The oxidant thimerosal modulates gating behavior of KCNQ1 by interaction with the channel outer shell.

Thimerosal (o-Ethylmercurithio)benzoic acid, TMS), a membrane-impermeable, sulfhydryl-oxidizing agent, has been described to increase the K+ current IKs in KCNE1-injected Xenopus laevis oocytes. Since there are no cysteine residues in the extracellular domain of KCNE1, it has been proposed that TMS interacts with its partner protein KCNQ1. The aim of this study was therefore to investigate the interaction of TMS with KCNQ1 and the respective K+current IK. In CHO cells stably transfected with KCNQ1/KCNE1, TMS increased IKs, whereas in CHO cells expressing KCNQ1 alone, TMS initially decreased IK. TMS also affected the cytosolic pH (pHi) and the cytosolic Ca2+ activity ([Ca2+]i) in these cells. TMS slowly decreased pHi. With a short delay, TMS increased [Ca2+]i by store depletion and capacitative influx. The time course of the effects of TMS on pHi and [Ca2+]i did not correlate with the effect of TMS on IK. We therefore anticipated a different mode of action by TMS and investigated the influence of TMS on cysteine residues of KCNQ1. For this purpose, KCNQ1wt and two mutants lacking a cysteine residue in the S6 or the S3 segment (KCNQ1C331A and KCNQ1C214A, respectively) were expressed in Xenopus laevis oocytes. A sustained current decrease was observed in KCNQ1wt and KCNQ1C331A, but not in KCNQ1C214A-injected oocytes. The analysis of tail currents, I/V curves and activation kinetics revealed a complex effect of TMS on the gating of KCNQ1wt and KCNQ1C331A. In another series we investigated the effect of TMS on IKs. TMS increased IKs of KCNQ1C214A/KCNE1-injected oocytes significantly less than IKs in KCNQ1wt/KCNE1- or KCNQ1C331A/KCNE1-injected cells. These results suggest that thimerosal interacts with the cysteine residue C214 in the S3 segment of KCNQ1, leading to a change of its gating properties. Our results support the idea that not only the inner shell, but also the outer shell of the channel is important for the gating behavior of voltage dependent K+ channels.

Properties and function of KCNQ1 K+ channels isolated from the rectal gland of Squalus acanthias.

KCNQ1 (KVLQT1) K+ channels play an important role during electrolyte secretion in airways and colon. KCNQ1 was cloned recently from NaCl-secreting shark rectal glands. Here we study the properties and regulation of the cloned sKVLQT1 expressed in Xenopus oocytes and Chinese hamster ovary (CHO) cells and compare the results with those obtained from in vitro perfused rectal gland tubules (RGT). The expression of sKCNQ1 induced voltage-dependent, delayed activated K+ currents, which were augmented by an increase in intracellular cAMP and Ca2+. The chromanol derivatives 293B and 526B potently inhibited sKCNQ1 expressed in oocytes and CHO cells, but had little effect on RGT electrolyte transport. Short-circuit currents in RGT were activated by alkalinization and were decreased by acidification. In CHO cells an alkaline pH activated and an acidic pH inhibited 293B-sensitive KCNQ1 currents. Noise analysis of the cell-attached basolateral membrane of RGT indicated the presence of low-conductance (<3 pS) K+ channels, in parallel with other K+ channels. sKCNQ1 generated similar small-conductance K+ channels upon expression in CHO cells and Xenopus oocytes. The results suggest the presence of low-conductance KCNQ1 K+ channels in RGT, which are probably regulated by changes in intracellular cAMP, Ca2+ and pH.

Regulation of slowly activating potassium current (I(Ks)) by secretin in rat pancreatic acinar cells.

