Search

Aims The apical membrane anion exchanger putative anion transporter-1 (Pat-1) is expressed at significant levels in the lower villus epithelium of murine duodenum. However, previous studies of Cl(−)/HCO₃(−) exchange in the lower villus have failed to demonstrate Pat-1 function. Those studies routinely included luminal glucose which induces Na(+) -coupled glucose transport and acidifies the villus epithelium. Since Pat-1 has been proposed to be an electrogenic 1Cl(−)/2HCO₃(−) exchanger, membrane depolarization or cell acidification during glucose transport may obscure Pat-1 activity. Therefore, we investigated the effects of luminal glucose on Cl(−)(IN)/HCO₃(−) (OUT) exchange activity in the lower villus epithelium. Methods Cl(−)(IN) /HCO (−) (OUT) exchange of villus epithelium in duodenal mucosa from Pat-1 knockout (KO), Slc26a3 [down-regulated in adenoma (Dra)] KO, cystic fibrosis transmembrane conductance regulator (Cftr) KO and wild-type (WT) littermate mice was measured using the pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. Short-circuit current (I(sc) ) was measured in Ussing chambers. Results During glucose absorption, Cl(−)(IN)/HCO₃(−) (OUT) exchange in the lower villus epithelium was abolished in the Dra KO and unaffected in the Pat-1 KO relative to WT. However, during electroneutral mannose absorption or electrogenic α-D-methyl glucoside absorption, Cl(−)(IN) /HCO₃(−) (OUT) exchange was reduced in both Pat-1 KO and Dra KO villi. Exposure to high [K(+)] abolished Cl(−)(IN) /HCO₃(−) (OUT) exchange in the Dra KO but not the Dra/Cftr double KO epithelium, suggesting that Pat-1 activity is little affected by membrane depolarization except in the presence of Cftr. Conclusions The metabolic and electrogenic activity of glucose transport obscures Cl(−)(IN) /HCO₃(−) (OUT) exchange activity of Pat-1 in the lower villus. The inhibitory effects of membrane depolarization on Pat-1 Cl(−)(IN) /HCO₃(−) (OUT) exchange may require concurrent membrane association with Cftr.

Cystic fibrosis is caused by mutations in CFTR, the cystic fibrosis transmembrane conductance regulator gene. Disruption of CFTR-mediated anion conductance results in defective fluid and electrolyte movement in the epithelial cells of organs such as the pancreas, airways and sweat glands, but the function of CFTR in salivary glands is unclear. Salivary gland acinar cells produce an isotonic, plasma-like fluid, which is subsequently modified by the ducts to produce a hypotonic, NaCl-depleted final saliva. In the present study we investigated whether submandibular salivary glands (SMGs) in ΔF508 mice (CftrΔF/ΔF) display ion transport defects characteristic of cystic fibrosis in other tissues. Immunolocalization and whole-cell recordings demonstrated that Cftr and the epithelial Na+ (ENaC) channels are co-expressed in the apical membrane of submandibular duct cells, consistent with the significantly higher saliva [NaCl] observed in vivo in CftrΔF/ΔF mice. In contrast, Cftr and ENaC channels were not detected in acinar cells, nor was saliva production affected in CftrΔF/ΔF mice, implying that Cftr contributes little to the fluid secretion process in the mouse SMG. To identify the source of the NaCl absorption defect in CftrΔF/ΔF mice, saliva was collected from ex vivo perfused SMGs. CftrΔF/ΔF glands secreted saliva with significantly increased [NaCl]. Moreover, pharmacological inhibition of either Cftr or ENaC in the ex vivo SMGs mimicked the CftrΔF/ΔF phenotype. In summary, our results demonstrate that NaCl absorption requires and is likely to be mediated by functionally dependent Cftr and ENaC channels localized to the apical membranes of mouse salivary gland duct cells.

Ischemic injury induces breakdown of the intestinal barrier. Recent studies in porcine postischemic tissues indicate that inhibition of NHE2 results in enhanced recovery of barrier function in vitro via a process involving interepithelial tight junctions. To further study this process, recovery of barrier function was assessed in wild-type (NHE2+/+) and NHE2−/− mice in vivo and wild-type mice in vitro. Mice were subjected to complete mesenteric ischemia in vivo, after which barrier function was measured by blood-to-lumen mannitol clearance over a 3-h recovery period or measurement of transepithelial electrical resistance (TER) in Ussing chambers immediately following ischemia. Tissues were assessed for expression of select junctional proteins. Compared with NHE2+/+ mice, NHE2−/− mice had greater intestinal permeability during the postischemic recovery process. In contrast to prior porcine studies, pharmacological inhibition of NHE2 in postischemic tissues from wild-type mice also resulted in significant reductions in TER. Mucosa from NHE2−/− mice displayed a shift of occludin and claudin-1 expression to the Triton-X-soluble membrane fractions and showed disruption of occludin and claudin-1 localization patterns following injury. This was qualitatively and quantitatively recovered in NHE2+/+ mice compared with NHE2−/− mice by the end of the 3-h recovery period. Serine phosphorylation of occludin and claudin-1 was downregulated in NHE2−/− postischemia compared with wild-type mice. These data indicate an important role for NHE2 in recovery of barrier function in mice via a mechanism involving tight junctions.

Basal HCO3− secretion across the duodenum has been shown in several species to principally involve the activity of apical membrane Cl−/HCO3− exchanger(s). To investigate the identity of relevant anion exchanger(s), experiments were performed using wild-type (WT) mice and mice with gene-targeted deletion of the following Cl−/HCO3− exchangers localized to the apical membrane of murine duodenal villi: Slc26a3 [down-regulated in adenoma (DRA)], Slc26a6 [putative anion transporter 1 (PAT-1)], and Slc4a9 [anion exchanger 4 (AE4)]. RT-PCR of the isolated villous epithelium demonstrated PAT-1, DRA, and AE4 mRNA expression. Using the pH-sensitive dye BCECF, anion exchange rates were measured across the apical membrane of epithelial cells in the upper villus of the intact duodenal mucosa. Under basal conditions, Cl−/HCO3− exchange activity was reduced by 65–80% in the PAT-1(−) duodenum, 30–40% in the DRA(−) duodenum, and 42−/HCO3− exchange was eliminated in the PAT-1(−) duodenum but was not affected in the DRA(−) and AE4(−) duodenum relative to the WT duodenum. Intracellular pH (pHi) was reduced in the PAT-1(−) villous epithelium but increased to WT levels in the absence of CO2/HCO3− or during methazolamide treatment. Further experiments under physiological conditions indicated active pHi compensation in the PAT-1(−) villous epithelium by combined activities of Na+/H+ exchanger 1 and Cl−-dependent transport processes at the basolateral membrane. We conclude that 1) PAT-1 is the major contributor to basal Cl−/HCO3− and SO42−/HCO3− exchange across the apical membrane and 2) PAT-1 plays a role in pHi regulation in the upper villous epithelium of the murine duodenum.

