Search

The aim of this study was to investigate the distribution, tolerance, and anticancer and antiviral activity of Zn-based physiometacomposites (PMCs). Manganese, iron, nickel and cobalt-doped ZnO, ZnS or ZnSe were synthesized. Cell uptake, distribution into 3D culture and mice, and biochemical and chemotherapeutic activity were studied by fluorescence/bioluminescence, confocal microscopy, flow cytometry, viability, antitumor and virus titer assays. Luminescence and inductively coupled plasma mass spectrometry analysis showed that nanoparticle distribution was liver >spleen >kidney >lung >brain, without tissue or blood pathology. Photophysical characterization as ex vivo tissue probes and LL37 peptide, antisense oligomer or aptamer delivery targeting RAS/Ras binding domain (RBD) was investigated. Treatment at 25 μg/ml for 48 h showed ≥98–99% cell viability, 3D organoid uptake, 3-log inhibition of β-Galactosidase and porcine reproductive respiratory virus infection. Data support the preclinical development of PMCs for imaging and delivery targeting cancer and infectious disease.

Goblet cell hyperplasia is an important manifestation of cystic fibrosis (CF) disease in epithelial-lined organs. Explants of CF airway epithelium show normalization of goblet cell numbers; therefore, we hypothesized that small intestinal enteroids from Cftr knockout (KO) mice would not exhibit goblet cell hyperplasia. Toll-like receptors 2 and 4 (Tlr2 and Tlr4) were investigated as markers of inflammation and influence on goblet cell differentiation. Ex vivo studies found goblet cell hyperplasia in Cftr KO jejunum compared with wild-type (WT) mice. IL-13, SAM pointed domain-containing ETS transcription factor (Spdef), Tlr2, and Tlr4 protein expression were increased in Cftr KO intestine relative to WT. In contrast, WT and Cftr KO enteroids did not exhibit differences in basal or IL-13-stimulated goblet cell numbers, or differences in expression of Tlr2, Tlr4, and Spdef. Ileal goblet cell numbers in Cftr KO/Tlr4 KO and Cftr KO/Tlr2 KO mice were not different from Cftr KO mice, but enumeration was confounded by altered mucosal morphology. Treatment with Tlr4 agonist LPS did not affect goblet cell numbers in WT or Cftr KO enteroids, whereas the Tlr2 agonist Pam3Csk4 stimulated goblet cell hyperplasia in both genotypes. Pam3Csk4 stimulation of goblet cell numbers was associated with suppression of Notch1 and Neurog3 expression and upregulated determinants of goblet cell differentiation. We conclude that goblet cell hyperplasia and inflammation of the Cftr KO small intestine are not exhibited by enteroids, indicating that this manifestation of CF intestinal disease is not epithelial-automatous but secondary to the altered CF intestinal environment. NEW & NOTEWORTHY Studies of small intestinal organoids from cystic fibrosis (CF) mice show that goblet cell hyperplasia and increased Toll-like receptor 2/4 expression are not primary manifestations of the CF intestine. Intestinal goblet cell hyperplasia in the CF mice was not strongly altered by genetic ablation of Tlr2 and Tlr 4, but could be induced in both wild-type and CF intestinal organoids by a Tlr2-dependent suppression of Notch signaling.

Small-molecule modulators of cystic fibrosis transmembrane conductance regulator (CFTR) biology show promise in the treatment of cystic fibrosis (CF). A Cftr knockout (Cftr KO) mouse expressing mutants of human CFTR would advance in vivo testing of new modulators. A bacterial artificial chromosome (BAC) carrying the complete hCFTR gene including regulatory elements within 40.1 kb of DNA 5′ and 25 kb of DNA 3′ to the gene was used to generate founder mice expressing hCFTR. Whole genome sequencing indicated a single integration site on mouse chromosome 8 (8qB2) with ~6 gene copies. hCFTR+ offspring were bred to murine Cftr KO mice, producing hCFTR+/mCftr− (H+/m−) mice, which had normal survival, growth and goblet cell function as compared to wild-type (WT) mice. Expression studies showed hCFTR protein and transcripts in tissues typically expressing mCftr. Functionally, nasal potential difference and large intestinal short-circuit (Isc) responses to cAMP stimulation were similar in magnitude to WT mice, whereas small intestinal cAMP ΔIsc responses were reduced. A BAC transgenic mouse with functional hCFTR under control of its regulatory elements has been developed to enable the generation of mouse models of hCFTR mutations by gene editing for in vivo testing of new CF therapies.

