CFTR Cl Research Articles

Cl- and regulation of pH by MDCK-C11 cells

The interaction between H+ extrusion via H+-ATPase and Cl- conductance was studied in the C11 clone of MDCK cells, akin to the intercalated cells of the collecting duct. Cell pH (pHi) was measured by fluorescence microscopy using the fluorescein-derived probe BCECF-AM. Control recovery rate measured after a 20 mM NH4Cl acid pulse was 0.136 ± 0.008 pH units/min (dpHi/dt) in Na+ Ringer and 0.032 ± 0.003 in the absence of Na+ (0 Na+). With 0 Na+ plus the Cl- channel inhibitor NPPB (10 µM), recovery was reduced to 0.014 ± 0.001 dpHi/dt. 8-Br-cAMP, known to activate CFTR Cl- channels, increased dpHi/dt in 0 Na+ to 0.061 ± 0.009 and also in the presence of 46 nM concanamycin and 50 µM Schering 28080. Since it is thought that the Cl- dependence of H+-ATPase might be due to its electrogenic nature and the establishment of a +PD (potential difference) across the cell membrane, the effect of 10 µM valinomycin at high (100 mM) K+ was tested in our cells. In Na+ Ringer, dpHi/dt was increased, but no effect was detected in 0 Na+ Ringer in the presence of NPPB, indicating that in intact C11 cells the effect of blocking Cl- channels on dpHi/dt was not due to an adverse electrical gradient. The effect of 100 µM ATP was studied in 0 Na+ Ringer solution; this treatment caused a significant inhibition of dpHi/dt, reversed by 50 µM Bapta. We have shown that H+-ATPase present in MDCK C11 cells depends on Cl- ions and their channels, being regulated by cAMP and ATP, but not by the electrical gradient established by electrogenic H+ transport.

Inhibition of CFTR Cl − channel function caused by enzymatic hydrolysis of sphingomyelin

Numerous mutations in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR, a Cl(-) channel) disrupt salt and fluid transport and lead to the formation of thick mucus in patients' airways. Obstruction by mucus predisposes CF patients to chronic infections and inflammation, which become gradually harder to control and eventually fatal. Aggressive antibiotic therapy and supportive measures have dramatically lengthened CF patients' lives. Here, we report that sphingomyelinases (SMase) from human respiratory pathogens strongly inhibit CFTR function. The hydrolysis of sphingomyelin by SMase makes it more difficult to activate CFTR by phosphorylation of its regulatory domain. By inhibiting CFTR currents, SMase-producing respiratory tract bacteria may not only aggravate pulmonary infection in some CF patients but may also elicit a condition, analogous to CFTR deficiency, in non-CF patients suffering from bacterial lung infection.

Seasonal changes in ionoregulatory variables of the glass eel Anguilla anguilla following estuarine entry: comparison with resident elvers

In the present study, glass eels Anguilla anguilla in the Minho River estuary (41·5° N, 8·5° W) decreased in size (standard length, LS and mass, M) from the beginning (autumn) to the end of the sampling season (summer). On the other hand elvers increased in LS and M from spring to summer and were significantly larger than glass eels in paired comparisons. Branchial Na+/K+‐ATPase and vacuolar (V‐type) proton ATPase (in vitro activities), two important ion transporting pumps, did not show significant seasonal changes in either glass eels or elvers although in glass eels Na+/K+‐ATPase (activity) expression was significantly higher than in elvers. In a single month comparison Na+/K+‐ATPase branchial mRNA expression was also higher in glass eels as was the protein level expression of both Na+/K+‐ATPase and NKCC (Na+:K+:2Cl− co‐transporter). Immunofluorescence microscopy indicated apical CFTR Cl− channel labelling in Na+/K+‐ATPase positive chloride cell in glass eels which was absent in elvers. Whole body sodium concentration and percentage water did not show significant seasonal differences in either glass eels or elvers although there were significant differences between these two groups during some months.