The NBC1 Na+/HCO3- cotransporter is expressed in many tissues, including kidney and intestinal epithelia. NBC1 mutations cause proximal renal tubular acidosis in humans, consistent with its role in HCO3- absorption in the kidney. In intestinal and colonic epithelia, NBC1 localizes to basolateral membranes and is thought to function in anion secretion. To test the hypothesis that NBC1 plays a role in transepithelial HCO3- secretion in the intestinal tract, null mutant (NBC1-/-) mice were prepared by targeted disruption of its gene (Slc4a4). NBC1-/- mice exhibited severe metabolic acidosis, growth retardation, reduced plasma Na+, hyperal-dosteronism, splenomegaly, abnormal dentition, intestinal obstructions, and death before weaning. Intracellular pH (pH(i)) was not altered in cAMP-stimulated epithelial cells of NBC1-/- cecum, but pH(i) regulation during sodium removal and readdition was impaired. Bioelectric measurements of NBC1-/- colons revealed increased amiloride-sensitive Na+ absorption. In Ringer solution containing both Cl- and HCO3-, the magnitude of cAMP-stimulated anion secretion was normal in NBC1-/- distal colon but increased in proximal colon, with the increase largely supported by enhanced activity of the basolateral NKCC1 Na+-K+-2Cl- cotransporter. Anion substitution studies in which carbonic anhydrase was inhibited and transepithelial anion conductance was limited to HCO3- revealed a sharp decrease in both cAMP-stimulated HCO3- secretion and SITS-sensitive current in NBC1-/- proximal colon. These results are consistent with the known function of NBC1 in HCO3- absorption in the kidney and demonstrate that NBC1 activity is a component of the basolateral mechanisms for HCO3- uptake during cAMP-stimulated anion secretion in the proximal colon.

Genistein, a naturally occurring isoflavone, augments in vitro epithelial anion transport via activation of the cystic fibrosis transmembrane conductance regulator chloride channel. In this study, we examined whether chronic dietary exposure to 600 mg/kg genistein (600 G) for 1 mo would stimulate anion secretion across wild-type (Wt, normal) murine intestine. Anion secretion was assessed in freshly excised segments of murine jejuna by measuring short circuit current (I(sc)) and comparing with jejunal segments from mice fed 0 mg/kg genistein (0 G). Basal and forskolin-stimulated anion secretions were augmented (P < 0.05) in female but not in male mice fed 600 G, compared with their counterparts fed 0 G. Serum genistein concentrations were greater in both female and male mice fed 600 G (approximately 3.5-6.9 micromol/L) than those fed 0 G (approximately 100 nmol/L). Anion substitution experiments and bumetanide-sensitivity demonstrated that chloride was the major anion mediating the increased secretion. A smaller bicarbonate component was not augmented by consumption of the genistein diet. These data indicate that chronic exposure to dietary genistein stimulates a sex-dependent increase in basal and forskolin-stimulated chloride secretion across murine intestine.

Villi of the proximal duodenum are situated for direct exposure to gastric acid chyme. However, little is known about active bicarbonate secretion across villi that maintains the protective alkaline mucus barrier, a process that may be compromised in cystic fibrosis (CF), i.e., in the absence of a functional CF transmembrane conductance regulator (CFTR) anion channel. We investigated Cl−/HCO3−exchange activity across the apical membrane of epithelial cells located at the midregion of villi in intact duodenal mucosa from wild-type (WT) and CF mice using the pH-sensitive dye BCECF. Under basal conditions, the Cl−/HCO3−exchange rate was reduced by ∼35% in CF compared with WT villous epithelium. Cl−/HCO3−exchange in WT and CF villi responded similarly to inhibitors of anion exchange, and membrane depolarization enhanced rates of Cl−out/HCO3−inexchange in both epithelia. In anion substitution studies, anionin/HCO3−outexchange rates were greater in WT epithelium using Cl−or NO3−, but decreased to the level of the CF epithelium using the CFTR-impermeant anion, SO42−. Similarly, treatment of WT epithelium with the CFTR-selective blocker glybenclamide decreased the Cl−/HCO3−exchange rate to the level of CF epithelium. The mRNA expression of Slc26a3 (downregulated in adenoma) and Slc26a6 (putative anion exchanger-1) was similar between WT and CF duodena. From these studies of murine duodenum, we conclude 1) characteristics of Cl−/HCO3−exchange in the villous epithelium are most consistent with Slc26a6 activity, and 2) Cl−channel activity of CFTR facilitates apical membrane Cl−in/HCO3−outexchange by providing a Cl−“leak” under basal conditions.

The major disease-causing mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of phenylalanine 508 (DeltaF508), which adversely affects processing and plasma membrane targeting of CFTR. Under conditions predicted to stabilize protein folding, DeltaF508 CFTR is capable of trafficking to the plasma membrane and retains cAMP-regulated anion channel activity. Overexpression is one factor that increases CFTR trafficking; therefore, we hypothesized that expression of a domain mimic of the first nucleotide-binding fold (NBF1) of CFTR, i.e., the site of F508, may be sufficient to overwhelm the quality control process or otherwise stabilize DeltaF508 CFTR and thereby restore cAMP-stimulated anion secretion. In epithelial cells expressing recombinant DeltaF508 human (h)CFTR, expression of wild-type NBF1 increased the amount of both core-glycosylated and mature protein to a greater extent than expression of DeltaF508 NBF1. Expression of wild-type NBF1 in the DeltaF508 hCFTR cells increased whole cell Cl(-) current density to approximately 50% of that in cells expressing wild-type hCFTR. Expression of NBF1 in polarized epithelial monolayers from a DeltaF508/DeltaF508 cystic fibrosis mouse (MGEF) restored cAMP-stimulated transepithelial anion secretion but not in monolayers from a CFTR-null mouse (MGEN). Restoration of anion secretion was sustained in NBF1-expressing MGEF for30 passages, whereas MGEN corrected with hCFTR progressively lost anion secretion capability. We conclude that expression of a NBF1 domain mimic may be useful for correction of the DeltaF508 CFTR protein trafficking defect in cystic fibrosis epithelia.

Studies of full-thickness, small intestinal preparations have shown that maximal anion secretion [indexed by short-circuit current ( Isc)] during intracellular cAMP (cAMPi) stimulation is transient and followed by a decline toward baseline. Declining Isc is preceded by decreases in transepithelial conductance (Gt), which in the small intestine reflects the lateral intercellular space (LIS) volume of the paracellular pathway. We hypothesized that decreases in LIS volume limit the magnitude and duration of cAMPi-stimulated anion secretion. Experimental manipulations to increase the patency of the LIS (assessed by Gt and electron microscopy) were investigated for an effect on the magnitude of cAMPi-stimulated anion secretion (assessed by the Isc and isotopic fluxes) across murine small intestine. In control studies, changes of Gt after cAMPi stimulation were associated with a morphological “collapse” of the LIS, which did not occur in intestine of CFTR-null mice. Removal of the outer intestinal musculature, exposure to a serosal hypertonic solution, or increased serosal hydrostatic pressure minimized reductions in Gt and increased the cAMPi-stimulated Isc response. Increased Isc primarily resulted from increased Cl− secretion that was largely bumetanide sensitive. However, bumetanide-insensitive Isc was also increased, and similar increases occurred in the Na+-K+-2Cl− cotransporter (NKCC1)-null intestine, indicating that activities of non-NKCC1 anion uptake proteins are also affected by LIS volume. Thus LIS patency is an important determinant of the magnitude and duration of CFTR-mediated anion secretion in murine small intestine. Decreases in LIS volume may limit the pool of available anions to basolateral transporters involved in transepithelial secretion.

