cGMP-dependent kinase 2, Na+/H+ exchanger NHE3, and PDZ-adaptor NHERF2 co-assemble in apical membrane microdomains.

AIM: Trafficking, membrane retention, and signal-specific regulation of the Na+/H+ exchanger 3 (NHE3) are modulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adapter proteins. This study explored the assembly of NHE3 and NHERF2 with the cGMP-dependent kinase II (cGKII) within detergent-resistant membrane microdomains (DRMs, "lipid rafts") during in vivo guanylate cycle C receptor (Gucy2c) activation in murine small intestine. METHODS: Small intestinal brush border membranes (siBBMs) were isolated from wild type, NHE3-deficient, cGMP-kinase II-deficient, and NHERF2-deficient mice, after oral application of the heat-stable Escherichia coli toxin (STa) analog linaclotide. Lipid raft and non-raft fractions were separated by Optiprep density gradient centrifugation of Triton X-solubilized siBBMs. Confocal microscopy was performed to study NHE3 redistribution after linaclotide application in vivo. RESULTS: In the WT siBBM, NHE3, NHERF2, and cGKII were strongly raft associated. The raft association of NHE3, but not of cGKII, was NHERF2 dependent. After linaclotide application to WT mice, lipid raft association of NHE3 decreased, that of cGKII increased, while that of NHERF2 did not change. NHE3 expression in the BBM shifted from a microvillar to a terminal web region. The linaclotide-induced decrease in NHE3 raft association and in microvillar abundance was abolished in cGKII-deficient mice, and strongly reduced in NHERF2-deficient mice. CONCLUSION: NHE3, cGKII, and NHERF2 form a lipid raft-associated signal complex in the siBBM, which mediates the inhibition of salt and water absorption by Gucy2c activation. NHERF2 enhances the raft association of NHE3, which is essential for its close interaction with the exclusively raft-associated activated cGKII.

SLC26A9 deficiency causes gastric intraepithelial neoplasia in mice and aggressive gastric cancer in humans.

BACKGROUND: Solute carrier family 26 member (SLC26A9) is a Cl- uniporter with very high expression levels in the gastric mucosa. Here, we describe morphological and molecular alterations in gastric mucosa of slc26a9-/- mice and in selective parietal cell-deleted slc26a9fl/fl/Atp4b-Cre mice and correlate SLC26A9 expression levels with morphological and clinical parameters in a cohort of gastric cancer (GC) patients. METHODS: The expression patterns of genes related to transport and enzymatic function, proliferation, apoptosis, inflammation, barrier integrity, metaplasia and neoplasia development were studied by immunohistochemistry (IHC), quantitative RT-PCR, in situ hybridization and RNA microarray analysis. SLC26A9 expression and cellular/clinical phenotypes were studied in primary human GC tissues and GC cell lines. RESULTS: We found that both complete and parietal cell-selective Slc26a9 deletion in mice caused spontaneous development of gastric premalignant and malignant lesions. Dysregulated differentiation of gastric stem cells in an inflammatory environment, activated Wnt signaling, cellular hyperproliferation, apoptosis inhibition and metaplasia were observed. Analysis of human gastric precancerous and cancerous tissues revealed that SLC26A9 expression progressively decreased from atrophic gastritis to GC, and that downregulation of SLC26A9 was correlated with patient survival. Exogenous expression of SLC26A9 in GC cells induced upregulation of the Cl-/HCO3- exchanger AE2, G2/M cell cycle arrest and apoptosis and suppressed their proliferation, migration and invasion. CONCLUSIONS: Our data indicate that SLC26A9 deletion in parietal cells is sufficient to trigger gastric metaplasia and the development of neoplastic lesions. In addition, we found that SLC26A9 expression decreases during human gastric carcinogenesis, and that exogenous SLC26A9 expression in GC cells reduces their malignant behavior.

Upregulation of antimicrobial peptide expression in slc26a3-/- mice with colonic dysbiosis and barrier defect.