Background & Aims Cystic fibrosis (CF) patients and CF mouse models have increased risk for gastrointestinal tumors. CF mice show augmented intestinal proliferation of unknown etiology and an altered intestinal environment. We examined the role of the cystic fibrosis transmembrane conductance regulator (Cftr) in Wnt/β-catenin signaling, stem cell proliferation, and its functional expression in the active intestinal stem cell (ISC) population. Dysregulation of intracellular pH (pHi) in CF ISCs was investigated for facilitation of Wnt/β-catenin signaling. Methods Crypt epithelia from wild-type (WT) and CF mice were compared ex vivo and in intestinal organoids (enteroids) for proliferation and Wnt/β-catenin signaling by standard assays. Cftr in ISCs was assessed by immunoblot of sorted Sox9enhanced green fluorescent protein(EGFP) intestinal epithelia and pHi regulation by confocal microfluorimetry of leucine-rich G-protein–coupled receptor 5 ISCs. Plasma membrane association of the Wnt transducer Dishevelled 2 (Dvl2) was assessed by fluorescence imaging of live enteroids from WT and CF mice crossed with Dvl2-EGFP/ACTB-tdTomato,-EGFP)Luo/J (RosamT/mG) mice. Results Relative to WT, CF intestinal crypts showed an ∼30% increase in epithelial and Lgr5+ ISC proliferation and increased Wnt/β-catenin signaling. Cftr was expressed in Sox9EGFPLo ISCs and loss of Cftr induced an alkaline pHi in ISCs. CF crypt-base columnar cells showed a generalized increase in plasma membrane Dvl2-EGFP association as compared with WT. Dvl2-EGFP membrane association was charge- and pH-dependent and increased in WT crypt-base columnar cells by Cftr inhibition. Conclusions CF intestine shows increased ISC proliferation and Wnt/β-catenin signaling. Loss of Cftr increases pHi in ISCs, which stabilizes the plasma membrane association of the Wnt transducer Dvl, likely facilitating Wnt/β-catenin signaling. Absence of Cftr-dependent suppression of ISC proliferation in the CF intestine may contribute to increased risk for intestinal tumors., Graphical abstract

The recent proposal that Dra/Slc26a3 mediates electrogenic 2Cl−/1HCO3− exchange suggests a required revision of classical concepts of electroneutral Cl− transport across epithelia such as the intestine. We investigated 1) the effect of endogenous Dra Cl−/HCO3− activity on apical membrane potential ( Va) of the cecal surface epithelium using wild-type (WT) and knockout (KO) mice; and 2) the electrical properties of Cl−/(OH−)HCO3− exchange by mouse and human orthologs of Dra expressed in Xenopus oocytes. Ex vivo 36Cl− fluxes and microfluorometry revealed that cecal Cl−/HCO3− exchange was abolished in the Dra KO without concordant changes in short-circuit current. In microelectrode studies, baseline Va of Dra KO surface epithelium was slightly hyperpolarized relative to WT but depolarized to the same extent as WT during luminal Cl− substitution. Subsequent studies indicated that Cl−-dependent Va depolarization requires the anion channel Cftr. Oocyte studies demonstrated that Dra-mediated exchange of intracellular Cl− for extracellular HCO3− is accompanied by slow hyperpolarization and a modest outward current, but that the steady-state current-voltage relationship is unaffected by Cl− removal or pharmacological blockade. Further, Dra-dependent 36Cl− efflux was voltage-insensitive in oocytes coexpressing the cation channels ENaC or ROMK. We conclude that 1) endogenous Dra and recombinant human/mouse Dra orthologs do not exhibit electrogenic 2Cl−/1HCO3− exchange; and 2) acute induction of Dra Cl−/HCO3− exchange is associated with secondary membrane potential changes representing homeostatic responses. Thus, participation of Dra in coupled NaCl absorption and in uncoupled HCO3− secretion remains compatible with electroneutrality of these processes, and with the utility of electroneutral transport models for predicting epithelial responses in health and disease.