Background & Aims: Unlike the intestine of normal subjects, small-intestinal epithelia of cystic fibrosis patients and cystic fibrosis transmembrane conductance regulator protein-null (CFTR − ) mice do not respond to stimulation of intracellular cyclic adenosine monophosphate with inhibition of electroneutral NaCl absorption. Because CFTR-mediated anion secretion has been associated with changes in crypt cell volume, we hypothesized that CFTR-mediated cell volume reduction in villus epithelium is required for intracellular cyclic adenosine monophosphate inhibition of Na + /H + exchanger (primarily Na + /H + exchanger 3) activity in the proximal small intestine. Methods: Transepithelial 22 Na flux across the jejuna of CFTR + , CFTR − , the basolateral membrane Na + /K + /2Cl − co-transporter protein NKCC1 + , and NKCC1 − mice were correlated with changes in epithelial cell volume of the midvillus region. Results: Stimulation of intracellular cyclic adenosine monophosphate resulted in cessation of Na + /H + exchanger-mediated Na + absorption (J ms NHE ) in CFTR + jejunum but had no effect on J ms NHE across CFTR − jejunum. Cell volume indices indicated an approximately 30% volume reduction of villus epithelial cells in CFTR + jejunum but no changes in CFTR − epithelium after intracellular cyclic adenosine monophosphate stimulation. In contrast, cell shrinkage induced by hypertonic medium inhibited J ms NHE in both CFTR + and CFTR − mice. Bumetanide treatment to inhibit Cl − secretion by blockade of the Na + /K + /2Cl − co-transporter, NKCC1, of stimulated CFTR + jejunum prevented maximal volume reduction of villus epithelium and recovered approximately 40% of J ms NHE . Likewise, J ms NHE and cell volume were unaffected by intracellular cyclic adenosine monophosphate stimulation in NKCC1 − jejuna. Conclusions: These findings show a previously unrecognized role of functional CFTR expressed in villus epithelium: regulation of Na + /H + exchanger 3-mediated Na + absorption by alteration of epithelial cell volume.

NHE3 is one of five plasma membrane Na+/H+ exchangers and is encoded by the mouse gene Slc9a3. It is expressed on apical membranes of renal proximal tubule and intestinal epithelial cells and is thought to play a major role in NaCl and HCO3- absorption. As the distribution of NHE3 overlaps with that of the NHE2 isoform in kidney and intestine, the function and relative importance of NHE3 in vivo is unclear. To analyse its physiological functions, we generated mice lacking NHE3 function. Homozygous mutant (Slc9a3-/-) mice survive, but they have slight diarrhoea and blood analysis revealed that they are mildly acidotic. HCO3- and fluid absorption are sharply reduced in proximal convoluted tubules, blood pressure is reduced and there is a severe absorptive defect in the intestine. Thus, compensatory mechanisms must limit gross perturbations of electrolyte and acid-base balance. Plasma aldosterone is increased in NHE3-deficient mice, and expression of both renin and the AE1 (Slc4a1) Cl-/HCO3- exchanger mRNAs are induced in kidney. In the colon, epithelial Na+ channel activity is increased and colonic H+,K+-ATPase mRNA is massively induced. These data show that NHE3 is the major absorptive Na+/H+ exchanger in kidney and intestine, and that lack of the exchanger impairs acid-base balance and Na+-fluid volume homeostasis.

Extracellular ATP applied to the luminal side of human airway epithelium (HAE) activates an apical membrane Cl- conductance and transepithelial Cl- secretion. However, in some HAE preparations, we have found that luminal ATP induces a change in short-circuit current (Isc), consistent with K+ secretion. Using intracellular microelectrodes and radioisotopic flux studies, we investigated whether extracellular ATP regulates transepithelial K+ secretion in primary HAE cultures. In physiological Ringer solution, HAE had a negligible electrochemical driving force for Cl- secretion (DFCl), and luminal ATP induced a change in Isc opposite in polarity to Cl- secretion. Intracellular microelectrode measurements indicated that the "reversed" Isc was associated with activation of a hyperpolarizing (K+) conductance in the apical membrane. Radioisotope studies of HAE pretreated with amiloride to induce a favorable DFCl revealed that luminal ATP stimulates a small 42K secretory flux concurrently with Cl- secretion. In ion-substituted Ringer solution, luminal ATP stimulated both the outward (K+) current and the inward (Cl-) current with approximately equal potency (approximately 10(-6) M). We conclude that luminal ATP activates an apical membrane K+ conductance and transepithelial K+ secretion across HAE.

Although loss of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl- channel function is common to all epithelia in cystic fibrosis (CF) patients, the severity of disease varies in different organs. We hypothesized that differences in disease severity in CF relate to the expression of an "alternative" plasma membrane Cl- conductance. In CF mice [Cftr(-/-); mice homozygous for Ser-489 to Xaa mutation], which do not express cAMP CFTR-mediated Cl- secretion, we surveyed organs that exhibit a range of disease severity for a Ca(2+)-mediated apical membrane epithelial Cl- conductance. This alternative conductance (Cl-a) was detected in epithelia of organs from CF mice that exhibit a mild disease phenotype (airway, pancreas) but not in epithelia with a severe phenotype (small, large intestine). We conclude that (i) there is an intracellular Ca(2+)-regulated Cl- conductance that is molecularly distinct from CFTR; and (ii) the level of expression of this alternative Cl- conductance in the epithelium is an important determinant of the severity of organ-level disease in CF.

The utility of a transgenic murine model of cystic fibrosis (CF) lung disease will likely depend on whether the mouse's proximal airway epithelium is characterized by Na(+)- and Cl(-)-conductive pathways comparable to those found in human airways. Therefore, the electrophysiological properties of primary cultures of mouse tracheal epithelium (MTE) were investigated using double-barreled, Cl(-)-selective microelectrodes. Epithelial cells isolated from freshly excised mouse tracheae formed confluent polarized monolayers on permeable collagen supports and developed significant transepithelial potential differences (approximately -10 mV) within 5-6 days postseeding. Under basal conditions, the MTE monolayers had an equivalent short-circuit current (Ieq) of -21.1 +/- 2.1 microA/cm2 and a transepithelial resistance of 424 +/- 49 omega.cm2. Intracellular measurements indicated that the apical (Va) and basolateral (Vb) membrane potential differences were -16.9 +/- 1.5 and -25.4 +/- 1.5 mV, respectively; apical membrane fractional resistance was 0.36 +/- 0.03; and intracellular Cl- activity was 56.1 +/- 2.3 mM. The presence of an apical Na+ conductance was demonstrated by luminal amiloride application (10(-4)M), which decreased Ieq, hyperpolarized Va, and increased the fractional resistance of the apical membrane. The presence of an apical Cl- conductance was demonstrated by substitution of Cl- with gluconate in the luminal bath, which decreased intracellular Cl- activity and increased the fractional resistance of the apical membrane. Luminal application of ATP (10(-4) M was also found to increase the rate of Cl- secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes an adenosine 39, 59-monophosphate (cyclic AMP)—activated chloride channel. In cystic fibrosis (CF) patients, loss of CFTR function because of a genetic mutation results in defective cyclic AMP—mediated chloride secretion across epithelia. Because of their potential role as an animal model for CF, mice with targeted disruption of the murine CFTR gene [CFTR(-/-)] were tested for abnormalities in epithelial chloride transport. In both freshly excised tissue from the intestine and in cultured epithelia from the proximal airways, the cyclic AMP—activated chloride secretory response was absent in CFTR(-/-) mice as compared to littermate controls. Thus, disruption of the murine CFTR gene results in the chloride transport abnormalities predicted from studies of human CF epithelia.