1. The secretagogue-activated K(+) conductance is indispensable for the electrogenic Cl(-) secretion in exocrine tissue. In this study, we investigated the effect of secretin and other cAMP-mediated secretagogues on the slowly activating voltage-dependent K(+) current (I(Ks)) of rat pancreatic acinar cells (RPAs) with the whole-cell patch clamp technique. 2. Upon depolarization, RPAs showed I(Ks) superimposed upon the instantaneous background outward current. Secretin (5 nM), vasoactive intestinal peptide (5 nM), forskolin (5 microM), isoprenaline (10 microM) or 3-isobutyl-1-methylxanthine (IBMX, 0.1 mM) increased the amplitude of I(Ks) two- to fourfold. 3. The physiological concentration of secretin (50 pM) had a relatively weak effect on I(Ks) (160 % increase), which was significantly enhanced by transient co-stimulation with carbachol (CCh) (10 microM). However, the secretin-induced production of cAMP, which was measured by enzyme-linked immunosorbent assay, was not augmented by co-stimulation with CCh. 4. This study is the first to demonstrate the regulation of K(+) channels in RPAs by cAMP-mediated agonists. The I(Ks) channel is a common target for both Ca(2+) and cAMP agonists. The vagal stimulation under the physiological concentration of secretin facilitates I(Ks), which provides an additional driving force for Cl(-) secretion.

Cloning and function of the rat colonic epithelial K+ channel KVLQT1.

KVLQT1 (KCNQ1) is a voltage-gated K+ channel essential for repolarization of the heart action potential that is defective in cardiac arrhythmia. The channel is inhibited by the chromanol 293B, a compound that blocks cAMP-dependent electrolyte secretion in rat and human colon, therefore suggesting expression of a similar type of K+ channel in the colonic epithelium. We now report cloning and expression of KVLQT1 from rat colon. Overlapping clones identified by cDNA-library screening were combined to a full length cDNA that shares high sequence homology to KVLQT1 cloned from other species. RT-PCR analysis of rat colonic musoca demonstrated expression of KVLQT1 in crypt cells and surface epithelium. Expression of rKVLQT1 in Xenopus oocytes induced a typical delayed activated K+ current, that was further activated by increase of intracellular cAMP but not Ca2+ and that was blocked by the chromanol 293B. The same compound blocked a basolateral cAMP-activated K+ conductance in the colonic mucosal epithelium and inhibited whole cell K+ currents in patch-clamp experiments on isolated colonic crypts. We conclude that KVLQT1 is forming an important component of the basolateral cAMP-activated K+ conductance in the colonic epithelium and plays a crucial role in diseases like secretory diarrhea and cystic fibrosis.

Cystic fibrosis and CFTR.

Cystic fibrosis (CF) is a complex disease affecting epithelial ion transport. There are not many diseases like CF that have triggered such intense research activities. The complexity of the disease is due to mutations in the CFTR protein, now known to be a Cl(-) channel and a regulator of other transport proteins. The various interactions and the large number of disease-causing CFTR mutations is the reason for a variable genotype-phenotype correlation and sometimes unpredictable clinical manifestation. Nevertheless, the research of the past 10 years has resulted in a tremendous increase in knowledge, not only in regard to CFTR but also in regard to molecular interactions and completely new means of ion channel and gene therapy.

Role of K(V)LQT1 in cyclic adenosine monophosphate-mediated Cl(-) secretion in human airway epithelia.

Ion transport defects underlying cystic fibrosis (CF) lung disease are characterized by impaired cyclic adenosine monophosphate (cAMP)-dependent Cl(-) conductance. Activation of Cl(-) secretion in airways depends on simultaneous activation of luminal Cl(-) channels and basolateral K(+) channels. We determined the role of basolateral K(+) conductance in cAMP- dependent Cl(-) secretion in native human airway epithelium obtained from non-CF and CF patients. CF tissues showed typical alterations of short-circuit currents with enhanced amiloride-sensitive Na(+) conductance and defective cAMP-mediated Cl(-) conductance. In non-CF tissues, Cl(-) secretion was significantly inhibited by the chromanol 293B (10 micromol/liter), a specific inhibitor of K(V)LQT1 K(+) channels. Inhibition was increased after cAMP-dependent stimulation. Similar effects were obtained with Ba(2+) (5 mmol/liter). In patch-clamp experiments with a human bronchial epithelial cell line, stimulation with forskolin (10 micromol/liter) simultaneously activated Cl(-) and K(+) conductance. The K(+) conductance was reversibly inhibited by Ba(2+) and 293B. Analysis of reverse-transcribed messenger RNA from non-CF and CF airways showed expression of human K(V)LQT1. We conclude that the K(+) channel K(V)LQT1 is important in maintaining cAMP-dependent Cl(-) secretion in human airways. Activation of K(V)LQT1 in CF airways in parallel with stimulation of residual CF transmembrane conductance regulator Cl(-) channel activity or alternative Cl(-) channels could help to circumvent the secretory defect.

