The gasotransmitter hydrogen sulphide decreases Na⁺ transport across pulmonary epithelial cells.

BACKGROUND AND PURPOSE The transepithelial absorption of Na(+) in the lungs is crucial for the maintenance of the volume and composition of epithelial lining fluid. The regulation of Na(+) transport is essential, because hypo- or hyperabsorption of Na(+) is associated with lung diseases such as pulmonary oedema or cystic fibrosis. This study investigated the effects of the gaseous signalling molecule hydrogen sulphide (H(2) S) on Na(+) absorption across pulmonary epithelial cells. EXPERIMENTAL APPROACH Ion transport processes were electrophysiologically assessed in Ussing chambers on H441 cells grown on permeable supports at air/liquid interface and on native tracheal preparations of pigs and mice. The effects of H(2)S were further investigated on Na(+) channels expressed in Xenopus oocytes and Na(+) /K(+)-ATPase activity in vitro. Membrane abundance of Na(+) /K(+)-ATPase was determined by surface biotinylation and Western blot. Cellular ATP concentrations were measured colorimetrically, and cytosolic Ca(2+) concentrations were measured with Fura-2. KEY RESULTS H(2)S rapidly and reversibly inhibited Na(+) transport in all the models employed. H(2)S had no effect on Na(+) channels, whereas it decreased Na(+) /K(+)-ATPase currents. H(2)S did not affect the membrane abundance of Na(+) /K(+)-ATPase, its metabolic or calcium-dependent regulation, or its direct activity. However, H(2)S inhibited basolateral calcium-dependent K(+) channels, which consequently decreased Na(+) absorption by H441 monolayers. CONCLUSIONS AND IMPLICATIONS H(2) S impairs pulmonary transepithelial Na(+) absorption, mainly by inhibiting basolateral Ca(2+)-dependent K(+) channels. These data suggest that the H(2)S signalling system might represent a novel pharmacological target for modifying pulmonary transepithelial Na(+) transport.

Toxoplasma gondii: changes of transepithelial ion transport in infected HT29/B6 cell monolayers.

An in vitro system was established to study the effect of coccidian parasites on ion transport systems in epithelial tissues using HT29/B6, a human colon carcinoma cell line, and Toxoplasma gondii as a model parasite. Ion transport was measured in perfusion chambers 5, 10 and 15 h post-infection using monolayers in which approximately 30% of the cells were parasitized. The infection had rapid effects on the conductance and unidirectional chloride fluxes of infected cell monolayers, which were two to three times higher than those of uninfected HT29/B6 cell monolayers throughout the observation period. However, the chloride net fluxes and short-circuit current were unaffected by the parasites, while the decrease of chloride seromucosal fluxes and conductance after addition of bumetanide were affected by the infection. The unidirectional mannitol fluxes, which correspond with water motion through paracellular pathways, were increased in infected HT29/B6 cell monolayers.

Effects of 8-cpt-cAMP on the epithelial sodium channel expressed in Xenopus oocytes.

Vasopressin stimulates the activity of the epithelial Na channel (ENaC) through the cAMP/PKA pathway in the cortical collecting tubule, or in similar amphibian epithelia, but the mechanism of this regulation is not yet understood. This stimulation by cAMP could not be reproduced with the rat or Xenopus ENaC expressed in Xenopus oocyte. Recently, it was shown that the alpha-subunit cloned from the guinea-pig colon (alpha gp) could confer the ability to be activated by the membrane-permeant cAMP analogue 8-chlorophenyl-thio-cAMP (cpt-cAMP) to channels produced by expression of alpha gp, beta rat and gamma rat ENaC subunits. In this study we investigate the mechanism of this activation. Forskolin treatment, endogenous production of cAMP by activation of coexpressed beta adrenergic receptors, or intracellular perfusion with cAMP did not increase the amiloride-sensitive Na current, even though these maneuvers stimulated CFTR (cystic fibrosis transmembrane conductance regulator)-mediated Cl currents. In contrast, extracellular 8-cpt-cAMP increased alpha gp, beta rat and gamma rat ENaC activity but had no effect on CFTR. Swapping intracellular domains between the cpt-cAMP-sensitive alpha gp and the cpt-cAMP-resistant alpha rat-subunit showed that neither the N-terminal nor the C-terminal of alpha ENaC was responsible for the effect of cpt-cAMP. The mechanisms of activation of ENaC by cpt-cAMP and of CFTR by the cAMP/PKA pathway are clearly different. cpt-cAMP seems to increase the activity of ENaC formed by alpha gp and beta gamma rat by interacting with the extracellular part of the protein.

Regulation of cAMP-sensitive colonic epithelial Na+ channel in oocyte expression system.