Basolateral Cl channels in primary airway epithelial cultures

Salt and water absorption and secretion across the airway epithelium are important for maintaining the thin film of liquid lining the surface of the airway epithelium. Movement of Cl across the apical membrane involves the CFTR Cl channel; however, conductive pathways for Cl movement across the basolateral membrane have been little studied. Here, we determined the regulation and single-channel properties of the Cl conductance (G(Cl)) in airway surface epithelia using epithelial cultures from human or bovine trachea and freshly isolated ciliated cells from the human nasal epithelium. In Ussing chamber studies, a swelling-activated basolateral G(Cl) was found, which was further stimulated by forskolin and blocked by N-phenylanthranilic acid (DPC) = sucrose > flufenamic acid = niflumic acid = glibenclamide > CdCl(2) = 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) = DIDS = ZnCl(2) > tamoxifen > 4,4'-dinitro-2,2'-stilbene-disulfonate disodium salt (DNDS). In whole cell patch-clamp experiments, three types of G(Cl) were identified: 1) a voltage-activated, DIDS- (but not Cd-) blockable and osmosensitive G(Cl); 2) an inwardly rectifying, hyperpolarization-activated and Cd-sensitive G(Cl); and 3) a forskolin-activated, linear G(Cl), which was insensitive to Cd and DIDS. In cell-attached patch-clamp recordings, the basolateral pole of isolated ciliated cells expressed three types of Cl channels: 1) an outwardly rectifying, swelling-activated Cl channel; 2) a strongly inwardly rectifying Cl channel; and 3) a forskolin-activated, low-conductance channel. We propose that, depending on the driving force for Cl across the apical membrane, basolateral Cl channels confine Cl(-) secretion or support transcellular Cl(-) absorption.

Plasticity of osmoregulatory function in the killifish intestine:drinking rates, salt and water transport, and gene expression after freshwater transfer

We have explored intestinal function in the euryhaline killifish Fundulus heteroclitus after transfer from brackish water (10% seawater) to fresh water. Plasma Na+ and Cl- concentrations fell at 12 h post-transfer, but recovered by 7 days. Drinking rate decreased substantially at 12 h (32% of control value) and remained suppressed after 3 and 7 days in fresh water (34 and 43%). By contrast, there was a transient increase in the capacity for water absorption measured across isolated intestines in vitro (3.3- and 2.6-fold at 12 h and 3 days), which returned to baseline after 7 days. These changes in water absorption could be entirely accounted for by changes in net ion flux: there was an extremely strong correlation (R2=0.960) between water absorption and the sum of net Na+ and net Cl- fluxes (3.42+/-0.10 microl water micromol(-1) ion). However, enhanced ion transport across the intestine in fresh water would probably not increase water uptake in vivo, because the drinking rate was far less than the capacity for water absorption across the intestine. The increased intestinal ion absorption after freshwater transfer may instead serve to facilitate ion absorption from food when it is present in the gut. Modulation of net ion flux occurred without changes in mRNA levels of many ion transporters (Na+/K+-ATPase alpha(1a), carbonic anhydrase 2, CFTR Cl- channel, Na+/K+/2Cl- cotransporter 2, and the signalling protein 14-3-3a), and before a measured increase in Na+/K+-ATPase activity at 3 days, suggesting that there is some other mechanism responsible for increasing ion transport. Interestingly, net Cl- flux always exceeded net Na+ flux, possibly to help maintain Cl- balance and/or facilitate bicarbonate excretion. Our results suggest that intestinal NaCl absorption from food is important during the period of greatest ionic disturbance after transfer to fresh water, and provide further insight into the mechanisms of euryhalinity in killifish.

The N-Terminus of the WD5 Repeat of Human RACK1 Binds to Airway Epithelial NHERF1