Effect of genistein on native epithelial tissue from normal individuals and CF patients and on ion channels expressed in Xenopus oocytes.

The flavonoid genistein has been shown to activate a Cl(-) conductance in various cell types expressing CFTR. We examined if similar effects can be observed when genistein is applied to native ex vivo tissues from human respiratory tract and rectum. We further compared the effects when genistein was applied to oocytes of Xenopus laevis expressing CFTR. In oocytes, both wtCFTR and DeltaF508-CFTR were activated by genistein while both cyclic AMP (K(v)LQT1) and Ca(2+) (SK4) activated K(+) channels were inhibited at high concentrations of genistein. Biopsies from nasal polyps and rectal mucosa were obtained from normal individuals (non-CF) and CF patients and in the presence of amiloride (10 micromol l(-1); mucosal side) the effects of genistein were assessed using a perfused Ussing chamber. In non-CF airway epithelia, genistein (50 micromol l(-1); mucosal side) increased lumen negative I(sc) but had no additional effects on tissues pre-stimulated with IBMX and forskolin (100 micromol l(-1) and 1 micromol l(-1); both sides). In non-CF rectal biopsies, in the presence of amiloride (10 micromol l(-1); mucosal side) and indomethacin (10 micromol l(-1); basolateral side), genistein increased lumen negative I(sc) and enabled cholinergic (carbachol; CCH, 100 micromol l(-1); basolateral side) stimulation of Cl(-) secretion indicating activation of luminal CFTR Cl(-) channels. However, after stimulation with IBMX/forskolin, genistein induced opposite effects and significantly inhibited CCH activated I(sc). In CF airway and intestinal tissues genistein failed to induce Cl(-) secretion. Thus, genistein is able to activate luminal CFTR Cl(-) conductance in non-CF tissues and mutant CFTR in oocytes. However, additional inhibitory effects on basolateral K(+) conductance and missing effects in native CF tissues do not support the use for pharmacological intervention in CF.

Role of CFTR in the colon.

In contrast to the airways, the defects in colonic function in cystic fibrosis (CF) patients are closely related to the defect in CFTR. The gastrointestinal phenotype of CF transgenic mice closely resembles the phenotype in CF patients, which clearly indicates the crucial role of CFTR in colonic Cl- secretion and the absence of an effective compensation. In the colon, stimulation of CFTR Cl- channels involves cAMP- or cGMP-dependent phosphorylation. Exocytosis is not involved. Activation of CFTR leads to coactivation of basolateral KVLQT1-type K+ channels and inhibition of luminal Na+ channels (ENaC). In contrast to cultured cells, Ca2+ does not activate luminal Cl- channels in intact enterocytes. It activates basolateral SK4-type K+ channels and luminal K+ channels, which provide additional driving force for Cl- exit. The magnitude of Cl- secretion, however, completely depends on the presence of at least a residual CFTR function in the luminal membrane. These findings have been clearly demonstrated by Ussing chamber experiments in colon epithelium biopsies of CF and normal individuals: Colonic Cl- secretion in CF patients is variable and reflects the genotype; a complete defect of CFTR is paralleled by the absence of Cl- secretion and unmasks Ca(2+)-regulated K+ channels in the luminal membrane; overabsorption of Na+ in CF reflects the absence of ENaC inhibition by CFTR; and the functional status of CF colon can be mimicked by the complete suppression of cAMP stimulation in enterocytes of healthy individuals.

Aquaporin 3 cloned from Xenopus laevis is regulated by the cystic fibrosis transmembrane conductance regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR) is essential for epithelial electrolyte transport and has been shown to be a regulator of epithelial Na(+), K(+), and Cl(-) channels. CFTR also enhances osmotic water permeability when activated by cAMP. This was detected initially in Xenopus oocytes and is also present in human airway epithelial cells, however, the mechanisms remain obscure. Here, we show that CFTR activates aquaporin 3 expressed endogenously and exogenously in oocytes of Xenopus laevis. The interaction requires stimulation of wild type CFTR by cAMP and an intact first nucleotide binding domain as demonstrated for other CFTR-protein interactions.