Genetic defects in SLC26A3 (DRA), an intestinal Cl-/HCO3- exchanger, result in congenital chloride diarrhea (CLD), marked by lifelong acidic diarrhea and a high risk of inflammatory bowel disease. Slc26a3-/- mice serve as a model to understand the pathophysiology of CLD and search for treatment options. This study investigates the microbiota changes in slc26a3-/- colon, the genotype-related causes for the observed microbiota alterations, its inflammatory potential, as well as the corresponding host responses. The luminal and the mucosa-adherent cecal and colonic microbiota of cohoused slc26a3-/- and wt littermates were analyzed by 16S rRNA gene sequencing. Fecal microbiota transfer from cohoused slc26a3-/- and wt littermates to germ-free wt mice was performed to analyze the stability and the inflammatory potential of the communities.The cecal and colonic luminal and mucosa-adherent microbiota of slc26a3-/- mice was abnormal from an early age, with a loss of diversity, of short-chain fatty acid producers, and an increase of pathobionts. The transfer of slc26a3-/- microbiota did not result in intestinal inflammation and the microbial diversity in the recipient mice normalized over time. A strong increase in the expression of Il22, Reg3ß/γ, Relmß, and other proteins with antimicrobial functions was observed in slc26a3-/- colon from juvenile age, while the mucosal and systemic inflammatory signature was surprisingly mild. The dysbiotic microbiota, low mucosal pH, and mucus barrier defect in slc26a3-/- colon are accompanied by a stark upregulation of the expression of a panel of antimicrobial proteins. This may explain the low inflammatory burden in the gut of these mice.

Loss of luminal carbonic anhydrase XIV results in decreased biliary bicarbonate output, liver fibrosis, and cholangiocyte proliferation in mice.

Carbonic anhydrase XIV (Car14) is highly expressed in the hepatocyte, with predominance in the canalicular membrane and its active site in the extracellular milieu. The aim of this study is to determine the physiological relevance of Car14 for biliary fluid and acid/base output, as well as its role in the maintenance of hepatocellular and cholangiocyte integrity. The common bile duct of anesthetized car14-/- and car14+/+ mice was cannulated and hepatic HCO3- output was measured by microtitration and bile flow gravimetrically before and during stimulation with intravenously applied tauroursodeoxycholic acid (TUDCA). Morphological alterations and hepatic damage were assessed histologically and immunohistochemically in liver tissue from 3- to 52-week-old car14-/- and car14+/+ mice, and gene and/or protein expression was measured for pro-inflammatory cytokines, fibrosis, and cholangiocyte markers. Biliary basal and more so TUDCA-stimulated HCO3- output were significantly reduced in car14-/- mice of all age groups, whereas bile flow and hepatic and ductular morphology were normal at young age. Car14-/- mice developed fibrotic and proliferative changes in the small bile ducts at advanced age, which was accompanied by a reduction in bile flow, and an upregulation of hepatic cytokeratin 19 mRNA and protein expression. Membrane-bound Car14 is essential for biliary HCO3- output, and its loss results in gradual development of small bile duct disease and hepatic fibrosis. Bile flow is not compromised in young adulthood, suggesting that Car14-deficient mice may be a model to study the protective role of biliary canalicular HCO3- against luminal noxi to the cholangiocyte.

Sodium/hydrogen-exchanger-2 modulates colonocyte lineage differentiation.

AIM: The sodium/hydrogen exchanger 2 (NHE2) is an intestinal acid extruder with crypt-predominant localization and unresolved physiological significance. Our aim was to decipher its role in colonic epithelial cell proliferation, differentiation and electrolyte transport. METHODS: Alterations induced by NHE2-deficiency were addressed in murine nhe2-/- and nhe2+/+ colonic crypts and colonoids, and NHE2-knockdown and control Caco2Bbe cells using pH-fluorometry, gene expression analysis and immunofluorescence. RESULTS: pHi -measurements along the colonic cryptal axis revealed significantly decreased intracellular pH (pHi ) in the middle segment of nhe2-/- compared to nhe2+/+ crypts. Increased Nhe2 mRNA expression was detected in murine colonoids in the transiently amplifying/progenitor cell stage (TA/PE). Lack of Nhe2 altered the differentiation programme of colonic epithelial cells with reduced expression of absorptive lineage markers alkaline phosphatase (iAlp), Slc26a3 and transcription factor hairy and enhancer-of-split 1 (Hes1), but increased expression of secretory lineage markers Mucin 2, trefoil factor 3 (Tff3), enteroendocrine marker chromogranin A and murine atonal homolog 1 (Math1). Enterocyte differentiation was found to be pHi dependent with acidic pHi reducing, and alkaline pHi stimulating the expression of enterocyte differentiation markers in Caco2Bbe cells. A thicker mucus layer, longer crypts and an expanded brush border membrane zone of sodium/hydrogen exchanger 3 (NHE3) abundance may explain the lack of inflammation and the normal fluid absorptive rate in nhe2-/- colon. CONCLUSIONS: The results suggest that NHE2 expression is activated when colonocytes emerge from the stem cell niche. Its activity increases progenitor cell pHi and thereby supports absorptive enterocyte differentiation.