Chloride secretion across cystic fibrosis (CF) airway epithelia is effectively regulated by pathways associated with intracellular Ca2+ metabolism, but not by mechanisms dependent on protein kinase A or C. In a search for therapeutically useful agonists, we investigated the effects of extracellular ATP on the Cl- secretory process in human normal and CF nasal epithelial cultures with double-barreled Cl- selective microelectrodes. When applied to the basolateral membrane of normal, but not CF, nasal epithelium, extracellular ATP (10(-4) M) stimulated a small increase in Cl- secretion that was primarily associated with a hyperpolarizing conductance in the basolateral membrane. In contrast, ATP applied to the apical (luminal) membrane of either normal or CF nasal epithelium stimulated a greater increase in Cl- secretion that was associated with activation of an apical membrane Cl- conductance. The increases in Cl- current and apical conductance were greater in CF tissues and attained maximal values similar to normal nasal epithelium. We conclude 1) that basolateral application of ATP indirectly induces Cl- secretion by activating a basolateral (K+) conductance and is an effective secretagogue only in normal nasal epithelium and 2) that luminally applied ATP is an effective Cl- secretagogue that activates the apical membrane Cl- conductance of normal and CF nasal epithelia to an equivalent level.

In cystic fibrosis, impaired secretion resulting from loss of activity of the cystic fibrosis transmembrane conductance regulator (CFTR) causes dehydration of intestinal contents and life-threatening obstructions. Conversely, impaired absorption resulting from loss of the NHE3 Na+/H+ exchanger causes increased fluidity of the intestinal contents and diarrhea. To test the hypothesis that reduced NHE3-mediated absorption could increase survival and prevent some of the intestinal pathologies of cystic fibrosis, Cftr/Nhe3 double heterozygous mice were mated and their offspring analyzed. Cftr-null mice lacking one or both copies of the NHE3 gene exhibited increased fluidity of their intestinal contents, which prevented the formation of obstructions and increased survival. Goblet cell hyperplasia was eliminated, but not the accumulation of Paneth cell granules or increased cell proliferation in the crypts. Microarray analysis of small intestine RNA from Cftr-null, NHE3-null, and double-null mice all revealed downregulation of genes involved in xenobiotic metabolism, including a cohort of genes involved in glutathione metabolism. Expression of energy metabolism genes was altered, but there were no changes in genes involved in inflammation. Total intracellular glutathione was increased in the jejunum of all of the mutants and the ratio of reduced to oxidized glutathione was reduced in Cftr-null mutants, indicating that CFTR deficiency affects intestinal glutathione metabolism. The data establish a major role for NHE3 in regulating the fluidity of the intestinal contents and show that reduced NHE3-mediated absorption reverses some of the intestinal pathologies of cystic fibrosis, thus suggesting that it may serve as a potential therapeutic target.

Cystic fibrosis is characterized by abnormal electrolyte transport across the epithelia of the airways. In particular, there is excessive sodium absorption and deficient chloride secretion. Drugs that block excessive sodium absorption may provide clinical benefit in cystic fibrosis, but there are no available therapeutic agents to improve chloride secretion. In vitro studies in cultured human-airway epithelia indicate that triphosphate nucleotides (ATP and UTP) induce chloride secretion through apical-membrane purinergic receptors.We tested the ability of nucleotides to induce chloride secretion in vivo in 9 normal subjects and 12 patients with cystic fibrosis by measuring responses of nasal transepithelial potential difference (PD) to superfusion of nucleotides. Changes in transepithelial bioelectric properties and the permeability of the apical membrane to chloride in response to extracellular (apical) UTP were determined with ion-selective microelectrodes in cultured nasal epithelia.ATP and UTP induced chloride secretion in vivo in both groups. At their maximal effective concentrations of 10(-4) M, ATP and UTP were more effective chloride secretagogues in the patients with cystic fibrosis (mean [+/- SE] change in PD, -19.8 +/- 1.4 mV and -15.0 +/- 1.7 mV, respectively) than in the normal subjects (-6.9 +/- 0.6 mV and -8.1 +/- 0.9 mV, respectively). Microelectrode studies established that extracellular UTP stimulated a larger increase in PD and chloride secretory current in epithelial cells from patients with cystic fibrosis than in cells from normal subjects, by actions localized to the apical membrane.Extracellular nucleotides are effective in vivo chloride secretagogues in the nasal epithelia of patients with cystic fibrosis. The equipotency of ATP and UTP suggests that the effect is mediated by P2 nucleotide receptors. Selected nucleotides, such as UTP or nucleotide analogues, should be investigated as therapeutic agents for lung disease in cystic fibrosis.

Background & Aims The current model of duodenal HCO 3 − secretion proposes that basal secretion results from Cl − /HCO 3 − exchange, whereas cyclic adenosine monophosphate (cAMP)-stimulated secretion depends on a cystic fibrosis transmembrane conductance regulator channel (Cftr)-mediated HCO 3 − conductance. However, discrepancies in applying the model suggest that Cl − /HCO 3 − exchange also contributes to cAMP-stimulated secretion. Of 2 candidate Cl − /HCO 3 − exchangers, studies of putative anion transporter-1 knockout (KO) mice find little contribution of putative anion transporter-1 to basal or cAMP-stimulated secretion. Therefore, the role of down-regulated in adenoma (Dra) in duodenal HCO 3 − secretion was investigated using DraKO mice. Methods Duodenal HCO 3 − secretion was measured by pH stat in Ussing chambers. Apical membrane Cl − /HCO 3 − exchange was measured by microfluorometry of intracellular pH in intact villous epithelium. Dra expression was assessed by immunofluorescence. Results Basal HCO 3 − secretion was reduced ∼55%–60% in the DraKO duodenum. cAMP-stimulated HCO 3 − secretion was reduced ∼50%, but short-circuit current was unchanged, indicating normal Cftr activity. Microfluorimetry of villi demonstrated that Dra is the dominant Cl − /HCO 3 − exchanger in the lower villous epithelium. Dra expression increased from villous tip to crypt. DraKO and wild-type villi also demonstrated regulation of apical Na + /H + exchange by Cftr-dependent cell shrinkage during luminal Cl − substitution. Conclusions In murine duodenum, Dra Cl − /HCO 3 − exchange is concentrated in the lower crypt-villus axis where it is subject to Cftr regulation. Dra activity contributes most basal HCO 3 − secretion and ∼50% of cAMP-stimulated HCO 3 − secretion. Dra Cl − /HCO 3 − exchange should be considered in efforts to normalize HCO 3 − secretion in duodenal disorders such as ulcer disease and cystic fibrosis.