Deoxycholic acid (DOC) affects the transport properties of distal colon.

Secondary bile acids can induce diarrhea. In the present study we examined the effects of deoxycholic acid (DOC) on equivalent short-circuit current (Isc) in rabbit colon and the cellular mechanisms involved in DOC action (rabbit and rat). Luminal DOC inhibited amiloride-sensitive Na+ absorption. In the presence of amiloride luminal DOC had a concentration dependent effect on Isc. Low concentrations (1-10 micromol/l) induced a lumen-positive current (51+/-3 microA/cm2, 10 micromol/l, n=7) which was inhibited by luminal Ba2+ suggesting the activation of a luminal K+ conductance. Higher luminal concentrations induced a lumen-negative current (-76+/-9 microA/cm2, 100 micromol/l, n=11). Basolateral application of DOC, also in the presence of amiloride, only induced lumen-negative Isc, (-58+/-10 microA/cm2, 100 micromol/l, n=6, EC50= 3 micromol/l). This current could be abolished completely by the K+ channel blocker 293B, a selective inhibitor of cAMP-dependent Cl- secretion. This action of DOC on Isc was additive to the effect of carbachol (CCH) but not additive to that of cAMP. In intact rat colon mucosa pre-treated with DOC a significant increase in cAMP production was observed. Fura-2 measurements of cytosolic Ca2+ activity ([Ca2+]i) in isolated colonic crypts (rabbit and rat) showed that 100 micromol/l DOC induced a weak [Ca2+]i increase. Whole-cell measurements of membrane voltage in isolated rat colonic crypts revealed a hyperpolarization by DOC (4.9+/-0.8 mV, 100 micromol/l, n=8) but a depolarization by prostaglandin E2 (PGE2, via cAMP) (24+/-7 mV, n=8). The present data show that DOC acts at more than one target in the colon: in the intact mucosa it activates luminal K+ channels and Cl- secretion and this is paralleled by an increase in cAMP production. In isolated crypts DOC probably activates a Ca(2+)-regulated K+ conductance but has no effect on cAMP. Hence DOC probably activates ion channels or channel-regulating factors in colonocytes and acts on non-epithelial cells to activate Cl- secretion indirectly.

Cyclosporin-A-induced effects on the free Ca2+ concentration in LLC-PK1-cells and their mechanisms.

Here we have examined the effects of Cyclosporin A (CyA) on the free intracellular Ca2+ concentration ([Ca2+]i) of LLC-PK1/PKE20 cells to evaluate mechanisms of CyA nephrotoxicity using Fura-2 microspectrofluorometry or digital fluorescence video imaging. The CyA-associated changes were compared to the effects of tacrolimus (Tac), a structurally unrelated immunosuppressant with similar cellular pathways which also causes nephrotoxicity. CyA (EC50(: 1 nmol/l, n=16) and Tac (EC50: 1 nmol/l, n=5) caused a concentration-dependent increase of [Ca2+]i which was substantially attenuated by reducing the external Ca2+ concentration (10(-6) mol/l). Similarly Cyclosporin H, a non-immunosuppressive analogue of CyA, stimulated a Ca2+ influx. Nicardipine (10(-6) mol/l) reduced the CyA- and the Tac-induced Ca2+ influx to 52+/-16% (n=10) and 13+/-10% (n=13) of control respectively. Diltiazem and verapamil (10(-6) mol/l) were also effective, but flufenamate (10(-4) mol/l), Gd3+ (10(-5) mol/l) and La3+ (10(-5) mol/l) were not. In the absence of extracellular Ca2+ CyA led to a small but significant [Ca2+]i increase, indicating additional release from internal stores. Depletion of inositol-1,4,5-trisphosphate-(InsP3-) sensitive Ca2+ stores by extracellular ATP (10(4) mol/l) in low-Ca2+ solution completely suppressed the CyA-induced [Ca2+]i rise. CyA had no effect on the cellular InsP3 concentration. Furthermore, inhibition of phospholipase-Cbeta (PLCbeta) by U73122 (2x10(-5) mol/l) did not alter the CyA-stimulated [Ca2+]i rise. A direct effect of CyA on InsP3-sensitive Ca2+ stores, the InsP3 receptor, the Ca2+ content of the stores or involvement of additional stores is assumed. Incubation with CyA for 1, 12 and 24 h enhanced the rise in [Ca2+]i peak induced by ATP, arginine vasopressin (AVP) and angiotensin II. In summary, CyA stimulated a [Ca2+]i increase in LLC-PK1 cells through Ca2+ release from InsP3-sensitive stores and Ca2+ influx via a nicardipine-sensitive pathway. The CyA-mediated [Ca2+]i increase is independent of PLCbeta activity and InsP3 metabolism. CyA caused long-term enhancement of the agonist-induced rise in [Ca2+]i. The effects of CyA on Ca2+ signaling appear to be independent of its immunosuppressive action.