Slc26a3 deletion alters pH-microclimate, mucin biosynthesis, microbiome composition and increases the TNFα expression in murine colon.

AIM: SLC26A3 (DRA) mediates the absorption of luminal Cl- in exchange for HCO3- in the distal intestine. Its expression is lost in congenital chloride diarrhoea (CLD) and strongly decreased in the presence of intestinal inflammation. To characterize the consequences of a loss of Slc26a3 beyond disturbed electrolyte transport, colonic mucus synthesis, surface accumulation and composition, pH microclimate, microbiome composition and development of inflammation was studied in slc26a3-/- mice. METHODS: The epithelial surface pH microclimate and the surface mucus accumulation in vivo was assessed by two photon microscopy in exteriorized mid colon of anaesthetized slc26a3-/- and wt littermates. Mucus synthesis, composition and inflammatory markers were studied by qPCR and immunohistochemistry and microbiome composition by 16S rRNA sequencing. RESULTS: Colonic pH microclimate was significantly more acidic in slc26a3-/- and to a lesser extent in cftr-/- than in wt mice. Goblet cell thecae per crypt were decreased in slc26a3-/- and increased in cftr-/- colon. Mucus accumulation in vivo was reduced, but much less so than in cftr-/- colon, which is possibly related to the different colonic fluid balance. Slc26a3-/- colonic luminal microbiome displayed strong decrease in diversity. These alterations preceded and maybe causally related to increased mucosal TNFα mRNA expression levels and leucocyte infiltration in the mid-distal colon of slc26a3-/- but not of cftr-/- mice. CONCLUSIONS: These findings may explain the strong increase in the susceptibility of slc26a3-/- mice to DSS damage, and offer insight into the mechanisms leading to an increased incidence of intestinal inflammation in CLD patients.

Deciphering ion transporters, kinases and PDZ-adaptor molecules that mediate guanylate cyclase C agonist-dependent intestinal fluid loss in vivo.

BACKGROUND: The molecular basis for heat-stable Escherichia coli enterotoxin (STa) action and its synthetic analogue linaclotide is well understood at the enterocyte level. Pharmacologic strategies to prevent STa-induced intestinal fluid loss by inhibiting its effector molecules, however, have achieved insufficient inhibition in vivo. AIMS AND EXPERIMENTAL APPROACH: To investigate whether the currently discussed effector molecules and signaling mechanisms of STa/linaclotide-induced diarrhea have similar relevance in vivo than at the enterocyte level, we studied the effect of 10-7M of the STa analogue linaclotide on short circuit current (Isc) of chambered isolated jejunal mucosa, and on the in vivo action on fluid transport in a perfused segment of proximal jejunum of anesthetized mice. The selected mice were deficient of transport (NHE3, CFTR, Slc26a3/a6), adaptor (NHERF1-3), or signal transduction molecules [cGMP-dependent kinase II (GKII)] considered to be downstream effectors after STa/linaclotide binding to guanylate cyclase C (GCC). Selective NHE3 inhibition by tenapanor was also employed. KEY RESULTS, CONCLUSIONS AND IMPLICATIONS: The comparison allowed the separation of effectors for stimulation of electrogenic anion secretion and for inhibition of electrolyte/fluid absorption in response to STa/linaclotide. The cGKII-NHERF1-CFTR and cGKII-NHERF2-NHE3 interactions are indeed major effectors of small intestinal fluid loss downstream of GCC activation in vitro and in vivo, but 50% of the linaclotide-induced fluid loss in vivo, while dependent on CFTR activation and NHE3 inhibition, does not involve cGKII, and 30% does not depend on NHERF1 or NHERF2. A combined NHERF1 and NHERF2 inhibition appears nevertheless a good pharmacological strategy against STa-mediated fluid loss.

Calcium-sensing receptor regulates intestinal dipeptide absorption via Ca2+ signaling and IKCa activation.