In amphibian epithelia and in cortical collecting duct the antidiuretic peptide arginine-vasopressin (AVP) stimulates activity of epithelial Na+ channels (ENaCs). Generally, the AVP action upon Na+ (re)absorption is believed to be a cAMP/protein-kinase-A mediated mechanism. In the Xenopus oocyte expression system, however, a clear stimulation of ENaC activity by cAMP could not be reproduced with channel subunits cloned from A6 cells or rat colon. We have recently shown that membrane-permeant 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) stimulates activity of a hybrid ENaC in Xenopus oocytes, that consists of an alpha-subunit cloned from guinea-pig colon and the beta- and gamma-subunit originating from rat colon (gpalpharbetagammaENaC). In the present study, we have further investigated the mechanisms by which cpt-cAMP upregulates gpalpharbetagammaENaC activity. Interestingly, we found AVP to stimulate the gpalpharbetagammaENaC in oocytes. Also, treatment with GTP-gamma-S largely activated this channel. In contrast, as a conflicting result, forskolin had no stimulatory effect on the cAMP-sensitive gpalpharbetagammaENaC. Experiments with Brefeldin A (BFA) or nocodazole suggested that only a minor part of cpt-cAMP-induced activation is probably due to an additional translocation of channel proteins into the oocyte membrane. In conclusion, the stimulatory effect of synthetic cpt-cAMP does not seem to be exclusively provided by classical cAMP/PKA-associated transduction mechanisms, i.e., as in A6 cells.

Inhibitory non-genomic effects of progesterone on Na+ absorption in epithelial cells from Xenopus kidney (A6).

The effect of the steroid hormone progesterone on transepithelial sodium transport was measured in confluent monolayers of the A6-cell line from Xenopus kidney. Apical permeabilization with Amphotericin B enabled us to measure the Na+/K+-pump current, and current-fluctuation analysis was used to analyze changes in apical channel density and gating characteristics. Basolateral progesterone (22.2 microM) had a rapid inhibitory effect on the Na+/K+-pump current, and a corresponding decrease in Na+ channel density. The effect occurred within some minutes and took about 50 min to reach a new steady state, in which 45% of the macroscopic current (ISC) was inhibited. Progesterone also inhibits the hypo-osmotic stimulation of Na+ channels which occurs in untreated monolayers. Compared with the known effects of adrenal steroids, our results show a rapid inhibitory action of a steroid hormone on Na+ absorption. The time profile of the progesterone effect suggests, at least in the first minutes, a non-genomic action of progesterone.

Regulation of Na(+) transport across leech skin by peptide hormones and neurotransmitters.

An increase in intracellular cyclic AMP concentration stimulates transepithelial Na(+) transport across the skin of the leech Hirudo medicinalis, but it is unclear how cytosolic cyclic AMP levels are elevated in vivo. In search of this external stimulus, we performed Ussing chamber experiments to test several peptide hormones and neurotransmitters for their effect on Na(+) transport across leech dorsal integument. Although all the peptide hormones under investigation significantly affected ion transport across leech integument, none of them mimicked the effect of an experimental rise in intracellular cyclic AMP level. The invertebrate peptides conopressin and angiotensin II amide inhibited short-circuit-current- (I(sc)) and amiloride-sensitive Na(+) transport (I(amil)), although to slightly different degrees. The vertebrate peptide hormones 8-arginine-vasopressin and 8-lysine-vasopressin both produced an inhibition of I(amil) comparable with that caused by angiotensin II amide. However, 8-lysine-vasopressin reduced I(sc), whereas 8-arginine-vasopressin induced a moderate increase in I(sc). The neurotransmitter dopamine, which occurs in the leech central nervous system in relatively large amounts, and its precursor l-dopamine both induced large decreases in I(sc) and I(amil). However, the reactions evoked by the catecholamines showed no pronounced similarity to the effects of intracellular cyclic AMP. Two other neurotransmitters known to occur in leeches, serotonin (5-hydroxytryptamine) and gamma-n-aminobutyric acid (GABA), had no influence on transepithelial ion transport in leech skin.

Detection of focal liver lesions at biphasic spiral CT: randomized double-blind study of the effect of iodine concentration in contrast materials.

PURPOSE: To evaluate the effect of iodine concentration on the detection of focal liver lesions at biphasic spiral computed tomography (CT). MATERIALS AND METHODS: One hundred two patients (64 men, 38 women) with neoplastic (n = 85) and nonneoplastic focal lesions (n = 17) were prospectively assigned to biphasic injection group A or B and received 180 mL of iopromide containing 370 or 300 mg of iodine per milliliter, respectively, during spiral CT. Comparison included assessment of quantitative and qualitative parameters. RESULTS: Hepatic time-attenuation curves and mean hepatic enhancement in the portal venous phase and aortic time-attenuation curves in both arterial and portal venous phases were statistically superior in group A compared with group B. There was no significant difference in the mean enhancement in all lesions in either group. In contrast, among patients with hepatocellular carcinoma, mean contrast enhancement in lesions in the arterial phase was significantly superior in group A compared with group B. Blinded readers classified hepatic attenuation and lesion visibility as very good and as improved significantly more often in group A than in group B. CONCLUSION: A decrease in iodine concentration significantly affects aortic and hepatic contrast enhancement and may impair the detectability of focal liver lesions during biphasic spiral CT.