Regulation of the CFTR Cl channel function involves a protein complex of activated protein kinase Cepsilon (PKCepsilon) bound to RACK1, a receptor for activated C kinase, and RACK1 bound to the human Na(+)/H(+) exchanger regulatory factor (NHERF1) in human airway epithelial cells. Binding of NHERF1 to RACK1 is mediated via a NHERF1-PDZ1 domain. The goal of this study was to identify the binding motif for human NHERF1 on RACK1. We examined the site of binding of NHERF1 on RACK1 using peptides encoding the seven WD40 repeat units of human RACK1. One WD repeat peptide, WD5, directly binds NHERF1 and the PDZ1 domain with similar EC(50) values, blocks binding of recombinant RACK1 and NHERF1, and pulls down endogenous RACK1 from Calu-3 cell lysate in a dose-dependent manner. The remaining WD repeat peptides did not block RACK1-NHERF1 binding. An 11-amino acid peptide encoding a site on the PDZ1 domain blocks binding of the WD5 repeat peptide with the PDZ1 domain. An N-terminal 12-amino acid segment of the WD5 repeat peptide, which comprises the first of four antiparallel beta-strands, dose-dependently binds to the PDZ1 domain of NHERF1 and blocks binding of the PDZ1 domain to RACK1. These results suggest that the binding site might form a beta-turn with topology sufficient for binding of NHERF1. Our results also demonstrate binding of NHERF to RACK1 at the WD5 repeat, which is distinct from the PKCepsilon binding site on the WD6 repeat of RACK1.

Mechanisms of Acid and Base Secretion by the Airway Epithelium

One of the main functions of the airway epithelium is to inactivate and remove infectious particles from inhaled air and thereby prevent infection of the distal lung. This function is achieved by mucociliary and cough clearance and by antimicrobial factors present in the airway surface liquid (ASL). There are indications that airway defenses are affected by the pH of the ASL and historically, acidification of the airway surfaces has been suggested as a measure of airway disease. However, even in health, the ASL is slightly acidic, and this acidity might be part of normal airway defense. Only recently research has focused on the mechanisms responsible for acid and base secretion into the ASL. Advances resulted from research into the airway disease associated with cystic fibrosis (CF) after it was found that the CFTR Cl(-) channel conducts HCO (3) (-) and, therefore, may contribute to ASL pH. However, the acidity of the ASL indicated parallel mechanisms for H(+) secretion. Recent investigations identified several H(+) transporters in the apical membrane of the airway epithelium. These include H(+) channels and ATP-driven H(+) pumps, including a non-gastric isoform of the H(+)-K(+) ATPase and a vacuolar-type H(+) ATPase. Current knowledge of acid and base transporters and their potential roles in airway mucosal pH regulation is reviewed here.

Alveolar Epithelial Ion and Fluid Transport

Studies of epithelial ion and fluid transport across the distal pulmonary epithelia have provided important new concepts regarding the resolution of pulmonary edema, specifically the removal of edema from the distal airspaces of the lung. Overall, there is convincing evidence that vectorial ion transport across the alveolar and distal airway epithelia is the primary determinant of alveolar fluid clearance (AFC). The general paradigm is that active Na and Cl transport drives net alveolar fluid clearance, as demonstrated in several different species, including the human lung. The objective of this article is to consider some areas of recent progress in the field of alveolar fluid transport under normal and pathologic conditions. More detailed reviews of this field including studies of the immature and the newborn lung are available (1–10). In the lung, as in other epithelia, ion transporters and other membrane proteins are asymmetrically distributed on opposing cell surfaces, conferring vectorial transport properties to the polarized epithelial cells (Figure 1). There are epithelial cells in the distal airway epithelia, such as Clara cells, that are capable of vectorial ion transport. The vast majority of the surface area available for transport in the distal lung is occupied by the alveolar epithelial type I (ATI) and type II cells (ATII). Tight junctions populate these epithelial cells near their apical surfaces, thereby sustaining apical and basolateral cell polarity (11). The permeability of tight junctions is dynamic and regulated, in part, by cytoskeletal proteins and intracellular Ca concentrations and possibly by ion channels (11). Figure 1. A section of an ARDS lung. Note the fluid-filled alveolar spaces with significant red blood cell infiltration. Insert demonstrates apical ENaC staining and basolateral Na,K-ATPase staining in lung epithelia. (Reprinted with permission from Ref. 3) Because ATII cells can be isolated from the lung and studied in vitro, they have been studied extensively. ATII cells are responsible for surfactant secretion (12) as well as vectorial Na and Cl transport. Na uptake occurs on the apical surface, partly through amiloride-sensitive and amiloride-insensitive channels. Subsequently, Na is pumped actively from the basolateral surface into the lung interstitium by the Na,K-ATPase. An epithelial Na channel (ENaC) participating in Na movement across the apical cell membrane has been cloned and well characterized (13, 14). Recent evidence indicates that the CFTR is expressed in ATII cells and plays a role in cAMP-mediated fluid transport (15–18). The role of ATI cells for AFC is less known, although several studies have established a possible contribution of ATI cells to vectorial fluid transport (19–22). ATI cells express aquaporin 5 (23), Na,K-ATPase (21), and ENaC (21, 22). The presence of Na,K-ATPase is consistent with a role for ATI cells in AFC, but is not conclusive since Na,K-ATPases are needed to maintain cell volume. However, one study using pharmacologic methods to inhibit the α2-subunit of the Na,K-ATPase suggested a role for Na,K-ATPase in driving fluid clearance across type I cells in vivo (24). Another study reported a role for the α2-Na,K-ATPase under cAMP-stimulated conditions, suggesting that type I cells may be involved, as the α2-subunit seems to be expressed only in type I cells (25). Detailed studies of type I cells have been limited to date because of difficulty maintaining them in cell culture although recent work has demonstrated functional ion channels in freshly isolated ATI cells with electrophysiologic evidence for Na channels (ENaC), K channels, and CFTR Cl channels (26). In addition, there is evidence for ENaC expression (21, 22) and a partial amiloride inhibition of 22Na-uptake in freshly isolated rat ATI cells (22). Evidence for β-adrenoceptor (βAR) expression in ATI cells has also been reported (20, 27). In addition, ATI cells may be involved in macromolecular transport due to the presence of vesicles and caveolin (28). The distal airway epithelium also actively transports Na (29–31).