Although absorption of di- and tripeptides into intestinal epithelial cells occurs via the peptide transporter 1 (PEPT1, also called solute carrier family 15 member 1 (SLC15A1)), the detailed regulatory mechanisms are not fully understood. We examined: (a) whether dipeptide absorption in villous enterocytes is associated with a rise in cytosolic Ca2+ ([Ca2+ ]cyt ), (b) whether the calcium sensing receptor (CaSR) is involved in dipeptide-elicited [Ca2+ ]cyt signaling, and (c) what potential consequences of [Ca2+ ]cyt signaling may enhance enterocyte dipeptide absorption. Dipeptide Gly-Sar and CaSR agonist spermine markedly raised [Ca2+ ]cyt in villous enterocytes, which was abolished by NPS-2143, a selective CaSR antagonist and U73122, an phospholipase C (PLC) inhibitor. Apical application of Gly-Sar induced a jejunal short-circuit current (Isc), which was reduced by NPS-2143. CaSR expression was identified in the lamina propria and on the basal enterocyte membrane of mouse jejunal mucosa in both WT and Slc15a1-/- animals, but Gly-Sar-induced [Ca2+ ]cyt signaling was significantly decreased in Slc15a1-/- villi. Clotrimazole and TRM-34, two selective blockers of the intermediate conductance Ca2+ -activated K+ channel (IKCa ), but not iberiotoxin, a selective blocker of the large-conductance K+ channel (BKCa ) and apamin, a selective blocker of the small-conductance K+ channel (SKCa ), significantly inhibited Gly-Sar-induced Isc in native tissues. We reveal a novel CaSR-PLC-Ca2+ -IKCa pathway in the regulation of small intestinal dipeptide absorption, which may be exploited as a target for future drug development in human nutritional disorders.

Slc26a3 deficiency is associated with epididymis dysplasia and impaired sperm fertilization potential in the mouse.

Members of the solute carrier 26 (SLC26) family have emerged as important players in mediating anions fluxes across the plasma membrane of epithelial cells, in cooperation with the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Among them, SLC26A3 acts as a chloride/bicarbonate exchanger, highly expressed in the gastrointestinal, pancreatic and renal tissues. In humans, mutations in the SLC26A3 gene were shown to induce congenital chloride-losing diarrhea (CLD), a rare autosomal recessive disorder characterized by life-long secretory diarrhea. In view of some reports indicating subfertility in some male CLD patients together with SLC26-A3 and -A6 expression in the male genital tract and sperm cells, we analyzed the male reproductive parameters and functions of SLC26A3 deficient mice, which were previously reported to display CLD gastro-intestinal features. We show that in contrast to Slc26a6, deletion of Slc26a3 is associated with severe lesions and abnormal cytoarchitecture of the epididymis, together with sperm quantitative, morphological and functional defects, which altogether compromised male fertility. Overall, our work provides new insight into the pathophysiological mechanisms that may alter the reproductive functions and lead to male subfertility in CLD patients, with a phenotype reminiscent of that induced by CFTR deficiency in the male genital tract.

IL-1ß-Induced Downregulation of the Multifunctional PDZ Adaptor PDZK1 Is Attenuated by ERK Inhibition, RXRα, or PPARα Stimulation in Enterocytes.

Background: The PDZ adaptor protein PDZK1 modulates the membrane expression and function of a variety of intestinal receptors and ion/nutrient transporters. Its expression is strongly decreased in inflamed intestinal mucosa of mice and IBD patients. Aim and Methods: We investigated whether the inflammation-associated PDZK1 downregulation is a direct consequence of proinflammatory cytokine release by treating intestinal Caco-2BBE cells with TNF-α, IFN-γ, and IL-1ß, and analysing PDZK1 promotor activity, mRNA and protein expression. Results: IL-1ß was found to significantly decrease PDZK1 promoter activity, mRNA and protein expression in Caco-2BBE cells. A distal region of the hPDZK1 promoter was identified to be important for basal expression and IL-1ß-responsiveness. This region harbors the retinoid acid response element RARE as well as binding sites for transcription factors involved in IL-ß downstream signaling. ERK1/2 inhibition by the specific MEK1/2 inhibitors PD98059/U0126 significantly attenuated the IL-1ß mediated downregulation of PDZK1, while NF-κB, p38 MAPK, and JNK inhibition did not. Expression of the nuclear receptors RXRα and PPARα was decreased in inflamed colonic-mucosa of ulcerative colitis patients and in IL-1ß-treated Caco2-BBE cells. Moreover, the RAR/RXR ligand 9-cis retinoic acid and the PPARα-agonist GW7647 stimulated PDZK1 mRNA and protein expression and attenuated IL-1ß-mediated inhibition. Conclusions: The strong decrease in PDZK1 expression during intestinal inflammation may be in part a consequence of IL-1ß-mediated RXRα and PPARα repression and can be attenuated by agonists for either nuclear receptor, or by ERK1/2 inhibition. The negative consequences of inflammation-induced PDZK1 downregulation on epithelial transport-function may thus be amenable to pharmacological therapy.