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.

The majority of dietary amino acids are absorbed via the H+-di-/tripeptide transporter Pept1 of the small intestine. Proton influx via Pept1 requires maintenance of intracellular pH (pHi) to sustain the driving force for peptide absorption. The apical membrane Na+/H+exchanger Nhe3 plays a major role in minimizing epithelial acidification during H+-di-/tripeptide absorption. However, the contributions of HCO3−-dependent transporters to this process have not been elucidated. In this study, we investigate the role of putative anion transporter-1 (Pat-1), an apical membrane anion exchanger, in epithelial pHiregulation during H+-peptide absorption. Using wild-type (WT) and Pat-1(−) mice, Ussing chambers were employed to measure the short-circuit current ( Isc) associated with Pept1-mediated glycyl-sarcosine (Gly-Sar) absorption. Microfluorometry was used to measure pHiand Cl−/HCO3−exchange in the upper villous epithelium. In CO2/HCO3−-buffered Ringers, WT small intestine showed significant Gly-Sar-induced Iscand efficient pHiregulation during pharmacological inhibition of Nhe3 activity. In contrast, epithelial acidification and reduced Iscresponse to Gly-Sar exposure occurred during pharmacological inhibition of Cl−/HCO3−exchange and in the Pat-1(−) intestine. Pat-1 interacts with carbonic anhydrase II (CAII), and studies using CAII(−) intestine or the pharmacological inhibitor methazolamide on WT intestine resulted in increased epithelial acidification during Gly-Sar exposure. Increased epithelial acidification during Gly-Sar exposure also occurred in WT intestine during inhibition of luminal extracellular CA activity. Measurement of Cl−/HCO3−exchange in the presence of Gly-Sar revealed an increased rate of Cl−OUT/HCO3−INexchange that was both Pat-1 dependent and CA dependent. In conclusion, Pat-1 Cl−/HCO3−exchange contributes to pHiregulation in the villous epithelium during H+-dipeptide absorption, possibly by providing a HCO3−import pathway.

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.

The Ussing chamber provides a physiological system to measure the transport of ions, nutrients, and drugs across various epithelial tissues. One of the most studied epithelia is the intestine, which has provided several landmark discoveries regarding the mechanisms of ion transport processes. Adaptation of this method to mouse intestine adds the dimension of investigating genetic loss or gain of function as a means to identify proteins or processes affecting transepithelial transport. In this review, the principles underlying the use of Ussing chambers are outlined including limitations and advantages of the technique. With an emphasis on mouse intestinal preparations, the review covers chamber design, commercial equipment sources, tissue preparation, step-by-step instruction for operation, troubleshooting, and examples of interpretation difficulties. Specialized uses of the Ussing chamber such as the pH stat technique to measure transepithelial bicarbonate secretion and isotopic flux methods to measure net secretion or absorption of substrates are discussed in detail, and examples are given for the adaptation of Ussing chamber principles to other measurement systems. The purpose of the review is to provide a practical guide for investigators who are new to the Ussing chamber method.

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.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), an anion channel providing a major pathway for Cl−and HCO3−efflux across the apical membrane of the epithelium. In the intestine, CF manifests as obstructive syndromes, dysbiosis, inflammation, and an increased risk for gastrointestinal cancer. Cftr knockout (KO) mice recapitulate CF intestinal disease, including intestinal hyperproliferation. Previous studies using Cftr KO intestinal organoids (enteroids) indicate that crypt epithelium maintains an alkaline intracellular pH (pHi). We hypothesized that Cftr has a cell-autonomous role in downregulating pHithat is incompletely compensated by acid-base regulation in its absence. Here, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein microfluorimetry of enteroids showed that Cftr KO crypt epithelium sustains an alkaline pHiand resistance to cell acidification relative to wild-type. Quantitative real-time PCR revealed that Cftr KO enteroids exhibit downregulated transcription of base (HCO3−)-loading proteins and upregulation of the basolateral membrane HCO3−-unloader anion exchanger 2 (Ae2). Although Cftr KO crypt epithelium had increased Ae2 expression and Ae2-mediated Cl−/HCO3−exchange with maximized gradients, it also had increased intracellular Cl−concentration relative to wild-type. Pharmacological reduction of intracellular Cl−concentration in Cftr KO crypt epithelium normalized pHi, which was largely Ae2-dependent. We conclude that Cftr KO crypt epithelium maintains an alkaline pHias a consequence of losing both Cl−and HCO3−efflux, which impairs pHiregulation by Ae2. Retention of Cl−and an alkaline pHiin crypt epithelium may alter several cellular processes in the proliferative compartment of Cftr KO intestine.