Defective cholinergic Cl(-) secretion and detection of K(+) secretion in rectal biopsies from cystic fibrosis patients.

Rectal biopsies from cystic fibrosis (CF) patients show defective cAMP-activated Cl(-) secretion and an inverse response of the short-circuit current (I(sc)) toward stimulation with carbachol (CCh). Alternative Cl(-) channels are found in airway epithelia and have been attributed to residual Cl(-) secretion in CF colon. The aim of the present study was to investigate ion conductances causing reversed I(sc) upon cholinergic stimulation. Furthermore, the putative role of an alternative Ca(2+)-dependent Cl(-) conductance in human distal colon was examined. Cholinergic ion secretion was assessed in the absence and presence of cAMP-dependent stimulation. Transepithelial voltage and I(sc) were measured in rectal biopsies from non-CF and CF individuals by means of a perfused micro-Ussing chamber. Under baseline conditions, CCh induced a positive I(sc) in CF rectal biopsies but caused a negative I(sc) in non-CF subjects. The CCh-induced negative I(sc) in non-CF biopsies was gradually reversed to a positive response by incubating the biopsies in indomethacin. The positive I(sc) was significantly enhanced in CF and was caused by activation of a luminal K(+) conductance, as shown by the use of the K(+) channel blockers Ba(2+) and tetraethylammonium. Moreover, a cAMP-dependent luminal K(+) conductance was detected in CF individuals. We conclude that the cystic fibrosis transmembrane conductance regulator is the predominant Cl(-) channel in human distal colon. Unlike human airways, no evidence was found for an alternative Cl(-) conductance in native tissues from CF patients. Furthermore, we demonstrated that both Ca(2+)- and cAMP-dependent K(+) secretion are present in human distal colon, which are unmasked in rectal biopsies from CF patients.

Physiology of renal sodium transport.

A wealth of studies performed with a spectrum of methods spanning simple clearance studies to the molecular identification of ion transporters has increased our understanding of how approximately 1.7 kg of NaCl and 180 L of H2O are absorbed by renal tubules in man and how the urinary excretion is fine-tuned to meet homeostatic requirements. This review will summarize our current understanding. In the proximal nephron, approximately 60 to 70% of the filtered Na+ and H2O is absorbed together with approximately 90% of the filtered HCO3-. The exact quantities are determined by many regulatory factors, such as glomerulotubular balance, angiotensin II, endothelin, sympathetic innervation, parathyroid hormone, dopamine, acid base status and others. The essential components of absorption are luminal membrane Na+/H+ exchange and the basolateral (Na+ + K+)-ATPase. In the thick ascending limb of the loop of Henle, 20 to 30% of the filtered NaCl is absorbed via Na+2Cl-K+ cotransport driven by the basolateral (Na+ + K+)-ATPase. No H2O is absorbed at this nephron site. The transport rate is determined by the Na+ load and by several hormones and neurotransmitters, including prostaglandins, parathyroid hormone, glucagon, calcitonin, arginine vasopressin (AVP), and adrenaline. In the distal tubule, some 5 to 10% of the filtered load is absorbed via Na+Cl- cotransport in the luminal membrane driven by the basolateral (Na+ + K+)-ATPase. The rate of transport is again determined by the delivered load and by several hormones and neurotransmitters. One of the tasks of the collecting duct is to control the absorption of approximately 10 to 15% of the filtered H2O, regulated by AVP, and just a few percent of the filtered Na+, controlled by aldosterone and natriuretic hormone. The water absorption proceeds through the luminal membrane via aquaporin 2 and through the basolateral membrane via aquaporin 3 channels and is driven by the osmotic gradient built up by the counter current concentrating system. The Na+ absorption occurs via Na+ channels present in the luminal membrane driven by the basolateral (Na+ + K+)-ATPase. With no pharmacological interference, urinary excretion of Na+ can vary between less than 0.1% and no more than 3% of the filtered load, and that of H2O can vary between 0.3 and 15%.