Na+ /H+ exchanger NHE1 and NHE2 have opposite effects on migration velocity in rat gastric surface cells.

Following superficial injury, neighbouring gastric epithelial cells close the wound by rapid cell migration, a process called epithelial restitution. Na+ /H+ exchange (NHE) inhibitors interfere with restitution, but the role of the different NHE isoforms expressed in gastric pit cells has remained elusive. The role of the basolaterally expressed NHE1 (Slc9a1) and the presumably apically expressed NHE2 (Slc9a2) in epithelial restitution was investigated in the nontransformed rat gastric surface cell line RGM1. Migration velocity was assessed by loading the cells with the fluorescent dye DiR and following closure of an experimental wound over time. Since RGM1 cells expressed very low NHE2 mRNA and have low transport activity, NHE2 was introduced by lentiviral gene transfer. In medium with pH 7.4, RGM1 cells displayed slow wound healing even in the absence of growth factors and independently of NHE activity. Growth factors accelerated wound healing in a partly NHE1-dependent fashion. Preincubation with acidic pH 7.1 stimulated restitution in a NHE1-dependent fashion. When pH 7.1 was maintained during the restitution period, migratory speed was reduced to ∼10% of the speed at pH 7,4, and the residual restitution was further inhibited by NHE1 inhibition. Lentiviral NHE2 expression increased the steady-state pHi and reduced the restitution velocity after low pH preincubation, which was reversible by pharmacological NHE2 inhibition. The results demonstrate that in RGM1 cells, migratory velocity is increased by NHE1 activation, while NHE2 activity inhibit this process. A differential activation of NHE1 and NHE2 may therefore, play a role in the initiation and completion of the epithelial restitution process.

Defective small intestinal anion secretion, dipeptide absorption, and intestinal failure in suckling NBCe1-deficient mice.

The electrogenic Na(+)HCO3 (-) cotransporter NBCe1 (Slc4a4) is strongly expressed in the basolateral enterocyte membrane in a villous/surface predominant fashion. In order to better understand its physiological function in the intestine, isolated mucosae in miniaturized Ussing chambers and microdissected intestinal villi or crypts loaded with the fluorescent pH-indicator BCECF were studied from the duodenum, jejunum, and colon of 14- to 17-days-old slc4a4-deficient (KO) and WT mice. NBCe1 was active in the basal state in all intestinal segments under study, most likely to compensate for acid loads imposed upon the enterocytes. Upregulation of other basolateral base uptake mechanism occurs, but in a segment-specific fashion. Loss of NBCe1 resulted in severely impaired Cl(-) and fluid secretory response, but not HCO3 (-) secretory response to agonist stimulation. In addition, NBCe1 was found to be active during transport processes that load the surface enterocytes with acid, such as Slc26a3 (DRA)-mediated luminal Cl(-)/HCO3 (-) exchange or PEPT1-mediated H(+)/dipeptide uptake. Possibly because of the high energy demand for hyperventilation in conjunction with the fluid secretory and nutrient absorptive defects and the relative scarcity of compensatory mechanisms, NBCe1-deficient mice developed progressive jejunal failure, worsening of metabolic acidosis, and death in the third week of life. Our data suggest that the electrogenic influx of base via NBCe1 maintains enterocyte anion homeostasis and pHi control. Its loss impairs small intestinal Cl(-) and fluid secretion as well as the neutralization of acid loads imposed on the enterocytes during nutrient and electrolyte absorption.

Evidence for a causal link between adaptor protein PDZK1 downregulation and Na⁺/H⁺ exchanger NHE3 dysfunction in human and murine colitis.