The association of feeding practices with the development of digestive disorders in horses has long been recognized, although the underlying mechanisms had been barely considered. The physiologic consequences of meal frequency may help to explain the relationship and prove to be of major significance in the induction of many conditions. Many Equidae kept for performance and leisure activities are fed high-energy, low-forage rations twice daily, with limited access to hay or grazing. Rapid ingestion of such meals stimulates a copious outpouring of upper alimentary secretions and results in transient hypovolemia (15% plasma volume loss). Subsequent activation of the renin-angiotensin-aldosterone system (RAAS) contributes to the preservation of circulatory status. Large meals may accelerate digesta passage to the cecum and, thereby, increase soluble carbohydrate availability for large intestinal fermentation. Intense periods of fermentation develop that require significant shifts of fluid into the colonic lumen. This is followed by net fluid absorption, which, in part, is dependent on postprandial increases of aldosterone. Potential consequences of these events include (1) imbalances in the RAAS response, which may promote conditions favorable to gastrointestinal disturbance, notably large intestinal impaction, and (2) changes in the gastrointestinal microflora, which may affect the intraluminal endotoxin pool and the population of enterotoxin-producing bacteria. In contrast to episodic feedings, similar changes are absent or greatly attenuated under simulated grazing conditions (for example, small, frequent meals). Thus, modification of management practices to facilitate a more continuous feeding pattern may significantly reduce the incidence of digestive problems in the stabled horse.

Intestinal disease in cystic fibrosis (CF) mice closely mirrors aspects of obstructive syndromes in CF patients. The pathogenesis involves accumulation of mucoid debris in the crypts that fuse with intestinal content to form obstructing mucofeculant impactions. Treatment involves modalities that increase the fluidity of the luminal content, such as osmotic laxatives and liquid diets. We investigated the effects of talniflumate (Lomucin, Genaera Corporation, Plymouth Meeting, PA), a compound that may be beneficial to treatment of CF intestinal disease based on three mechanisms of action: mucus synthesis inhibition by blockade of the murine calcium-activated chloride channel 3 (mCLCA3), nonsteroidal anti-inflammatory effects, and inhibition of Cl(-)/HCO (-)(3) exchanger(s) involved in intestinal NaCl absorption. Cohorts of CF mice were fed control diet or diets containing either talniflumate (0.4 mg/g chow) or ibuprofen (0.4 mg/g chow) for 21 days to assess survival. Talniflumate significantly increased CF mouse survival from 26 to 77%, whereas ibuprofen had no effect (22% survival). Oral talniflumate did not alter crypt goblet cell numbers or change intestinal expression of mCLCA3 but tended to decrease crypt mucoid impaction. Ussing chamber studies indicated that talniflumate slightly increased the basal short-circuit current of CF intestine, but the change was not sensitive to secretagogue stimulation or bumetanide inhibition. In contrast, intracellular pH measurements of intact intestinal villous epithelium indicated that talniflumate significantly inhibited apical membrane Cl(-)/HCO (-)(3) exchange by >50%. We conclude that oral talniflumate increases the survival of CF mice, possibly by the beneficial effects of decreasing small intestinal NaCl absorption through the inhibition of apical membrane Cl(-)/HCO (-)(3) exchanger(s).

Until the mid 20th century, children with cystic fibrosis (CF) died at a young age from a combination of malnutrition and suppurative lung disease. In the past three to four decades, coincident with new treatments for pulmonary complications and the use of high-calorie diets without restrictions on fat intake, there has been marked and progressive improvement in lung function, nutritional status and survival. However, treatment for pancreatic insufficiency, which affects most individuals with CF, remains largely unchanged, and there have been few advances in our understanding and management of gastrointestinal problems in this population. Pancreatic Enzyme Replacement Therapy (PERT), principally using enzyme extracts of porcine origin, has been the mainstay for treating maldigestion resulting from pancreatic insufficiency (PI) for over a century. Powdered PERT was instituted before the 1931 creation of the United States Food and Drug Administration (FDA) and the subsequent Federal Food, Drug and Cosmetic Act of 1938 that required proof of safety and efficacy before approval of drugs for marketing. Enteric-coated microcapsules, the most commonly used PERT, have never been FDA approved. After the epidemic of fibrosing colonopathy in the early 1990s, a condition that was clearly related to high doses of exogenous pancreatic enzymes, the FDA initiated a process of re-evaluating the safety and efficacy of PERT. In 2004, the FDA released a draft of a new Guidance Document for public comment that stated that within the next 4 years, all manufacturers of current enzyme products will need to submit a New Drug Application to the FDA with proof of safety in manufacturing, stability and efficacy. The coefficient of fat absorption (CFA) is the traditional method of testing the clinical efficacy of PERT. CF clinicians have long recognized that there is a very wide range of CFA in patients with CF. Less well known is that there is marked variability in the actual amount of enzyme in each capsule. Capsules are overfilled so that the stated dosage reflects the least amount of enzyme activity present after a six-month shelf life. There is little correlation between CFA and enzyme dose when subjects are receiving a dose of PERT considered optimal by the caregiver and/or patient. Furthermore, a recent cross-sectional study showed no correlation between enzyme dose and growth or self-reported symptoms. Consequently, the common belief that PERT dose can be titrated to correct malabsorption and/or maldigestion using patient-reported relief of abdominal symptoms or stool bulk and consistency appears to be questionable. PERT may not completely correct PI even if given in adequate doses because the release and onset of activity is not synchronous with the presence of food in the proximal intestine, the site for optimal absorption. Furthermore, as discussed below, it is becoming evident that poorly understood nonpancreatic intestinal and/or hepatobiliary factors likely contribute to incomplete and variable nutrient assimilation in patients with CF. However, we lack tools to distinguish PERT-related factors that might contribute to treatment failure from other confounding factors. Ironically, as mouse models of CF were developed in the late 20th century, these animals did not develop lung or severe pancreatic manifestations. However, the mice have characteristic CF-like bowel obstruction and are proving to be excellent models for study of nonpancreatic gastrointestinal manifestations of CF. The combination of new ways to study gastrointestinal disease in CF that further our understanding of the pathophysiology of CF and the need to evaluate novel methods to study pancreatic and nonpancreatic causes of maldigestion and malabsorption led the CF Foundation to sponsor a workshop, which was held in Baltimore in May 2005. The goals of this workshop were to evaluate what is known about gastrointestinal issues leading to malabsorption, to consider other factors that may contribute to malabsorption and to identify ways to improve care of patients with CF. We set an agenda for further clinical and laboratory-based investigations that would help to elucidate the spectrum of causes of maldigestion and malabsorption. The workshop participants included clinicians and investigators with a variety of interests, members of the US and European regulatory agencies and individuals from industry who were involved in producing traditional PERT or developing novel PERT or diagnostic tests. © 2005 Lippincott Williams & Wilkins, Inc.