Synergic action of insulin and genistein on Na+/K+/2Cl‐ cotransporter (NKCC) in renal epithelium

We studied the effect of insulin and quercetin on Cl- secretion in renal epithelial A6 cells by estimating Cl- channel activity and Cl− secretion by measuring NPPB-sensitive transepithelial conductance (Gt) and short-circuit current (Isc). Pretreatment with 1mM insulin for 24h had no effects on Gt or Isc, but increased the Isc with no effects on Gt in A6 cells treated with MG132 (100mM for 2h) that inhibits endocytosis of proteins at the plasma membrane into the cytosolic space. Genistein (100mM) induced sustained increases in Gt and Isc, which were diminished by brefeldin A. Co-application of insulin and genistein synergically stimulated the Isc without any effects on Gt. These observations suggest that: 1) insertion and endocytosis of NKCC are stimulated by insulin, 2) the insulin-induced stimulation of NKCC insertion into the basolateral membrane is offset by the stimulatory action on NKCC endocytosis from the basolateral membrane, 3) genistein stimulates both insertion of CFTR Cl− channel into the apical membrane and insertion of NKCC into the basolateral membrane, and 4) insulin and genistein synergically stimulated NKCC insertion into the basolateral membrane. Supported by JSPS (17390057, 17590191).

Characterization of H+and HCO3-transporters in CFPAC-1 human pancreatic duct cells

To characterize H+ and HCO3- transporters in polarized CFPAC-1 human pancreatic duct cells, which were derived from a cystic fibrosis patient with the DeltaF508 CFTR mutation. CFPAC-1 cells were seeded at high density onto permeable supports and grown to confluence. The cells were loaded with the pH-sensitive fluorescent dye BCECF, and mounted into a perfusion chamber, which allowed the simultaneous perfusion of the basolateral and apical membranes. Transmembrane base flux was calculated from the changes in intracellular pH and the buffering capacity of the cells. Our results showed differential permeability to HCO3-/CO2 at the apical and basolateral membranes of CFPAC-1 cells. Na+/ HCO3- co-transporters (NBCs) and Cl-/ HCO3- exchangers (AEs) were present on the basolateral membrane, and Na+/H+ exchangers (NHEs) on both the apical and basolateral membranes of the cells. Basolateral HCO3- uptake was sensitive to variations of extracellular K+ concentration, the membrane permeable carbonic anhydrase (CA) inhibitors acetazolamide (100 micromol/L) and ethoxyzolamide (100 micromol/L), and was partially inhibited by H2-DIDS (600 micromol/L). The membrane-impermeable CA inhibitor 1-N-(4-sulfamoylphenylethyl)-2,4,6-trimethylpyridine perchlorate did not have any effect on HCO3- uptake. The basolateral AE had a much higher activity than that in the apical membrane, whereas there was no such difference with the NHE under resting conditions. Also, 10 micromol/L forskolin did not significantly influence Cl-/ HCO3- exchange on the apical and basolateral membranes. The administration of 250 micromol/L H2-DIDS significantly inhibited the basolateral AE. Amiloride (300 micromol/L) completely inhibited NHEs on both membranes of the cells. RT-PCR revealed the expression of pNBC1, AE2, and NHE1 mRNA. These data suggest that apart from the lack of CFTR and apical Cl-/ HCO3- exchanger activity, CFPAC-1 cells express similar H+ and HCO3- transporters to those observed in native animal tissue.