A dysfunction of the Na(+)/H(+) exchanger isoform 3 (NHE3) significantly contributes to the reduced salt absorptive capacity of the inflamed intestine. We previously reported a strong decrease in the NHERF family member PDZK1 (NHERF3), which binds to NHE3 and regulates its function in a mouse model of colitis. The present study investigates whether a causal relationship exists between the decreased PDZK1 expression and the NHE3 dysfunction in human and murine intestinal inflammation. Biopsies from the colon of patients with ulcerative colitis, murine inflamed ileal and colonic mucosa, NHE3-transfected Caco-2BBe colonic cells with short hairpin RNA (shRNA) knockdown of PDZK1, and Pdzk1-gene-deleted mice were studied. PDZK1 mRNA and protein expression was strongly decreased in inflamed human and murine intestinal tissue as compared to inactive disease or control tissue, whereas that of NHE3 or NHERF1 was not. Inflamed human and murine intestinal tissues displayed correct brush border localization of NHE3 but reduced acid-activated NHE3 transport activity. A similar NHE3 transport defect was observed when PDZK1 protein content was decreased by shRNA knockdown in Caco-2BBe cells or when enterocyte PDZK1 protein content was decreased to similar levels as found in inflamed mucosa by heterozygote breeding of Pdzk1-gene-deleted and WT mice. We conclude that a decrease in PDZK1 expression, whether induced by inflammation, shRNA-mediated knockdown, or heterozygous breeding, is associated with a decreased NHE3 transport rate in human and murine enterocytes. We therefore hypothesize that inflammation-induced loss of PDZK1 expression may contribute to the NHE3 dysfunction observed in the inflamed intestine.

Loss of Slc26a9 anion transporter alters intestinal electrolyte and HCO3(-) transport and reduces survival in CFTR-deficient mice.

Slc26a9 is an anion transporter that is strongly expressed in the stomach and lung. Slc26a9 variants were recently found associated with a higher incidence of meconium ileus in cystic fibrosis (CF) infants, raising the question whether Slc26a9 is expressed in the intestine and what its functional role is. Slc26a9 messenger RNA (mRNA) was found highly expressed in the mucosae of the murine and human upper gastrointestinal tract, with an abrupt decrease in expression levels beyond the duodenum. Absence of SLC26a9 expression strongly increased the intestinally related mortality in cystic fibrosis transmembrane conductance regulator (CFTR)-deficient mice. Proximal duodenal JHCO3(-) and fluid secretion were reduced in the absence of Slc26a9 expression. In the proximal duodenum of young Slc26a9 KO mice, the glands and villi/crypts were elongated and proliferation was enhanced. This difference was lost with ageing, as were the alterations in fluid movement, whereas the reduction in JHCO3(-) remained. Laser dissection followed by qPCR suggested Slc26a9 expression to be crypt-predominant in the duodenum. In summary, deletion of Slc26a9 caused bicarbonate secretory and fluid absorptive changes in the proximal duodenal mucosa and increased the postweaning death rates in CFTR-deficient mice. Functional alterations in the duodenum were most prominent at young ages. We assume that the association of meconium ileus and Slc26a9 variants may be related to maldigestion and impaired downstream signaling caused by loss of upper GI tract digestive functions, aggravating the situation of lack of secretion and sticky mucus at the site of obstruction in CF intestine.

An unusually powerful mode of low-frequency sound interference due to defective hair bundles of the auditory outer hair cells.

A detrimental perceptive consequence of damaged auditory sensory hair cells consists in a pronounced masking effect exerted by low-frequency sounds, thought to occur when auditory threshold elevation substantially exceeds 40 dB. Here, we identified the submembrane scaffold protein Nherf1 as a hair-bundle component of the differentiating outer hair cells (OHCs). Nherf1(-/-) mice displayed OHC hair-bundle shape anomalies in the mid and basal cochlea, normally tuned to mid- and high-frequency tones, and mild (22-35 dB) hearing-threshold elevations restricted to midhigh sound frequencies. This mild decrease in hearing sensitivity was, however, discordant with almost nonresponding OHCs at the cochlear base as assessed by distortion-product otoacoustic emissions and cochlear microphonic potentials. Moreover, unlike wild-type mice, responses of Nherf1(-/-) mice to high-frequency (20-40 kHz) test tones were not masked by tones of neighboring frequencies. Instead, efficient maskers were characterized by their frequencies up to two octaves below the probe-tone frequency, unusually low intensities up to 25 dB below probe-tone level, and growth-of-masker slope (2.2 dB/dB) reflecting their compressive amplification. Together, these properties do not fit the current acknowledged features of a hypersensitivity of the basal cochlea to lower frequencies, but rather suggest a previously unidentified mechanism. Low-frequency maskers, we propose, may interact within the unaffected cochlear apical region with midhigh frequency sounds propagated there via a mode possibly using the persistent contact of misshaped OHC hair bundles with the tectorial membrane. Our findings thus reveal a source of misleading interpretations of hearing thresholds and of hypervulnerability to low-frequency sound interference.