Estrogen receptor α (ERα) is essential for male fertility. Its activity is responsible for maintaining epithelial cytoarchitecture in efferent ductules and the reabsorption of fluid for concentrating sperm in the head of the epididymis. These discoveries and others have helped to establish estrogen's bisexual role in reproductive importance. Reported here is the molecular mechanism to explain estrogen's role in fluid reabsorption in the male reproductive tract. It is shown that estrogen regulates expression of the Na + /H + exchanger-3 (NHE3) and the rate of 22 Na + transport, sensitive to an NHE3 inhibitor. Immunohistochemical staining for NHE3, carbonic anhydrase II (CAII), and aquaporin-I (AQP1) was decreased in ERα knockout (αERKO) efferent ductules. Targeted gene-deficient mice were compared with αERKO, and the NHE3 knockout and CAII -deficient mice showed αERKO-like fluid accumulation, but only the NHE3 knockout and αERKO mice were infertile. Northern blot analysis showed decreases in mRNA for NHE3 in αERKO and antiestrogen-treated mice. The changes in AQP1 and CAII in αERKO seemed to be secondary because of the disruption of apical cytoarchitecture. Ductal epithelial ultrastructure was abnormal only in αERKO mice. Thus, in the male, estrogen regulates one of the most important epithelial ion transporters and maintains epithelial morphological differentiation in efferent ductules of the male, independent of its regulation of Na + transport. Finally, these data raise the possibility of targeting ERα in developing a contraceptive for the male.

Although abnormal hard tissue mineralization is a recognized complication of cystic fibrosis (CF), the pathogenesis leading from the defective cystic fibrosis transmembrane conductance regulator (CFTR) protein is poorly understood. We hypothesized that CFTR plays a direct role in the mineralization of bone and teeth and tested the hypothesis using CF mouse models [CFTR(-) mice]. In vivo measurements by dual-emission X-ray absorpitometry (DEXA) indicated that bone mineral density (BMD) was reduced in CF mice as compared to gender-matched littermates. However, no change was evident after correction of BMD for the covariant of body weight. The latter finding was confirmed in isolated femurs and nasal bones by standard dry-ashing and instrumental neutron activation analysis (INAA). INAA of the continuously growing hypsodont incisor teeth from CFTR(-) mice revealed reduced Ca and normal P in the enamel layer--a finding consistent with changes in the deciduous teeth of CF children. Interestingly, enamel fluoride was increased in the CFTR(-) incisors and may associate with abnormal enamel crystallite formation. The iron content of the incisor enamel was reduced, explaining the loss of yellow pigmentation in CFTR(-) incisors. In contrast to the incisors, the mineral content of the slow-growing brachydont molar teeth was not different between CFTR(-) and CFTR(+) mice. It was concluded that CFTR does not play a direct role in the mineralization of bones or brachydont teeth in mice. Functional CFTR is apparently required for normal mineralization of the hypsodont incisors. However, multiple changes in the mineral composition of the CF incisors suggest an indirect role for CFTR, perhaps by maintaining a normal salivary environment for continuous tooth eruption.

To investigate the role of the pro alpha 2(I) collagen chains of type I collagen in mineralization we used the oim (osteogenesis imperfecta model) mouse as our model system. The oim/oim mouse (homozygous for a null mutation in its COL1A2 gene of type I collagen) fails to synthesize functional pro alpha 2(I) collagen chains, synthesizing only homotrimers of pro alpha 1(I) collagen chains. To evaluate the role of pro alpha 2(I) collagen in type I collagen structure/function in mineralized tissues, we examined age-matched oim/oim, heterozygous (oim/+), and wild-type (+/+) mouse femurs and incisors for mineral composition (calcium, phosphorus, magnesium, fluoride, sodium, potassium, and chloride) by neutron activation analyses (NAA), and bone mineral content (BMC) and bone mineral density (BMD) by dual-energy X-ray absorptiometry (DEXA) in a longitudinal study (7 weeks to 16 months of age). NAA demonstrated that oim/oim femurs had significant differences in magnesium, fluoride, and sodium content as compared with +/+ mouse femurs, and oim/oim teeth had significant differences in magnesium content as compared to +/+ teeth. The ratio of calcium to phosphate was also significantly reduced in the oim/oim mouse femurs (1.58 +/- 0.01) compared with +/+ femurs (1.63 +/- 0.01). DEXA demonstrated that oim/oim mice had significantly reduced BMC and BMD as compared to oim/+ and +/+ mice. Serum and urine calcium, magnesium, and phosphorus levels, and Ca(47) absorption across the gut were equivalent in oim/oim and +/+ mice, with no evidence of hypercalciuria. These studies suggest that the known decreased biomechanical properties of oim/oim bone reflect both altered mineral composition as well as the decreased BMD, which further suggests that the presence of alpha2(I) chains plays an important role in mineralization.

The loss of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transepithelial HCO3 − secretion contributes to the pathogenesis of pancreatic and biliary disease in cystic fibrosis (CF) patients. Recent studies have investigated P2Y2 nucleotide receptor agonists, e.g., UTP, as a means to bypass the CFTR defect by stimulating Ca2+-activated Cl− secretion. However, the value of this treatment in facilitating transepithelial HCO3 − secretion is unknown. Gallbladder mucosae from CFTR knockout mice were used to isolate the Ca2+-dependent anion conductance during activation of luminal P2Y2receptors. In Ussing chamber studies, UTP stimulated a transient peak in short-circuit current ( I sc) that declined to a stable plateau phase lasting 30–60 min. The plateau I sc after UTP was Cl− independent, HCO3 − dependent, insensitive to bumetanide, and blocked by luminal DIDS. In pH stat studies, luminal UTP increased both I sc and serosal-to-mucosal HCO3 − flux ( J s→m) during a 30-min period. Substitution of Cl− with gluconate in the luminal bath to inhibit Cl−/HCO3 −exchange did not prevent the increase in J s→mand I sc during UTP. In contrast, luminal DIDS completely inhibited UTP-stimulated increases in J s→m and I sc. We conclude that P2Y2 receptor activation results in a sustained (30–60 min) increase in electrogenic HCO3 − secretion that is mediated via an intracellular Ca2+-dependent anion conductance in CF gallbladder.

In chloride-secretory epithelia, the basolateral Na-K-2Cl cotransporter (NKCC1) is thought to play a major role in transepithelial Cl(-) and fluid transport. Similarly, in marginal cells of the inner ear, NKCC1 has been proposed as a component of the entry pathway for K(+) that is secreted into the endolymph, thus playing a critical role in hearing. To test these hypotheses, we generated and analyzed an NKCC1-deficient mouse. Homozygous mutant (Nkcc1(-/-)) mice exhibited growth retardation, a 28% incidence of death around the time of weaning, and mild difficulties in maintaining their balance. Mean arterial blood pressure was significantly reduced in both heterozygous and homozygous mutants, indicating an important function for NKCC1 in the maintenance of blood pressure. cAMP-induced short circuit currents, which are dependent on the CFTR Cl(-) channel, were reduced in jejunum, cecum, and trachea of Nkcc1(-/-) mice, indicating that NKCC1 contributes to cAMP-induced Cl(-) secretion. In contrast, secretion of gastric acid in adult Nkcc1(-/-) stomachs and enterotoxin-stimulated fluid secretion in the intestine of suckling Nkcc1(-/-) mice were normal. Finally, homozygous mutants were deaf, and histological analysis of the inner ear revealed a collapse of the membranous labyrinth, consistent with a critical role for NKCC1 in transepithelial K(+) movements involved in generation of the K(+)-rich endolymph and the endocochlear potential.