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.

Paneth cells of intestinal crypts contribute to host defense by producing antimicrobial peptides that are packaged as granules for secretion into the crypt lumen. Here, we provide evidence using light and electron microscopy that postsecretory Paneth cell granules undergo limited dissolution and accumulate within the intestinal crypts of cystic fibrosis (CF) mice. On the basis of this finding, we evaluated bacterial colonization and expression of two major constituents of Paneth cells, i.e., α-defensins (cryptdins) and lysozyme, in CF murine intestine. Paneth cell granules accumulated in intestinal crypt lumens in both untreated CF mice with impending intestinal obstruction and in CF mice treated with an osmotic laxative that prevented overt clinical symptoms and mucus accretion. Ultrastructure studies indicated little change in granule morphology within mucus casts, whereas granules in laxative-treated mice appear to undergo limited dissolution. Protein extracts from CF intestine had increased levels of processed cryptdins compared with those from wild-type (WT) littermates. Nonetheless, colonization with aerobic bacteria species was not diminished in the CF intestine and oral challenge with a cryptdin-sensitive enteric pathogen, Salmonella typhimurium, resulted in greater colonization of CF compared with WT intestine. Modest downregulation of cryptdin and lysozyme mRNA in CF intestine was shown by microarray analysis, real-time quantitative PCR, and Northern blot analysis. Based on these findings, we conclude that antimicrobial peptide activity in CF mouse intestine is compromised by inadequate dissolution of Paneth cell granules within the crypt lumens.

Electrolyte transport processes of small intestinal epithelia maintain a balance between hydration of the luminal contents and systemic fluid homeostasis. Under basal conditions, electroneutral Na+absorption mediated by Na+/H+exchanger 3 (NHE3) predominates; under stimulated conditions, increased anion secretion mediated by CFTR occurs concurrently with inhibition of Na+absorption. Homeostatic adjustments to diseases that chronically affect the activity of one transporter (e.g., cystic fibrosis) may include adaptations in the opposing transport process to prevent enterosystemic fluid imbalance. To test this hypothesis, we measured electrogenic anion secretion (indexed by the short-circuit current) across NHE3-null [NHE3(−)] murine small intestine and electroneutral Na+absorption (by radioisotopic flux analysis) across small intestine of mice with gene-targeted disruptions of the anion secretory pathway, i.e., CFTR-null [CFTR(−)] or Na+-K+-2Cl−cotransporter-null [NKCC1(−)]. Protein expression of NHE3 and CFTR in the intestinal epithelia was measured by immunoblotting. In NHE3(−), compared with wild-type small intestine, maximal and bumetanide-sensitive anion secretion following cAMP stimulation was significantly reduced, and there was a corresponding decrease in CFTR protein expression. In CFTR(−) and NKCC1(−) intestine, Na+absorption was significantly reduced compared with wild-type. NHE3 protein expression was decreased in the CFTR(−) intestine but was unchanged in the NKCC1(−) intestine, indicating that factors independent of expression also downregulate NHE3 activity. Together, these data support the concept that absorptive and secretory processes determining NaCl and water movement across the intestinal epithelium are regulated in parallel to maintain balance between the systemic fluid volume and hydration of the luminal contents.

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.

