an increase inintracellular cAMP resulting in a secondary activation ofCftr channels in villus cells. One implication of these resultsis that villus cells can contribute to anion secretion ininflamed tissue.

Discussion

During jejunal and ileal inflammation, it is widely acceptedthat absorption of nutrients, electrolytes and water byvillus cells is significantly decreased (Sundaram & West,1997b). In addition, fluid secretion by crypts may alsobe enhanced. Thus, the fine balance between absorptionand secretion is altered, resulting in fluid accumulationand the subsequent diarrhoea. Our present findings ofincreased I sc after anti-CD3 treatment suggests that fluidsecretion may indeed be increased during acute ilealinflammation. Increased I sc in anti-CD3 treated mice wasinhibited by 100 μm NPPB, a non-specific anion channelblocker, and 300 μm glibenclamide, a relatively specificCFTR blocker. To exclude an effect of glibenclamide onapical membrane K+ channels, 5 mm barium was added tothe luminal solution as shown previously (Vidyasagar et al.2004). 8-Br-cAMP-stimulated HCO3

− secretion measuredin the luminal Cl−-free solution, failed to show significantdifference in the presence 5 mm barium (4.7 ± 0.06 versus4.4 ± 0.08 μEq h−1 cm2, n = 4). These studies indicatethat anti-CD3 treatment results in an increased anionconductance in mice that probably arises via Cftr channels.However, the cAMP-stimulated I sc in anti-CD3 mice waslower when compared with normal mice (Table 2). Sincethe basal I sc was significantly higher in treated mice, Cftrchannels may already be in a state of maximal conductance,

A B C

0

600

1200

1800

2400

*

Sham ShamFSK Sham FSK

Sham AntiCD3 Anti-CD3

Sham FSK

pm

ol/m

g p

rote

in

Crypt

AntiCD3 AntiCD3 FSK

*

NS

0

400

800

1200

pm

ol/m

g p

rote

in

Anti-CD3 FSK

*

NS

Whole Fraction

#

0

200

400

600

800

AntiCD3 FSK

*$ NS

Villus

pm

ol/m

g p

rote

in

Figure 5. Colorimetric immunoassay for intracellular cAMPThe values are from n = 4 different mice repeated in triplicate. cAMP levels were standardized to protein levelsfrom respective fractions and expressed in pmol (mg protein)−1. Anti-CD3-treated crypt cells did not increasecAMP levels (A). Forskolin treatment significantly increased intracellular cAMP levels in crypt and villus cells andunfractionated cells from sham-injected and anti-CD3 mice (A–C). Anti-CD3 treatment resulted in a significantincrease in cAMP levels in unfractionated cells (C) and villus cells (B) but not in crypt cells (A). Addition of forskolin toanti-CD3-treated mice resulted in significant in crease in cAMP levels in crypt but not villus and unfractionated cells.Columns represent the mean values and bars show the S.E.M. ∗P < 0.001 compared with group after addition offorskolin; $P < 0.001 and #P < 0.02 comparison between sham- and anti-CD3-injected mice. NS = not significant(Bonferroni’s multiple comparisons).

resulting in an attenuated response to further stimulationby cAMP.

In contrast to our studies showing increased I sc

with anti-CD3 treatment, Musch et al. (2002) did notobserve significant alterations in jejunum I sc after asimilar anti-CD3 mAb treatment. Similarly, Musch et al.(2002) using the same model had shown a possibledefect in the epithelial barrier function. However, thetransepithelial measurement of conductance in Ussingchamber studies did not show an increase in conductancein anti-CD3-injected mice. These differing observationsmay reflect segmental heterogeneity and/or differences intissue-stripping techniques. In particular, our ileal tissuewas completely stripped of all muscle layers (Fig. 1A), thusensuring that any observed changes in I sc were mostly dueto responses from epithelial cells (Fig. 1B). Furthermore,compared with the ileum, fluid and electrolyte absorptionis significantly higher in the jejunum.