A constitutively open potassium channel formed by KCNQ1 and KCNE3.

Mutations in all four known KCNQ potassium channel alpha-subunit genes lead to human diseases. KCNQ1 (KvLQT1) interacts with the beta-subunit KCNE1 (IsK, minK) to form the slow, depolarization-activated potassium current I(Ks) that is affected in some forms of cardiac arrhythmia. Here we show that the novel beta-subunit KCNE3 markedly changes KCNQ1 properties to yield currents that are nearly instantaneous and depend linearly on voltage. It also suppresses the currents of KCNQ4 and HERG potassium channels. In the intestine, KCNQ1 and KCNE3 messenger RNAs colocalized in crypt cells. This localization and the pharmacology, voltage-dependence and stimulation by cyclic AMP of KCNQ1/KCNE3 currents indicate that these proteins may assemble to form the potassium channel that is important for cyclic AMP-stimulated intestinal chloride secretion and that is involved in secretory diarrhoea and cystic fibrosis.

Regulation of the Na+2Cl-K+ cotransporter in isolated rat colon crypts.

The Na(+2)Cl(-)K+ cotransporter accepts NH4+ at its K+-binding site. Therefore, the rate of cytosolic acidification after NH4+ addition to the bath (20 mmol/l) measured by BCECF fluorescence can be used to quantify the rate of this cotransporter. In isolated colon crypts of rat distal colon (RCC) addition of NH4+ led to an initial alkalinization, corresponding to NH3 uptake. This was followed by an acidification, corresponding to NH4+ uptake. The rate of this uptake was quantified by exponential curve fitting and is given in arbitrary units (delta fluorescence ratio units/1000 s). In pilot experiments it was shown that the pH signal caused by the Na(+)2Cl(-)K+ co-transporter could be amplified if the experiments were carried out in the presence of bath Ba2+ to inhibit NH4+ uptake via K+ channels. Therefore all subsequent experiments were performed in the presence of 1 mmol/l Ba2+. In the absence of any secretagogue, preincubation of RCC in a low-Cl- solution (4 mmol/l) for 10 min enhanced the uptake rate significantly from 1.70+/-0.11 to 2.54+/-0.27 U/1000 s (n=20). The addition of 100 mmol/l mannitol (hypertonic solution) enhanced the rate significantly from 1.93+/-0.17 to 2.84+/-0.43 U/1000 s (n=5). Stimulation of NaCl secretion by a solution containing 100 micromol/l carbachol (CCH) led to a small but significant increase in NH4+ uptake rate from 2.06+/-0.34 to 2.40+/-0.30 U/1000 s (n= 11). The increase in uptake rate observed with stimulation of the cAMP pathway by isobutylmethylxanthine (IBMX) and forskolin (100 micromol/l and 5 micromol/l, respectively) was from 2.39+/-0.24 to 3.06+/-0.36 U/1000 s (n=24). Whatever the mechanism used to increase the NH4+ uptake rate, azosemide (500 micromol/l) always reduced this rate to control values. Hence three manoeuvres enhanced loop-diuretic-inhibitable uptake rates of the Na(+)2Cl(-)K+ cotransporter: (1) lowering of cytosolic Cl- concentration; (2) cell shrinkage; (3) activation of NaCl secretion by carbachol and (4) activation of NaCl secretion by cAMP. The common denominator of all four activation pathways may be a transient fall in cell volume.

CFTR-mediated inhibition of epithelial Na+ conductance in human colon is defective in cystic fibrosis.