Intracellular calcium and pH conditions of cultured cells infected with Eimeria bovis or E. separata.

Loading of Eimeria bovis-infected Vero cells with membrane-permeant acetoxymethyl esters (AM-esters) of ion-sensitive dyes provided us with a noninvasive method for investigation of the permeability of the parasitophorous vacuole membrane (PVM) and simultaneous measurement of Ca2+ and H+ concentrations in different compartments of the infected cells. The distribution patterns of the cleaved membrane-impermeant dyes argue against the existence of nonselective pores in the PVM. There is also no indication of a parasitophorous duct connecting the vacuolar space with extracellular media. The pH inside the parasitophorous vacuole (PV) was lower than that in the cytoplasm of the host cell or the parasite, whereas the [Ca2+] in these compartments did not differ significantly. In HT29 cells infected with E. separata for 24 h the Ca2+ response to extracellular adenosine triphosphate (ATP) was significantly reduced, indicating influences on the host cell's intracellular signaling.

cAMP sensitivity conferred to the epithelial Na+ channel by alpha-subunit cloned from guinea-pig colon.

The rate of Na+ (re)absorption across tight epithelia such as in distal kidney nephron and colon is to a large extent controlled at the level of the epithelial Na+ channel (ENaC). In kidney, antidiuretic hormone (ADH, vasopressin) stimulates the expression/activity of this channel by a cAMP/protein-kinase-A- (PKA-) mediated pathway. However, a clear upregulation of ENaC function by cAMP could not be reproduced with cloned channel subunits in the Xenopus oocyte expression system, suggesting the hypothesis that an additional factor is missing. In contrast, we show here that membrane-permeant cAMP can activate ENaC expressed in Xenopus oocytes (3.8-fold) upon replacement of the rat alpha-subunit by a new alpha-subunit cloned from guinea-pig colon (gpalpha). This alpha-subunit is 76% identical with its rat orthologue originating from ADH-insensitive rat colon. The biophysical fingerprints of the hybrid ENaC formed by this guinea-pig alpha-subunit together with rat beta- and gamma-subunits are indistinguishable from those of rat ENaC (rENaC). Injection of the PKA inhibitor PKI-(6-22)-amide into the oocyte had no effect on the basal activity of rat ENaC but inhibited the activity of gpalpha-containing hybrid ENaC and greatly decreased its stimulation by cAMP. This suggests that, unlike for rat ENaC, tonic PKA activity is required for basal function of gpalpha-containing ENaC and that PKA mediates its cAMP-induced activation. This regulatory behaviour is not common to all ENaCs containing an alpha-subunit cloned from an ADH-responsive tissue since xENaC, which was cloned from the ADH-sensitive Xenopus laevis A6 epithelia, is, when expressed in oocytes, resistant to cAMP, similar to rat ENaC. This study demonstrates that the PKA sensitivity of ENaC can depend on the nature of the ENaC alpha-subunit and raises the possibility that cAMP can stimulate ENaCs by different mechanisms.

Capacitance measurements reveal different pathways for the activation of CFTR.

We used the Xenopus laevis oocyte expression system to characterize adenosine 3',5'-cyclic monophosphate (cAMP) activation of the cystic fibrosis transmembrane conductance regulator (CFTR). With conventional two-microelectrode voltage-clamp techniques, we recorded transmembrane conductance (Gm) and membrane current (Im). Using five different sine wave frequencies, we also monitored changes of the plasma membrane surface area by recording continuously membrane capacitance (Cm) under voltage-clamp conditions. Impedance spectra recorded in the frequency range 0.1-500 Hz showed that, at least up to 200 Hz, Cm is independent of the frequency. In control oocytes, cAMP (100 microM) treatment did not affect Gm or Im but evoked a small, slowly occurring increase in Cm, probably mediated by cAMP-stimulated exocytosis. However, in oocytes expressing CFTR, large simultaneous increases of Gm, Im and Cm occurred after stimulation with cAMP. Oocytes injected with the delta F508 CFTR mutant behaved like control oocytes and cAMP had no additional effects on Gm, Im or Cm. In oocytes injected with wild-type CFTR, adenosine 5'-triphosphate (ATP, 100 microM) did not activate the cAMP-induced augmentation of Im, Gm or Cm further. On the other hand, cAMP-induced increases in Cm were reduced significantly by the specific blockers of protein kinase A (PKA) KT5720 and N-[2-(methylamino-9-ethyl]-5-isoquinolinesulphonamide hydrochloride (H8), whereas the increases in Gm and Im were essentially unaffected by these agents. Reducing intracellular Ca2+ by injection of a Ca2+ chelator 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) prevented PKA-dependent exocytosis while activation of Im and Gm of already-inserted CFTR still could be detected. The specific cAMP antagonist adenosine 3',5'-cyclic monophosphothioate Rp diastereomer (RpcAMPS) completely suppressed the effects of cAMP on all parameters. These findings are consistent with the concept of different pathways of CFTR activation by cAMP: already-inserted CFTR Cl- channels are activated directly by cAMP, while traffic of CFTR proteins from an intracellular pool to the plasma membrane and functional insertion into the plasma membrane occurs via cAMP- and Ca(2+)-dependent PKA-mediated exocytosis.