Also in the pH stat studies with a large serosal-to-mucosal gradient for HCO3

−, no bicarbonate secretionwas detected in the absence of luminal Cl−. This rulesout the possibility of an increased HCO3

− secretionthat could occur via the paracellular route due tothe large transepithelial HCO3

− gradient. If HCO3−

secretion occurred via a paracellular route, a luminalCl−-independent HCO3

− secretion should have beendetected in anti-CD3-treated mice. Our studies thereforeshow no evidence for increased barrier permeability inanti-CD3-injected mice.

As Cl− secretion is the driving force for fluid secretionin the small intestine, the increased Cl− secretion duringacute inflammation may be an adaptive measure to flushthe toxins and/or the elevated proinflammatory cytokines



from the lumen. In addition to Cl− secretion, net fluidsecretion, which is associated with most acute and chronicclinical diarrhoeas and those induced experimentally invivo, also contains HCO3

−-rich, plasma-like solution(Fordtran, 1967). Under normal physiological states, thesmall 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 manybiological functions. Pathophysiological conditions suchas acute severe diarrhoea and chronic persistent diarrhoea,result in significant HCO3

− loss and often lead to metabolicacidosis.

Our present findings in the normal mice ileumdemonstrates that HCO3

− secretion is luminal Cl−

dependent and DIDS sensitive, indicating that ilealCl−–HCO3

− activity is mediated by AE and is electro-neutral. However, during acute inflammation, HCO3

−

secretion was significantly decreased. The subsequentremoval of luminal Cl− did not completely abolishHCO3

− secretion in anti-CD3-treated mice. Furthermore,the Cl−-independent HCO3

− secretion was blockedby glibenclamide but insensitive to DIDS (Table 3).Thus, during acute inflammation an electrogenic HCO3

−

component is upregulated, and this is probably mediatedby Cftr channels. Similar experiments done usinganti-CD3-injected Cftr−/− mice showed minimal HCO3

−

secretion when compared to that in sham-injected Cftr+/+

mice, indicating the role of Cftr channels in mediatingCl−-independent HCO3

− secretion (Table 4). Indeed,CFTR has been shown to be permeable to HCO3

− in avariety 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-expressingcells, HCO3

− secretion was not enhanced by increases incAMP, suggesting that CFTR does not conduct HCO3

−

(Shumaker et al. 1999; Soleimani & Ulrich, 2000). Inthe present studies, increases in cAMP levels enhancedluminal Cl−-independent HCO3

− secretion in bothnormal and anti-CD3-treated mice, and at the sametime inhibited luminal Cl−-dependent HCO3

− secretion.Similar findings with cAMP were reported in the ratdistal colon (Vidyasagar et al. 2004; Vidyasagar et al.2005). However, the cAMP-stimulated increase in totalHCO3

− secretion in anti-CD3 mAb-injected mice wassignificantly lower than that in normal mice (Table 3).The HCO3

− transport studies correlated with the I sc

data and support the fact that HCO3− contributes to net

anion secretion seen during inflammation. Thus, one ofthe major transport alterations during inflammation isinhibition of electroneutral Cl−–HCO3

− exchange andstimulation of electrogenic anion secretion which includesluminal Cl−-independent HCO3

− secretion.Increased anion conductance during acute

inflammation may result from increased Cftr expression,

increased recruitment to the membrane, and/or anincreased conductance, possibly via stimulation ofintracellular cAMP levels. We used Western blot analysisof mucosal tissue from control and anti-CD3-injectedmice to rule out increased Cftr protein levels (Figs 4,n = 6), though this approach does not rule outmobilization of Cftr from intracellular organelles. Cftrprotein levels were not increased in anti-CD3-injectedmice. Changes in protein levels might be a late responseto inflammation as a 3 h anti-CD3 treatment time may beinsufficient for translation of mRNA and post-translationof protein to sufficiently increase Cftr protein level inthe cell and its express on the membrane. Changes withacute inflammation may not represent changes seenwith chronic inflammation. This could also explainthe increased CFTR expression reported in humancrypts during chronic ulcerative colitis (Sundaram &West, 1997a), and may reflect an effect of long-terminflammation on modifications on protein expression.In addition, hyperproliferated mouse colonic cryptsexhibited increased cAMP-stimulated Cl− secretion andCftr expression (Umar et al. 2000).