In comparing product bioavailability across animal species, it is not unusual to observe marked interspecies differences. For many compounds, these differences reflect presystemic drug metabolism. However, a host of other variables must also be considered such as in vivo drug solubility, gastric transit time, intestinal permeability, diet, and species-by-formulation interactions. By combining information on drug solubility and intestinal permeability with an understanding of the interrelationship between pH, product dissolution and gastrointestinal physiology, we attempt to define those conditions under which in vitro dissolution data may be used as a surrogate for data on in vivo bioavailability. We consider the likely physiological causes for species-related differences in the absolute and relative bioavailability of orally administered pharmaceuticals, and examine the potential for these normal interspecies differences to reflect bioavailability changes that can occur with various human pathologies.

UTP activates P2Y2 receptors in both 1321N1 cell transfectants expressing the P2Y2 receptor and human HT-29 epithelial cells expressing endogenous P2Y2 receptors with an EC50 of 0.2- 1.0 μM. Pretreatment of these cells with UTP diminished the effectiveness of a second dose of UTP (the IC50 for UTP-induced receptor desensitization was 0.3 - 1.0 μM for both systems). Desensitization and down-regulation of the P2Y2 nucleotide receptor may limit the effectiveness of UTP as a therapeutic agent. The present studies investigated the phenomenon of P2Y2 receptor desensitization in human 1321N1 astrocytoma cells expressing recombinant wild type and C-terminal truncation mutants of the P2Y,2 receptor. In these cells, potent P2Y2 receptor desensitization was observed after a 5 min exposure to UTP. Full receptor responsiveness returned 5-10 min after removal of UTP. Thapsigargin, an inhibitor of Ca2+-ATPase in the endoplasmic reticulum, induced an increase in the intracellular free calcium concentration, [Ca2+]i, after addition of desensitizing concentrations of UTP, indicating that P2Y2 receptor desensitization is not due to depletion of calcium from intracellular stores. Single cell measurements of increases in [Ca2+]i induced by UTP in 1321N1 cell transfectants expressing the P2Y2 receptor indicate that time- and UTP concentration-dependent desensitization occurred uniformly across a cell population. Other results suggest that P2Y2 receptor phosphorylation/dephosphorylation regulate receptor desensitization/resensitization. A 5 min preincubation of 1321N1 cell transfectants with the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), reduced the subsequent response to UTP by about 50% whereas co-incubation of PMA with UTP caused a greater inhibition in the response. The protein phosphatases - 1 and -2A inhibitor, okadaic acid, partially blocked resensitization of the receptor. Furthermore, C-terminal truncation mutants of the P2Y2 receptor that eliminated several potential phosphorylation sites including two for PKC were resistant to UTP-, but not phorbol ester-induced desensitization. Down regulation of protein kinase C isoforms prevented phorbol ester-induced desensitization but had no effect on agonist-induced desensitization of wild type or truncation mutant receptors. These results suggest that phosphorylation of the C-terminus of the P2Y2 receptor by protein kinases other than protein kinase C mediates agonist-induced receptor desensitization. A better understanding of the molecular mechanisms of P2Y2 nucleotide receptor desensitization may help optimize a promising cystic fibrosis pharmacotherapy based on the activation of anion secretion in airway epithelial cells by P2Y2 receptor agonists.

We report the cloning of a murine ClC-2 chloride channel cDNA from duodenal epithelium by reverse transcriptase-polymerase chain reaction (RT-PCR) using degenerate primers and by rapid amplification of cDNA ends (RACE)-PCR. Other than CFTR, this represents the first cloned chloride channel from intact intestine. The ClC-2 cDNA predicts encoding of a 908 amino acid polypeptide with a calculated M(r) of 99,373. The amino acid sequence of the murine ClC-2 chloride channel is over 94% identical to the ClC-2 chloride channel proteins of other species. Of interest is the finding that the ClC-2 mRNA is expressed about the same level in duodena from both CFTR knockout and wild-type mice. This is in keeping with the suggestion that ClC-2 might be a therapeutic target in cystic fibrosis.