Acidic extracellular pH-activated outwardly rectifying chloride current in mammalian cardiac myocytes

Extracellular acidic pH was found to induce an outwardly rectifying Cl- current (I(Cl,acid)) in mouse ventricular cells, with a half-maximal activation at pH 5.9. The current showed the permeability sequence for anions to be SCN- > Br- > I- > Cl- > F- > aspartate, while it exhibited a time-dependent activation at large positive potentials. Similar currents were also observed in mouse atrial cells and in atrial and ventricular cells from guinea pig. Some Cl- channel blockers (DIDS, niflumic acid, and glibenclamide) inhibited ICl,acid, whereas tamoxifen had little effect on it. Unlike volume-regulated Cl- current (ICl,vol) and CFTR Cl- current (ICl,CFTR), ICl,acid was independent of the presence of intracellular ATP. Activation of ICl,acid appeared to be also independent of intracellular Ca2+ and G protein. ICl,acid and ICl,vol could develop in an additive fashion in acidic hypotonic solutions. Isoprenaline-induced ICl,CFTR was inhibited by acidification in a pH-dependent manner in guinea pig ventricular cells. Our results support the view that ICl,acid and ICl,vol stem from two distinct populations of anion channels and that the ICl,acid channels are present in cardiac cells. ICl,acid may play a role in the control of action potential duration or cell volume under pathological conditions, such as ischemia-related cardiac acidosis.

Pseudomonas aeruginosa inhibits endocytic recycling of CFTR in polarized human airway epithelial cells

The most common mutation in the CFTR gene in individuals with cystic fibrosis (CF), DeltaF508, leads to the absence of CFTR Cl(-) channels in the apical plasma membrane, which in turn results in impairment of mucociliary clearance, the first line of defense against inhaled bacteria. Pseudomonas aeruginosa is particularly successful at colonizing and chronically infecting the lungs and is responsible for the majority of morbidity and mortality in patients with CF. Rescue of DeltaF508-CFTR by reduced temperature or chemical means reveals that the protein is at least partially functional as a Cl(-) channel. Thus current research efforts have focused on identification of drugs that restore the presence of CFTR in the apical membrane to alleviate the symptoms of CF. Because little is known about the effects of P. aeruginosa on CFTR in the apical membrane, whether P. aeruginosa will affect the efficacy of new drugs designed to restore the plasma membrane expression of CFTR is unknown. Accordingly, the objective of the present study was to determine whether P. aeruginosa affects CFTR-mediated Cl(-) secretion in polarized human airway epithelial cells. We report herein that a cell-free filtrate of P. aeruginosa reduced CFTR-mediated transepithelial Cl(-) secretion by inhibiting the endocytic recycling of CFTR and thus the number of WT-CFTR and DeltaF508-CFTR Cl(-) channels in the apical membrane in polarized human airway epithelial cells. These data suggest that chronic infection with P. aeruginosa may interfere with therapeutic strategies aimed at increasing the apical membrane expression of DeltaF508-CFTR.

Disruption of CFTR chloride channel alters mechanical properties and cAMP-dependent Cl- transport of mouse aortic smooth muscle cells.