However, an immunoassay for intracellular cAMPshowed significant increases in basal cAMP levels inboth villus cell and unfractionated cells isolated fromanti-CD3-treated mice. Similar changes were not observedin a crypt cell fraction (Fig. 5A). Addition of forskolinto villus and unfractionated cells of anti-CD3-treatedmice did not result in a further increase in intra-cellular cAMP level, while crypt cell fractions on theother hand showed significantly increased intracellularcAMP levels in anti-CD3-treated mice. These studiestherefore indicate that increased anion secretion seenwith anti-CD3 treatment may result from increasedintracellular cAMP. These observations correlate wellwith our electrophysiology data, where forskolin didnot result in further increase in I sc in anti-CD3-treatedmice, as opposed to the sham-treated mice (Table 2).Although cAMP-stimulated anion secretion is associatedwith crypt cells under normal conditions (Barrett & Keely,2000), the present studies show that villus cells may alsocontribute to anion secretion in both forskolin-stimulatedand anti-CD3-treated mice. This may reflect a pre-existingpool of unutilized Cftr in ileal villus cells under normalconditions.

A family of anion exchangers (AEs) including apicalAE1 and AE3 and basolateral AE2 has been describedin the small intestine (Alper et al. 1999, 2002; Simpsonet al. 2005; Wang et al. 2005; Tuo et al. 2006). Themajority of the studies investigating AEs in the smallintestine have focused on the duodenum, with relativelylittle information in the ileum. Furthermore, functionalstudies using pH stat and Ussing chamber techniquescannot accurately delineate between the different types ofHCO3

− secretory mechanisms. Thus, the exact identity



of the ileal HCO3− transporter(s) modulating HCO3

−

movement remains to be resolved. The SLC26A3 gene ismutated in congenital chloride diarrhoea (CLD) (Hoglundet al. 1996). CLD is characterized by defects in intestinalHCO3

− secretion and massive loss of Cl− in the stool.These defects result in acidic stool and systemic metabolicalkalosis. Cl− loss along with acidic stool increases theelectrochemical gradient for protons, and secondarilyimpairs the intestinal absorption of Na+ resulting in lossof both sodium and chloride in the stool (Holmberg et al.1975). SLC26A3 has been observed in villus and surfaceepithelium of ileum and colon, respectively, and is thoughtto mediate Na+-independent Cl−–OH− and Cl−–HCO3

−

exchange (Hoglund et al. 1996; Melvin et al. 1999; Moseleyet al. 1999).

Taken together, these studies demonstrate that acuteinflammation induces a host of complex changes atthe cellular and molecular levels. In particular, theincreased anion secretion together with a shift fromelectroneutral to electrogenic process may represent anadaptive mechanism to ‘flush’ harmful bacteria and/orthe circulating pro-inflammatory cytokines in the ilealmucosa. Thus, our studies indicate that acute ilealinflammation downregulates electroneutral Cl−–HCO3

−

exchange, simultaneously inducing an electrogenic anionsecretion that includes HCO3−. These alterationsin epithelial electrolyte movement lead to decreasedabsorption of electrolytes with a concomitant increasein anion secretion resulting in diarrhoea. Further studiesare, however, needed to resolve the complex interactionbetween inflammatory mediators and altered epithelialtransport.

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Acknowledgements

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.

Supplemental material

Online supplemental material for this paper can be accessed at:

http://jp.physoc.org/cgi/content/full/jphysiol.2007.129262/DC1

and

http://www.blackwell-synergy.com/doi/suppl/10.1113/jphysiol.

2007.129262


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