Cystic fibrosis (CF) patients show characteristic defects in epithelial ion transport, such as failure in cAMP-dependent Cl- secretion. Because the cystic fibrosis transmembrane conductance regulator (CFTR) also functions as a downregulator of epithelial Na+ channels (ENaC), enhanced Na+ conductance was found in the airways of CF patients. Here, we examined whether enhanced epithelial Na+ conductance is also present in the colonic epithelium of CF patients and examined the underlying mechanisms. Thus transepithelial voltages were measured, and equivalent short-circuit currents (I(sc-eq)) were determined by means of a novel type of Ussing chamber. Non-CF tissues demonstrated cAMP-dependent Cl- secretion that was absent in biopsies of CF patients. Correspondingly, Isc-eq was inhibited in non-CF but not in CF epithelia when synthesis of endogenous prostaglandins was blocked by indomethacin. In the presence of indomethacin, a larger portion of amiloride-sensitive Isc-eq was detected in CF tissues, suggesting enhanced ENaC conductance in colonic mucosa of CF patients. Increase of intracellular cAMP by forskolin and IBMX inhibited amiloride-sensitive ENaC currents in non-CF tissues but not in CF biopsies. Therefore, enhanced epithelial Na+ conductance is present in the CF colon and is probably due to missing downregulation by CFTR.

The cellular mechanisms of Cl- secretion induced by C-type natriuretic peptide (CNP). Experiments on isolated in vitro perfused rectal gland tubules of Squalus acanthias.

We have examined the mechanism whereby C-type natriuretic peptide (CNP), an agonist acting through the second messenger cGMP, enhances NaCl secretion in the rectal gland of Squalus acanthias. Single rectal gland tubules (RGT) were dissected manually, perfused in vitro and equivalent short-circuit current [Isc=transepithelial voltage/transepithelial resistance (Rte)] as well as basolateral membrane voltage (Vbl) were measured. CNP was added to luminal and basolateral perfusates at concentrations between 1 and 1000 nmol/l and its effects on the above parameters were compared to those of a "stimulation cocktail" (Stim, containing dibutyryl cAMP, adenosine and forskolin) that maximally enhances cytosolic cAMP, and other agonists and hormones such as guanylin, vasoactive intestinal peptide (VIP), and adenosine. CNP had no effect from the luminal side (n=6). Its effects from the basolateral side consisted of a substantial increase in Isc (-31.6+/-7.7 to -316+/-82.2 microA/cm2, n=15). CNP significantly depolarized the luminal membrane from -87. 4+/-1.0 to -82.3+/-2.6 mV (n=12). Vbl was not changed (n=12) but the fractional conductance for K+ was increased (n=3). These effects were qualitatively and even quantitatively comparable to those of other agonists acting via cytosolic cAMP, but were less marked than those caused by Stim (n=64). The effects of VIP and CNP on Isc were not additive (n=5). The cytosolic Ca2+ concentration ([Ca2+]i) was monitored using the fura-2 fluorescence ratio (FFR 340/380 nm) and it was found that CNP, like agonists acting via cAMP, enhances FFR significantly from 1.02+/-0.05 to 1.32+/-0.05 (n=8) with a time constant in the 1-2 min in range. Our data suggest that CNP, acting via the second messenger cGMP, induces a marked increase in Isc in the rectal gland. The concomitant fall in Rte corresponds to increases in the luminal membrane Cl- conductance and in the basolateral membrane K+ conductance. The latter effect is probably due to an increase in [Ca2+]i.

The Na+2Cl-K+ cotransporter in the rectal gland of Squalus acanthias is activated by cell shrinkage.