Pseudomonas aeruginosa reduces the expression of CFTR via post-translational modification of NHERF1.

Pseudomonas aeruginosa infections of the airway cells decrease apical expression of both wild-type (wt) and F508del CFTR through the inhibition of apical endocytic recycling. CFTR endocytic recycling is known to be regulated by its interaction with PDZ domain containing proteins. Recent work has shown that the PDZ domain scaffolding protein NHERF1 finely regulates both wt and F508delCFTR membrane recycling. Here, we investigated the effect of P. aeruginosa infection on NHERF1 post-translational modifications and how this affects CFTR expression in bronchial epithelial cells and in murine lung. Both in vitro in bronchial cells, and in vivo in mice, infection reduced CFTR expression and increased NHERF1 molecular weight through its hyper-phosphorylation and ubquitination as a consequence of both bacterial pilin- and flagellin-mediated host-cell interaction. The ability of P. aeruginosa to down-regulate mature CFTR expression was reduced both in vivo in NHERF1 knockout mice and in vitro after silencing NHERF1 expression or mutations blocking its phosphorylation at serines 279 and 301. These studies provide the first evidence that NHERF1 phosphorylation may negatively regulate its action and, therefore, the assembly and function of multiprotein NHERF1 complexes in response to infection. The identification of molecular mechanisms responsible for these effects could identify novel targets to block potential P. aeruginosa interference with the efficacy of potentiator and/or corrector compounds.

Short-term regulation of murine colonic NBCe1-B (electrogenic Na+/HCO3(-) cotransporter) membrane expression and activity by protein kinase C.

The colonic mucosa actively secretes HCO3(-), and several lines of evidence point to an important role of Na+/HCO3(-) cotransport (NBC) as a basolateral HCO3(-) import pathway. We could recently demonstrate that the predominant NBC isoform in murine colonic crypts is electrogenic NBCe1-B, and that secretagogues cause NBCe1 exocytosis, which likely represents a component of NBC activation. Since protein kinase C (PKC) plays a key role in the regulation of ion transport by trafficking events, we asked whether it is also involved in the observed NBC activity increase. Crypts were isolated from murine proximal colon to assess PKC activation as well as NBC function and membrane abundance using fluorometric pHi measurements and cell surface biotinylation, respectively. PKC isoform translocation and phosphorylation occurred in response to PMA-, as well as secretagogue stimulation. The conventional and novel PKC inhibitors Gö6976 or Gö6850 did not alter NBC function or surface expression by themselves, but stimulation with forskolin (10(-5) M) or carbachol (10(-4) M) in their presence led to a significant decrease in NBC-mediated proton flux, and biotinylated NBCe1. Our data thus indicate that secretagogues lead to PKC translocation and phosphorylation in murine colonic crypts, and that PKC is necessary for the increase in NBC transport rate and membrane abundance caused by cholinergic and cAMP-dependent stimuli.

Na⁺/H⁺ exchanger regulatory factor 3 is critical for multidrug resistance protein 4-mediated drug efflux in the kidney.

Na(+)/H(+) exchanger regulatory factor 3 (NHERF3) is a PSD-95/discs large/ZO-1 (PDZ)-based adaptor protein that regulates several membrane-transporting proteins in epithelia. However, the in vivo physiologic role of NHERF3 in transepithelial transport remains poorly understood. Multidrug resistance protein 4 (MRP4) is an ATP binding cassette transporter that mediates the efflux of organic molecules, such as nucleoside analogs, in the gastrointestinal and renal epithelia. Here, we report that Nherf3 knockout (Nherf3(-/-)) mice exhibit profound reductions in Mrp4 expression and Mrp4-mediated drug transport in the kidney. A search for the binding partners of the COOH-terminal PDZ binding motif of MRP4 among several epithelial PDZ proteins indicated that MRP4 associated most strongly with NHERF3. When expressed in HEK293 cells, NHERF3 increased membrane expression of MRP4 by reducing internalization of cell surface MRP4 and consequently, augmented MRP4-mediated efflux of adefovir, a nucleoside-based antiviral agent and well known substrate of MRP4. Examination of wild-type and Nherf3(-/-) mice revealed that Nherf3 is most abundantly expressed in the kidney and has a prominent role in modulating Mrp4 levels. Deletion of Nherf3 in mice caused a profound reduction in Mrp4 expression at the apical membrane of renal proximal tubules and evoked a significant increase in the plasma and kidney concentrations of adefovir, with a corresponding decrease in the systemic clearance of this drug. These results suggest that NHERF3 is a key regulator of organic transport in the kidney, particularly MRP4-mediated clearance of drug molecules.