Molecular determinants of P2Y2 receptor desensitization and sequestration have been investigated. Wild-type P2Y2 receptors and a series of five C-terminal truncation mutants of the receptor were epitope-tagged and stably expressed in 1321N1 cells. These constructs were used to assess the importance of the intracellular C terminus on 1) UTP-stimulated increases in intracellular calcium concentration, 2) homologous desensitization of the receptor, and 3) agonist-induced decreases in cell-surface density (receptor sequestration) of epitope-tagged receptors using fluorescence-activated cell sorting. The potency and efficacy of UTP were similar for the wild-type and all mutant P2Y2 receptors. Truncation of 18 or more amino acids from the C terminus increased by approximately 30-fold the concentration of UTP necessary to desensitize the receptor. Both the rate and magnitude of UTP-induced receptor sequestration were decreased with progressively larger truncations of the C terminus. Furthermore, the recovery from sequestration was slower for the most extensively truncated receptor. Complete desensitization was obtained with >50% of the original receptor complement remaining on the cell surface. Protein kinase C activation, which desensitizes the P2Y2 receptor, had no effect on sequestration, consistent with the ideas that desensitization and sequestration are discrete events and that agonist occupancy is required for receptor sequestration.

A genetically engineered “knockout gene” mouse model for human cystic fibrosis (CF) has been utilized to study bone mineralization. In CF, the so-called cystic fibrosis transmembrane conductance regulator (CFTR) protein, a chloride ion channel, is either absent or defective. To produce the animal model the murine CFTR gene has been inactivated producing CF symptoms in the homozygotic progeny. CF results in abnormal intestinal absorption of minerals and nutrients which presumably results in substandard bone mineralization. The objective of this study was to determine the feasibility of using whole-body thermal and fast neutron activation analysis to determine mineral and trace-element differences between homozygote controls (+/+) and CF (−/−), murine siblings. Gender-matched juvenile +/+ and −/− litter mates were lyophilized and placed in a BN capsule to reduce thermal-neutron activation and irradiated for 10 seconds at φfast ≈ 1·1013 n·cm−2·s−1 using the MURR pneumatic-tube facility. Phosphorus was measured via the31P15(n,α)28Al13 reaction. After several days decay, the whole-body specimens were re-irradiated in the same facility, but without thermal-neutron shielding, for 5 seconds and the gamma-ray spectrum was recorded at two different decay periods allowing measurement of77mSe,24Na,27Mg,38Cl,42K,49Ca,56Mn,66Cu and80Br from the corresponding radiative-capture reactions.

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.

Multiple isoforms of the Na+/H+ exchanger (NHE) are expressed at high levels in gastric epithelium, but the physiological role of individual isoforms is unclear. To study the function of NHE2, which is expressed in mucous, zymogenic, and parietal cells, we prepared mice with a null mutation in the NHE2 gene. Homozygous null mutants exhibit no overt disease phenotype, but the cellular composition of the oxyntic mucosa of the gastric corpus is altered, with parietal and zymogenic cells reduced markedly in number. Net acid secretion in null mutants is reduced slightly relative to wild-type levels just before weaning and is abolished in adult animals. Although mature parietal cells are observed, and appear morphologically to be engaged in active acid secretion, many of the parietal cells are in various stages of degeneration. These results indicate that NHE2 is not required for acid secretion by the parietal cell, but is essential for its long-term viability. This suggests that the unique sensitivity of NHE2 to inhibition by extracellular H+, which would allow upregulation of its activity by the increased interstitial alkalinity that accompanies acid secretion, might enable this isoform to play a specialized role in maintaining the long-term viability of the parietal cell.

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.

Cystic fibrosis (CF) intestinal disease is associated with the pathological manifestation mucoviscidosis, which is the secretion of tenacious, viscid mucus that plugs ducts and glands of epithelial-lined organs. Goblet cells are the principal cell type involved in exocytosis of mucin granules; however, little is known about the exocytotic process of goblet cells in the CF intestine. Using intestinal organoids from a CF mouse model, we determined that CF goblet cells have altered exocytotic dynamics, which involved intrathecal granule swelling that was abruptly followed by incomplete release of partially decondensated mucus. Some CF goblet cells exhibited an ectopic granule location and distorted cellular morphology, a phenotype that is consistent with retrograde intracellular granule movement during exocytosis. Increasing the luminal concentration of bicarbonate, which mimics CF transmembrane conductance regulator-mediated anion secretion, increased spontaneous degranulation in WT goblet cells and improved exocytotic dynamics in CF goblet cells; however, there was still an apparent incoordination between granule decondensation and exocytosis in the CF goblet cells. Compared with those within WT goblet cells, mucin granules within CF goblet cells had an alkaline pH, which may adversely affect the polyionic composition of the mucins. Together, these findings indicate that goblet cell dysfunction is an epithelial-autonomous defect in the CF intestine that likely contributes to the pathology of mucoviscidosis and the intestinal manifestations of obstruction and inflammation.