Desensitization of P2Y2receptor-activated anion secretion may limit the usefulness of extracellular nucleotides in secretagogue therapy of epithelial diseases, e.g., cystic fibrosis (CF). To investigate the desensitization process for endogenous P2Y2receptors, freshly excised or cultured murine gallbladder epithelia (MGEP) were mounted in Ussing chambers to measure short-circuit current ( Isc), an index of electrogenic anion secretion. Luminal treatment with nucleotide receptor agonists increased the Iscwith a potency profile of ATP = UTP > 2-methylthioATP >> α,β-methylene-ATP. RT-PCR revealed the expression of P2Y2receptor mRNA in the MGEP cells. The desensitization of anion secretion required a 10-min preincubation with the P2Y2receptor agonist UTP and increased in a concentration-dependent manner (IC50≈ 10−6M). Approximately 40% of the anion secretory response was unaffected by maximal desensitizing concentrations of UTP. Recovery from UTP-induced desensitization was rapid (50(1.9 × 10−6M) but required progressively longer time periods at greater concentrations. UTP-induced total inositol phosphate production and intracellular Ca2+mobilization desensitized with a concentration dependence similar to that of anion secretion. In contrast, maximal anion secretion induced by Ca2+ionophore ionomycin was unaffected by preincubation with a desensitizing concentration of UTP. It was concluded that 1) desensitization of transepithelial anion secretion stimulated by the P2Y2receptor agonist UTP is time and concentration dependent; 2) recovery from desensitization is prolonged (>90 min) at UTP concentrations >10−5M; and 3) UTP-induced desensitization occurs before the operation of the anion secretory mechanism.

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in cAMP-stimulated HCO3- secretion across the murine duodenum was investigated. Serosal-to-mucosal flux of HCO3- (Js--m, in mu eq.cm-2.h-1) and short-circuit current (Isc; in mu eq.cm-2.h-1) were measured by the pH stat method in duodenum from CFTR knockout [CFTR(-)] and normal [CFTR(+)] mice. Under control conditions, forskolin increased Js--m and Isc (+1.7 and +3.5, respectively) across the CFTR(+) but not CFTR(-) duodenum. Both the forskolin-stimulated delta Js--m and delta Isc were abolished by the CFTR channel blocker 5-nitro-2-(3-phenylpropylamino)benzoate, whereas inhibition of luminal Cl-/HCO3- exchange by luminal Cl- removal or DIDS reduced the Js--m by approximately 18% without a consistent effect on the delta Isc. Methazolamide also reduced the Js--m by 39% but did not affect the delta Isc. When carbonic anhydrase-dependent HCO3- secretion was isolated by using a CO2-gassed, HCO3(-)-free Ringer bath, forskolin stimulated the Js--m and Isc (+0.7 and +2.0, respectively) across CFTR(+) but not CFTR(-) duodenum. Under these conditions, luminal Cl- substitution or DIDS abolished the Js--m but not the delta Isc. It was concluded that cAMP-stimulated HCO3- secretion across the duodenum involves 1) electrogenic secretion via a CFTR HCO3- conductance and 2) electroneutral secretion via a CFTR-dependent Cl-/HCO3- exchange process that is closely associated with the carbonic anhydrase activity of the epithelium.

Uroguanylin is an intestinal peptide hormone that may regulate epithelial ion transport by activating a receptor guanylyl cyclase on the luminal surface of the intestine. In this study, we examined the action of uroguanylin on anion transport in different segments of freshly excised mouse intestine, using voltage-clamped Ussing chambers. Uroguanylin induced larger increases in short-circuit current (Isc) in proximal duodenum and cecum compared with jejunum, ileum, and distal colon. The acidification of the lumen of the proximal duodenum (pH 5.0-5.5) enhanced the stimulatory action of uroguanylin. In physiological Ringer solution, a significant fraction of the Isc stimulated by uroguanylin was insensitive to bumetanide and dependent on HCO3- in the bathing medium. Experiments using pH-stat titration revealed that uroguanylin stimulates serosal-to-luminal HCO3- secretion (Js--lHCO3-) together with a larger increase in Isc. Both Js--lHCO3- and Isc were significantly augmented when luminal pH was reduced to pH 5.15. Uroguanylin also stimulated the Js--lHCO3- and Isc across the cecum, but luminal acidity caused a generalized decrease in the bioelectric responsiveness to agonist stimulation. In cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice, the duodenal Isc response to uroguanylin was markedly reduced, but not eliminated, despite having a similar density of functional receptors. It was concluded that uroguanylin is most effective in acidic regions of the small intestine, where it stimulates both HCO3- and Cl-secretion primarily via a CFTR-dependent mechanisms.

Acute adenosine 3',5'-cyclic monophosphate (cAMP) stimulation of intestinal epithelium induces net transepithelial Cl- secretion and inhibits neutral coupled NaCl absorption. To investigate the role that the cystic fibrosis transmembrane conductance regulator (CFTR) plays in these events, we measured bioelectric changes and radioisotopic NaCl flux across jejunal tissues from gene-targeted cftr "knockout" mice [cftr(-/-) homozygotes] and their normal littermates [cftr(+/+) homozygotes and cftr(+/-) heterozygotes]. Before stimulation, the short-circuit current (Isc, an index of Cl- secretion) of the cftr(-/-) jejunum was essentially zero and significantly less than in the cftr(+/+) or cftr(+/-) intestine. Acute cAMP stimulation had little effect on the bioelectric parameters of the cftr(-/-) intestine but induced a marked increase of Isc and decrease of total tissue conductance in both the cftr(+/+) and cftr(+/-) intestine. Differences in the magnitude of the cAMP-induced Isc between the cftr(+/+) and cftr(+/-) intestine were only observed when the cell-to-lumen anion concentration gradient was maximized by removal of permeant anions from the luminal bath. Radioisotope flux measurements revealed that Na+ and Cl- were absorbed equally across the cftr(-/-) jejunum under basal conditions. In cftr(+/+) and cftr(+/-) intestine, Na+ was absorbed at a similar rate, but net Cl- absorption was reduced from that in cftr(-/-) intestine by an amount approximating the Isc. Acute cAMP stimulation of the cftr(+/+) and cftr(+/-) intestine abolished net NaCl absorption and induced electrogenic Cl- secretion. In contrast, net NaCl absorption was unchanged from the preceding flux period in the cftr(-/-) jejunum. The data suggest that CFTR not only mediates cAMP-induced transepithelial Cl- secretion but is also required for cAMP inhibition of neutral NaCl absorption in the intestine.

The mechanism by which receptors coupled to phospholipase C (PLC) induce Cl- secretion in amiloride-pretreated cultures of human nasal epithelial (HNE) cultures was investigated. Histamine (10(-4) M, basolateral administration) stimulated a rapid increase in equivalent short-circuit current, an index of Cl- secretion, that returned to baseline within 5 min. Intracellular recordings with double-barreled Cl(-)-selective microelectrodes showed that the apical and basolateral membrane potentials rapidly hyperpolarized, the fractional resistance of the apical membrane increased, and the transepithelial resistance decreased in response to histamine. Intracellular Cl- activity remained constant. Equivalent circuit analysis revealed that the early portion (< 0.9 min) of the Cl- secretory response was driven by an activation of a hyperpolarizing basolateral conductance, likely K+, whereas the later (> 0.9 min) phase of Cl- secretion reflects activation of the apical membrane Cl- conductance. Histamine raised intracellular Ca2+ (Ca2+i) measured by fura-2 in HNE with a potency similar to that observed for induction of Cl- secretion. Both intracellular release and plasma membrane influx pathways were identified, typical of receptor-mediated activation of PLC. The intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (15 microM), coupled with reduced bathing solution Ca2+, blunted the rise in Ca2+i and the net transepithelial Cl- secretory response to histamine. We conclude that 1) histamine induced Cl- secretion in HNE by a sequential mechanism: the rapid initial component reflects activation of the basolateral K+ conductance, and the later component reflects activation of an apical Cl- conductance; and 2) the level of Ca2+i may participate in the activation of both the basolateral and apical conductances.