Effects of cAMP on Cl- secretion, intracellular Cl- activity and cell volume were studied in isolated perfused rectal gland tubules (RGT) of Squalus acanthias with electrophysiological and fluorescence methods. Recording of equivalent short-circuit current (Isc) showed that cAMP stimulates Na+Cl- secretion in a biphasic manner. The first and rapid phase corresponds to Cl- exit via the respective protein-kinase-A- (PKA-) phosphorylated Cl- conductance. The inhibitory effect of the loop diuretic furosemide (0.5 mmol/l, n=12) indicates that second phase reflects the delayed (1-2 min) activation of the Na+2Cl-K+ cotransporter. During the first phase cytosolic Cl- activity, as monitored by 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) fluorescence, fell to 78% (n=23) of the control value. Concomitantly, a transient fall in cell volume was recorded by calcein fluorescence to 92% (n=5) of the control value. Preincubation of the RGT with phalloidin (0.1 mmol/l, n=6) or cytochalasin D (0.1 mmol/l, n=4) almost completely prevented the development of the second phase of Isc activation. When cytosolic Cl- activity was increased by exposing the RGT to a high K+ concentration (25 mmol/l), in the presence of mannitol to prevent volume increases, stimulation was unaffected and biphasic. In contrast, when cell volume was clamped to an increased value (115%, n=8) by removing extracellular NaCl, the second phase was abolished completely (n=11). These data suggest that the primary and key process for triggering the Na+2Cl-K+ cotransport is transient cell shrinkage.

Cystic fibrosis transmembrane conductance regulator inhibits epithelial Na+ channels carrying Liddle's syndrome mutations.

Epithelial Na+ channels (ENaC) are inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) upon activation by protein kinase A. It is, however, still unclear how CFTR regulates the activity of ENaC. In the present study we examined whether CFTR interacts with ENaC by interfering with the Nedd4- and ubiquitin-mediated endocytosis of ENaC. Various C-terminal mutations were introduced into the three alpha-, beta-, and gamma-subunits of the rat epithelial Na+ channel, thereby eliminating PY motifs, which are important binding domains for the ubiquitin ligase Nedd4. When expressed in Xenopus oocytes, most of the ENaC stop (alpha-H647X, beta-P565X, gamma-S608X) or point (alpha-P671A, beta-Y618A, gamma-P(624-626)A) mutations induced enhanced Na+ currents when compared with wild type alpha,beta,gamma-rENaC. However, ENaC currents formed by either of the mutant alpha-, beta-, or gamma-subunits were inhibited during activation of CFTR by forskolin (10 micromol/l) and 3-isobutyl-1-methylxanthine (1 mmol/l). Antibodies to dynamin or ubiquitin enhanced alpha,beta,gamma-rENaC whole cell Na+ conductance but did not interfere with inhibition of ENaC by CFTR. Another mutant, beta-T592M,T593A-ENaC, also showed enhanced Na+ currents, which were down-regulated by CFTR. Moreover, activation of ENaC by extracellular proteases and xCAP1 does not disturb CFTR-dependent inhibition of ENaC. We conclude that regulation of ENaC by CFTR is distal to other regulatory limbs and does not involve Nedd4-dependent ubiquitination.

Lack of correlation between CFTR expression, CFTR Cl- currents, amiloride-sensitive Na+ conductance, and cystic fibrosis phenotype.

Cystic fibrosis (CF) is characterized by defective Cl- and enhanced Na+ conductance, both due to malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in airway epithelial cells. In the present study we examined whether expression of CFTR mRNA (CFTR messenger ribonucleic acid) is different in airway epithelia derived from either CF patients or healthy volunteers. Moreover, we tried to correlate differences in epithelial Cl- and Na+ conductance with the level of CFTR mRNA expression and studied whether these properties correlate to the clinical phenotype of CF patients. To that end, CFTR mRNA was determined by means of quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and cyclic adenosine monophosphate (cAMP)-activated Cl- and epithelial Na+ conductances were examined in airway epithelial cells using microelectrode techniques. Complementary in vitro data were obtained from cultured CF and non-CF airway epithelial cell lines. Genotype and Shwachman score were assessed for each patient. We found variable levels of CFTR mRNA expression in airway cells of both CF patients and healthy volunteers. As expected, epithelial Na+ conductance was enhanced and CFTR Cl- conductance was absent in airway cells from CF patients. However, CFTR mRNA expression did not correlate with either electrophysiological properties or Shwachman scores obtained from CF patients. In addition, CFTR mRNA expression did not correlate to Cl- conductance in cultured CF and non-CF airway epithelial cells. These results indicate a lack of correlation between levels of CFTR mRNA and CFTR function, and that only small amounts of CFTR are required for expression of the CFTR Cl- conductance.