to the editor: We appreciate the interest that Dr. Kaunitz and colleagues ([1][1]) have expressed regarding our recent article. One aspect of the manuscript was our conjecture that sustained alkalinity in the crypt epithelium would support intestinal hyperproliferation, a statement based on numerous

Physiological studies of intact crypt epithelium have been limited by problems of accessibility in vivo and dedifferentiation in standard primary culture. Investigations of murine intestinal stem cells have recently yielded a primary intestinal culture in three-dimensional gel suspension that recapitulates crypt structure and epithelial differentiation (Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, Van Es JH, Abo A, Kujala P, Peters PJ, Clevers H. Nature 459: 262–265, 2009). We investigated the utility of murine intestinal crypt cultures (termed “enteroids”) for physiological studies of crypt epithelium by focusing on the transport activity of the cystic fibrosis transmembrane conductance regulator Cftr. Enteroids had multiple crypts with well-differentiated goblet and Paneth cells that degranulated on exposure to the muscarinic agonist carbachol. Modified growth medium provided a crypt proliferation rate, as measured by 5-ethynyl-2′-deoxyuridine labeling, which was similar to proliferation in vivo. Immunoblots demonstrated equivalent Cftr expression in comparisons of freshly isolated crypts with primary and passage 1 enteroids. Apparent enteroid differences in mRNA expression of other transporters were primarily associated with villous epithelial contamination of freshly isolated crypts. Microelectrode analysis revealed cAMP-stimulated membrane depolarization in enteroid epithelium from wild-type (WT) but not Cftr knockout (KO) mice. Morphological and microfluorimetric studies, respectively, demonstrated Cftr-dependent cell shrinkage and lower intracellular pH in WT enteroid epithelium in contrast to Cftr KO epithelium or WT epithelium treated with Cftr inhibitor 172. We conclude that crypt epithelium of murine enteroids exhibit Cftr expression and activity that recapitulates crypt epithelium in vivo. Enteroids provide a primary culture model that is suitable for physiological studies of regenerating crypt epithelium.

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.

Bicarbonate serves many functions in our body. It is the predominant buffer maintaining a physiological pH in the blood and within our cells. It is also essential for proper digestion of nutrients and solubilization of complex protein mixtures, such as digestive enzymes and mucins, in epithelial secretions. Transepithelial HCO3- transport also drives net fluid secretion in many epithelial tissues including those in the gastrointestinal and reproductive tracts as well as the airways. Indeed, defective bicarbonate secretion is a hallmark of the pathophysiology in the pancreas of most patients suffering from cystic fibrosis. Some, but not all, disease-causing mutations in the CF gene lead to impaired bicarbonate transport when expressed in heterologous systems. Recently developed pharmacological modulators of mutant CFTR have demonstrated an ability to activate chloride transport but little is known about whether they also increase the secretion of bicarbonate. It is therefore essential to assay bicarbonate transport when studying the effect of small molecules on CFTR function. However, due to the chaotropic nature of the ion, the measurement of the absolute bicarbonate concentration and its permeability through CFTR is far from trivial. In this chapter we will review some of the techniques available to measure bicarbonate transport through single ion channels, individual cells, and intact epithelial layers.