Chloride (Cl(-)) channels expressed in vascular smooth muscle cells (VSMC) are important to control membrane potential equilibrium, intracellular pH, cell volume maintenance, contraction, relaxation and proliferation. The present study was designed to compare the expression, regulation and function of CFTR Cl(-) channels in aortic VSMC from Cftr(+/+) and Cftr(-)(/)(-) mice. Using an iodide efflux assay we demonstrated stimulation of CFTR by VIP, isoproterenol, cAMP agonists and other pharmacological activators in cultured VSMC from Cftr(+/+). On the contrary, in cultured VSMC from Cftr(-)(/)(-) mice these agonists have no effect, showing that CFTR is the dominant Cl(-) channel involved in the response to cAMP mediators. Angiotensin II and the calcium ionophore A23187 stimulated Ca(2)(+)-dependent Cl(-) channels in VSMCs from both genotypes. CFTR was activated in myocytes maintained in medium containing either high potassium or 5-hydroxytryptamine (5-HT) and was inhibited by CFTR(inh)-172, glibenclamide and diphenylamine-2,2'-dicarboxylic acid (DPC). We also examined the mechanical properties of aortas. Arteries with or without endothelium from Cftr(-/-) mice became significantly more constricted (approximately 2-fold) than that of Cftr(+/+) mice in response to vasoactive agents. Moreover, in precontracted arteries of Cftr(+/+) mice, VIP and CFTR activators induced vasorelaxation that was altered in Cftr(-/-) mice. Our findings suggest a novel mechanism for regulation of the vascular tone by cAMP-dependent CFTR chloride channels in VSMC. To our knowledge this study is the first to report the phenotypic consequences of the loss of a Cl(-) channel on vascular reactivity.

Ionic transport in tall columnar epithelial (TCE) cells obtained by nasal brushing from non-cystic fibrosis (CF) individuals.

Tall columnar epithelial (TCE) cells can be obtained by a non-invasive procedure through brushing the inferior turbinate and the adjacent lateral nasal wall. Here, we present results from the functional study of epithelial cells, thus obtained by using the patch-clamp technique. By patch-clamping the sub-apical region of TCE cells, we were able to identify at least three different groups of Cl- channels, namely: a) one with large conductance, rectifying, which was the most frequently found type of Cl- channel; b) a second type of small conductance, activated by cAMP and IBMX, in excised inside-out patches and voltage independent; c) a third type with a conductance around 25 pS, voltage independent, with a linear IV relationship, that could be observed in the excised inside-out configuration. The study of CFTR Cl- channel and its role in airway cell physiology has generally been conducted in cultured cells, most of which not polarized. This experimental work using freshly obtained TCE cells from the nasal epithelium, demonstrates that such cells may be one valid tool to study Cl- channels (most probably ORCC and CFTR Cl- channels) as a model for the lower respiratory epithelium.

Protection of ischemic injury by activation of CFTR Cl− channel in heart in vivo

Protection of ischemic injury by activation of CFTR Cl− channel in heart in vivo

Vasopressin‐stimulated CFTR Cl− currents are increased in the renal collecting duct cells of a mouse model of Liddle's syndrome

Liddle's syndrome is a genetic form of hypertension linked to Na(+) retention caused by activating mutations in the COOH terminus of the beta or gamma subunit of the epithelial sodium channel (ENaC). In this study, we used the short-circuit current (I(sc)) method to investigate the effects of deamino-8-d-arginine vasopressin (dDAVP) on Na(+) and Cl(-) fluxes in primary cultures of cortical collecting ducts (CCDs) microdissected from the kidneys of mice with Liddle's syndrome carrying a stop codon mutation, corresponding to the beta-ENaC R(566) stop mutation (L) found in the original pedigree. Compared to wild-type (+/+) CCD cells, untreated L/+ and L/L CCD cells exhibited 2.7- and 4.2-fold increases, respectively, in amiloride-sensitive (Ams) I(sc), reflecting ENaC-dependent Na(+) absorption. Short-term incubation with dDAVP caused a rapid and significant increase (approximately 2-fold) in Ams I(sc) in +/+, but not in L/+ or L/L CCD cells. In sharp contrast, dDAVP induced a greater increase in 5-nitro-2-(3-phenylpropamino)benzoate (NPPB)-inhibited apical Cl(-) currents in amiloride-treated L/L and L/+ cells than in their +/+ counterparts. I(sc) recordings performed under apical ion substituted conditions revealed that the dDAVP-stimulated apical secretion of Cl(-), which was absent in cultured CCDs lacking CFTR, was 1.8-fold greater in L/+ and 3.7-fold greater in L/L CCD cells than in their +/+ CCD counterparts. After the basal membrane had been permeabilized with nystatin and a basal-to-apical Cl(-) gradient had been imposed, dDAVP also stimulated larger Cl(-) currents across L/L and L/+ CCD layers than +/+ CCD layers. These findings demonstrate that vasopressin stimulates greater apical CFTR Cl(-) conductance in the renal CCD cells of mice with Liddle's syndrome than in wild-type mice. This effect could contribute to the enhanced NaCl reabsorption observed in the distal nephron of patients with Liddle's syndrome.