The distinct roles of anion transporters Slc26a3 (DRA) and Slc26a6 (PAT-1) in fluid and electrolyte absorption in the murine small intestine.

The mixing of gastric and pancreatic juice subjects the jejunum to unique ionic conditions with high luminal CO2 tension and HCO3 − concentration. We investigated the role of the small intestinal apical anion exchangers PAT-1 (Slc26a6) and DRA (Slc26a3) in basal and CO2/HCO3 −-stimulated jejunal fluid absorption. Single pass perfusion of jejunal segments was performed in anaesthetised wild type (WT) as well as in mice deficient in DRA, PAT-1, Na+/H+ exchanger 3 (NHE3) or NHE2, and in carbonic anhydrase II (CAII). Unbuffered saline (pH 7.4) perfusion of WT jejunum resulted in fluid absorption and acidification of the effluent. DRA-deficient jejunum absorbed less fluid than WT, and acidified the effluent more strongly, consistent with its action as a Cl−/HCO3 − exchanger. PAT-1-deficient jejunum also absorbed less fluid but resulted in less effluent acidification. Switching the luminal solution to a 5 % CO2/HCO3 − buffered solution (pH 7.4), resulted in a decrease in jejunal enterocyte pHi in all genotypes, an increase in luminal surface pH and a strong increase in fluid absorption in a PAT-1- and NHE3- but not DRA-, CAII, or NHE2-dependent fashion. Even in the absence of luminal Cl−, luminal CO2/HCO3 − augmented fluid absorption in WT, CAII, NHE2- or DRA-deficient, but not in PAT-1- or NHE3-deficient mice, indicating the likelihood that PAT-1 serves to import HCO3 − and NHE3 serves to import Na+ under these circumstances. The results suggest that PAT-1 plays an important role in jejunal Na+HCO3 ­ reabsorption, while DRA absorbs Cl− and exports HCO3 − in a partly CAII-dependent fashion. Both PAT-1 and DRA significantly contribute to intestinal fluid absorption and enterocyte acid/base balance but are activated by different ion gradients.

The role of pancreatic ductal secretion in protection against acute pancreatitis in mice*.

OBJECTIVES: A common potentially fatal disease of the pancreas is acute pancreatitis, for which there is no treatment. Most studies of this disorder focus on the damage to acinar cells since they are assumed to be the primary target of multiple stressors affecting the pancreas. However, increasing evidence suggests that the ducts may also have a crucial role in induction of the disease. To test this hypothesis, we sought to determine the specific role of the duct in the induction of acute pancreatitis using well-established disease models and mice with deletion of the Na/H exchanger regulatory factor-1 that have selectively impaired ductal function. DESIGN: Randomized animal study. SETTING: Animal research laboratory. SUBJECTS: Wild-type and Na/H exchanger regulatory factor-1 knockout mice. INTERVENTIONS: Acute necrotizing pancreatitis was induced by i.p. administration of cerulein or by intraductal administration of sodium taurocholate. The pancreatic expression of Na/H exchanger regulatory factor-1 and cystic fibrosis transmembrane conductance regulator (a key player in the control of ductal secretion) was analyzed by immunohistochemistry. In vivo pancreatic ductal secretion was studied in anesthetized mice. Functions of pancreatic acinar and ductal cells as well as inflammatory cells were analyzed in vitro. MEASUREMENTS AND MAIN RESULTS: Deletion of Na/H exchanger regulatory factor-1 resulted in gross mislocalization of cystic fibrosis transmembrane conductance regulator, causing marked reduction in pancreatic ductal fluid and bicarbonate secretion. Importantly, deletion of Na/H exchanger regulatory factor-1 had no deleterious effect on functions of acinar and inflammatory cells. Deletion of Na/H exchanger regulatory factor-1, which specifically impaired ductal function, increased the severity of acute pancreatitis in the two mouse models tested. CONCLUSIONS: Our findings provide the first direct evidence for the crucial role of ductal secretion in protecting the pancreas from acute pancreatitis and strongly suggest that improved ductal function should be an important modality in prevention and treatment of the disease.