1. We have characterized an adenosine receptor subtype present in human airway epithelial cells by measuring the changes in the intracellular levels of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and the rate of transepithelial Cl- secretion. 2. Primary cultures of human nasal epithelium obtained from excised surgical airway epithelial tissues and the cell lines BEAS39 and CF/T43 derived from human airway epithelium were grown on plastic dishes and labelled with [3H]-adenine for measurement of intracellular cyclic AMP accumulation. Primary cultures were loaded with the calcium indicator fura-2 to measure [Ca2+]i and studied as polarized, ion transporting epithelia on collagen matrix supports for measurement of Cl- secretion. 3. Adenosine analogues stimulated cyclic AMP accumulation with a rank order of potency characteristic of an A2-receptor: 5-N-ethyl-carboxamidoadenosine (NECA) greater than adenosine greater than R-phenylisopropyladenosine (R-PIA), 6-N-cyclopentyladenosine (CPA) greater than S-PIA. NECA increased cyclic AMP accumulation in normal and cystic fibrosis (CF) primary cells as well as in the CF/T43 and BEAS39 cell lines with K0.5 values ranging from 0.3 to 3 microM. Preincubation with NECA resulted in the homologous desensitization of airway epithelial cells. The effect of NECA was specifically inhibited by the adenosine receptor antagonist, aminophylline, in a competitive manner. 4. The A1-adenosine receptor agonists CPA and R-PIA did not inhibit isoprenaline-stimulated cyclic AMP accumulation in CF/T43 cells, and potentiating effects of the adenosine analogues were observed on forskolin-stimulated cyclic AMP accumulation. Adenosine analogues did not cause significant changes in intracellular Ca2+ ([Ca2+]i) in airway epithelium.5. Adenosine analogues, applied to either the serosal or mucosal side of the polarized amiloride pretreated primary cultures, induced changes in I,, with a rank order of potency of agonists similar to that observed for stimulation of cyclic AMP accumulation. Intracellular microelectrode studies indicated that the locus of action was the apical membrane Cl- conductance. Adenosine failed to stimulate C1- secretion in CF airway epithelium.6. These results provide evidence for the existence of an A2-adenosine receptor that modulates intracellular levels of cyclic AMP in human airway epithelium. Activation of this receptor might lead to stimulation of Cl- secretion in amiloride pretreated normal but not CF cells.

The rate of Cl- secretion by human airway epithelium is determined, in part, by apical cell membrane Cl- conductance. In cystic fibrosis airway epithelia, defective regulation of Cl- conductance decreases the capability to secrete Cl-. Here we report that extracytosolic ATP in the luminal bath of cultured human airway epithelia increased transepithelial Cl- secretion and apical membrane Cl- permeability. Single-channel studies in excised membrane patches revealed that ATP increased the open probability of outward rectifying Cl- channels. The latter effect occurs through a receptor mechanism that requires no identified soluble second messengers and is insensitive to probes of G protein function. These results demonstrate a mode of regulation of anion channels by binding ATP at the extracellular surface. Regulation of Cl- conductance by external ATP is preserved in cystic fibrosis airway epithelia.

Human nasal epithelium (HNE) is a Na+ absorptive epithelium but establishes a baseline Cl- secretory current in the presence of amiloride (10(-4) M, luminal). We compared the effects of an inflammatory mediator, bradykinin (BK), on ion transport in primary cultures of HNE using double-barreled Cl(-)-selective microelectrodes. In untreated HNE, BK (10(-5) M) transiently increased the equivalent short-circuit current (Ieq). Maximal Ieq occurred with hyperpolarization of the transepithelial potential difference (Vt), which was associated with hyperpolarization and decreased resistance of the basolateral membrane; a subsequent depolarization of Vt was observed that was associated with depolarization and decreased resistance of the apical membrane. Removal of bath Cl- did not affect the BK-induced Ieq response. In amiloride-treated HNE, the electrical pattern of the BK-induced response was identical, but the magnitude of the Ieq was reduced by 54% and the change in Ieq could be abolished by removal of bath Cl-. Equivalent-circuit analysis of the response in amiloride-treated tissues indicated activation of a hyperpolarizing conductance in the basolateral membrane, followed 20-30 s later by activation of an apical Cl- conductance. We conclude that BK stimulates both Na+ absorption in untreated HNE and Cl- secretion in amiloride-treated HNE by activating a basolateral (K+) conductance. Analysis of the entire Ieq response under both conditions also suggested that BK induces a delayed activation of apical membrane Na+ and Cl- conductances.

Dra is the major apical membrane Cl/HCO3 exchanger in the murine small intestine. Studies of recombinant DRA have proposed an electrogenic 2Cl:1HCO3 stoichiometry (J. Gen. Physiol. 127: 511, 2006). In Dra knockout jejunum (DraKO), loss of Clabsorption is associated with an increase in the baseline short-circuit current (Isc) that may result from loss of an inward anion current due to electrogenic Cl/HCO3 exchange. Therefore, we investigated the ionic basis of the increased Isc in isolated DraKO jejunal preparations using intracellular pH (pHi) microfluorimetry and Ussing chamber studies. As described previously (Gastroenterology 136: 1645, 2008), pHi of the DraKO villous epithelium was alkaline relative to wild type (WT), ostensibly due to loss of Cl/HCO3 exchange. Studies also revealed little Cl-/OHexchange in villous epithelium by measurements performed in the nominal absence of extracellular HCO3. In Ussing chamber studies, removal of extracellular Cleliminated the elevated baseline Isc whereas removal of extracellular HCO3 had no effect in DraKO jejunum, indicating that Cl/HCO3 exchange was not responsible for the elevated Isc. The contribution of the cystic fibrosis transmembrane conductance regulator (Cftr) anion channel was investigated usingDra/Cftr double knockout micewhich demonstrated a baseline Isc that was reduced by 40% relative to the DraKO jejunum. Epithelial alkalinity in the DraKO jejunal epithelium may increase the electrical gradient for Clsecretion by hyperpolarization secondary to activation of basolateral K+ channels (Am. J. Physiol. 258:C336, 1990). Therefore, epithelial acidification was induced by exposing the luminal surface to pH 6.4 by replacing HCO3 with TES buffer in the presence of 5% CO2: 95% O2. Under this condition, the elevated baseline Isc in the DraKO was abolished. On-going studies are investigating the effect of basolateral K+ channel blockers in the DraKO jejunum. We conclude that the elevated baseline Isc in the DraKO intestine does not result from loss of electrogenic Cl-/ HCO3 exchange but from increased anion secretion associated with epithelial alkalinity that may activate basolateral membrane K+ channels. Supported by NIDDK and the Cystic Fibrosis Foundation.