A novel extract SB-300 from the stem bark latex of Croton lechleri inhibits CFTR-mediated chloride secretion in human colonic epithelial cells

An oligomeric proanthocyanidin (SP-303) extracted from the bark latex of the tree Croton lechleri (family Euphorbiaceae) is a potent inhibitor of cholera toxin-induced fluid accumulation and chloride secretion. The manufacturing process for SP-303 was optimized and simplified to produce an increased yield of the herbal extract. The novel extract (named SB-300) contained on average 70.6±7.2% SP-303 by weight (mean±S.D.; n=56 lots). Here, we describe the effectiveness of SB-300 on cAMP-regulated chloride secretion, which is mediated by the cystic fibrosis transmembrane conductance regulator Cl − channel (CFTR) in human colonic T84 cells. Exposure of the apical surface to SB-300 blocked forskolin-stimulated Cl − secretion by 92.2±3.0% with a half-maximal inhibition constant ( K B) of 4.8±0.8 μM. For SP-303, stimulated Cl − currents were decreased by 98.0±7.2% and K B averaged 4.1±1.3 μM. There was no significant difference between the blocking kinetics of SP-303 and SB-300. Forskolin-stimulated whole cell Cl − currents were effectively blocked by extracellular addition of SB-300 (63±8.5%; n=3) and to a similar extent by SP-303 (83 ± 0.6%; n=2; at 50 μM each). Both extracts inhibited a time- and voltage-independent Cl − conductance, which indicated the involvement of CFTR Cl − channels. We conclude that both SP-303 (used in Provir ®) and SB-300 (used in NSF Normal Stool Formula™) are novel natural products that target the CFTR Cl − channel. SB-300 is a low cost herbal extract and may present a complementary and alternative medicine approach for the treatment of fluid loss in watery diarrhea.

Osmoregulatory plasticity of the glass eel ofAnguilla anguilla: freshwater entry and changes in branchial ion-transport protein expression

The glass-eel stage of the life history of Anguillid eels (Anguilla spp.) makes the important physiological transition from seawater (SW) to freshwater (FW) osmoregulation. Central to successful adaptation to fresh water is the switch from active hypoosmoregulation (ion excretion) to active hyperosmoregulation (ion uptake) to counter passive fluxes imposed by concentration gradients. Anguilla anguilla (L.) glass eels, captured from the lower Minho River estuary and maintained in brackish water (BW; 24‰), were fully capable of adapting to FW following acute transfer. In a comparison between SW- (34‰) and FW-adapted glass eels, gill Na+/K+-ATPase and Na+:K+:2Cl–cotransporter (NKCC) expression were both markedly reduced in the FW group. Branchial mitochondria-rich chloride cells (MRCs) were also significantly larger in SW-adapted glass eels. Apical CFTR Cl–channel immunoreactivity associated with branchial MRCs was also only present in SW-adapted glass eels. The expression of these three ion-transport proteins is strongly associated with active ion excretion in SW teleost fishes. In FW-adapted glass eels, cortisol treatment increased branchial Na+/K+-ATPase expression but was without effect on NKCC expression. Glucocorticoid receptor blockade by RU-486 had no effect on Na+/K+-ATPase or NKCC expression.

CFTR Cl− channel function in native human colon correlates with the genotype and phenotype in cystic fibrosis

CFTR Cl− channel function in native human colon correlates with the genotype and phenotype in cystic fibrosis

Modulation of cardiac CFTR Cl current by extracellular ATP through PKC pathway

Modulation of cardiac CFTR Cl current by extracellular ATP through PKC pathway