Bicarbonate serves many functions in our body. It is the predominant buffer maintaining a physiological pH in the blood and within our cells. It is also essential for proper digestion of nutrients and solubilization of complex protein mixtures, such as digestive enzymes and mucins, in epithelial secretions. Transepithelial HCO3- transport also drives net fluid secretion in many epithelial tissues including those in the gastrointestinal and reproductive tracts as well as the airways. Indeed, defective bicarbonate secretion is a hallmark of the pathophysiology in the pancreas of most patients suffering from cystic fibrosis. Some, but not all, disease-causing mutations in the CF gene lead to impaired bicarbonate transport when expressed in heterologous systems. Recently developed pharmacological modulators of mutant CFTR have demonstrated an ability to activate chloride transport but little is known about whether they also increase the secretion of bicarbonate. It is therefore essential to assay bicarbonate transport when studying the effect of small molecules on CFTR function. However, due to the chaotropic nature of the ion, the measurement of the absolute bicarbonate concentration and its permeability through CFTR is far from trivial. In this chapter we will review some of the techniques available to measure bicarbonate transport through single ion channels, individual cells, and intact epithelial layers.

In cystic fibrosis (CF), numerous epithelial cell functions are abnormal, including Cl- conductance, sodium absorption, mucin sulphation and enzyme secretion. Although the CF gene product, the cystic fibrosis transmembrane conductance regulator (CFTR), functions as a small linear Cl- channel, it is difficult to attribute such pleiotropic disease manifestations solely to a defect in Cl- conductance. This has led to speculation that CFTR regulates the activity of other proteins. One possible example is the protein kinase A activation of outward rectifying Cl- channels (ORCC), which is defective in membrane patches excised from CF cells. Whether CFTR regulates the activity of an independent anion channel is debatable, because ORCC occur exclusively in excised membrane patches and could be an excision-induced molecular derivative of CFTR. 'Knockout' mice that lack CFTR provide a means to define the relationship between CFTR and ORCC. Here we report that ORCC are present in CFTR(-/-) mouse nasal epithelial cells and thus cannot be a derivative of the CFTR molecule. Also ORCC were regulated by protein kinase A in membrane patches from normal but not CFTR(-/-) cells. These observations are the first, to our knowledge definitive demonstration that CFTR regulates the activity of another protein.

The respiratory epithelium lining the airway relies on mucociliary clearance and a complex network of inflammatory mediators to protect the lung. Alterations in the composition and volume of the periciliary liquid layer, as occur in cystic fibrosis (CF), lead to impaired mucociliary clearance and persistent airway infection. Moreover, the respiratory epithelium releases chemoattractants after infection, inciting airway inflammation. However, characterizing the inflammatory response of primary human airway epithelial cells to infection can be challenging because of genetic heterogeneity. Using well-characterized, differentiated, primary murine tracheal cells grown at an air–liquid interface, which provides an in vitro polarized epithelial model, we compared inflammatory gene expression and secretion in wild-type and ΔF508 CF airway cells after infection with Pseudomonas aeruginosa. The expression of several CXC-chemokines, including macrophage inflammatory protein–2, small inducible cytokine subfamily member 2, lipopolysaccharide-induced chemokine, and interferon-inducible cytokine–10, was markedly increased after infection, and these proinflammatory mediators were asymmetrically released from the airway epithelium, predominantly from the basolateral surface. Equal amounts of CXC-chemokines were released from wild-type and CF cells. Secreted mediators were concentrated in the thin, periciliary fluid layer, and the dehydrated apical microenvironment of CF airway epithelial cells amplified the inflammatory signal, potentially resulting in high chemokine concentration gradients across the epithelium. Consistent with this observation, the enhanced chemotaxis of wild-type neutrophils was detected in CF airway epithelial cultures, compared with wild-type cells. These data suggest that P. aeruginosa infection of the airway epithelium induces the expression and polarized secretion of CXC-chemokines, and the increased concentration gradient across the CF airway leads to an exaggerated inflammatory response.

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.

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 F508 mice (Cftr(F/F)) 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 Cftr(F/F) mice. In contrast, Cftr and ENaC channels were not detected in acinar cells, nor was saliva production affected in Cftr(F/F) 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 Cftr(F/F) mice, saliva was collected from ex vivo perfused SMGs. Cftr(F/F) glands secreted saliva with significantly increased [NaCl]. Moreover, pharmacological inhibition of either Cftr or ENaC in the ex vivo SMGs mimicked the Cftr(F/F) 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.