Minor role of Cl- secretion in non-cystic fibrosis and cystic fibrosis human nasal epithelium.

Na+ and Cl- currents were studied in primary cultures of human nasal epithelium derived from non-cystic fibrosis (non-CF) and cystic fibrosis (CF) patients. We found that Na+ absorption dominates transepithelial transport and the Na+ current contains an amiloride-sensitive and amiloride-insensitive component. In non-CF tissue both components contribute about equally to the entire short-circuit current (ISC), whereas in CF tissues the major part of the current is amiloride-sensitive. Na+ removal reduced ISC to values close to zero. Several Cl- channel blockers were used to identify the remaining tiny Na+-independent current. Under unstimulated, physiological conditions in the presence of Cl- on both sides and amiloride on the apical side of the epithelium diphenylamine-2-carboxic acid (DPC), 4,4'-diisothiocyanatostilbene-2, 2'- disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) failed to induce clearcut inhibition of ISC. cAMP as well as ATP did not affect ISC either in CF or in non-CF epithelia. Reduction of apical Cl- increased ISC and depolarized transepithelial potential; however, the observed increase was insensitive to DIDS, DPC and NPPB. From these data we conclude that Cl- conductances in primary cultures of human nasal epithelium derived from CF patients as well as from non-CF patients are present only in low numbers or do not contribute significantly to transepithelial ion transport.

[Ion transport in nasal and paranasal sinus mucosa in mucoviscidosis and chronic sinusitis]. / Ionentransporte über die Nasen- und Nasennebenhöhlenschleimhaut bei Mukoviszidose und chronischer Sinusitis.

Cystic fibrosis (CF) is the most commonly inherited disease in Caucasians and is caused by a mutation in the gene encoding a membrane transport protein. This cystic fibrosis transmembrane conductance regulator (CFTR) is thought to be an apical Cl- channel activated by intracellular cAMP. Most recent findings suggest that CFTR is more than a pure Cl- channel and might be involved in the regulation of other transport systems. In the present study we show that CFTR as a Cl- channel plays only a minor role in primary cultured human nasal epithelium derived from non-CF and CF patients. These findings are especially of interest for non-CF human nasal epithelia in which CFTR is correctly inserted. In both tissues Cl- secretion is negligible as compared with Na+ absorption. We confirm and expand our previous observations that Na+ absorption in human nasal epithelium is the dominant ion transport process and that Cl- secretion is detectable in both CF and non-CF tissue. Moreover, we show that cAMP and ATP were not able to stimulate any silent Cl- channels in CF or non-CF human nasal epithelial cells. We further give evidence that in human nasal CF and non-CF epithelium Na+ absorption is mediated by epithelial Na+ channels (ENaC) that are either different from those of other epithelia or which exhibit altered regulation. These differences between Na+ channels of human nasal epithelium and "classical" epithelial Na+ channels include lack of activation by the intracellular second messenger cAMP and the steroid hormone aldosterone. We show further that human nasal Na+ channels are inhibited by Cl- channel blockers and exhibit a different pharmacology towards common Na+ channel blockers.

Potassium conductances in isolated single cells from Xenopus laevis colonic epithelium.

The patch-clamp technique was employed in whole cells to analyze K+ conductances of amphibian colonic cells. Xenopus laevis colonic epithelium was dissected, and single epithelial cells were isolated using Ca(2+)-free solution and mild enzyme treatment. Vital epithelial cells had a round shape, and a distinction between apical and basolateral poles was no longer possible. Their epithelial origin was, however, verified by antibodies against keratin. The average resting potential of the colonocytes was -37.6 +/- 1 mV (n = 220) and the resulting membrane current was strongly potassium selective. Further characterization of this conductance was achieved by current-voltage relationship in the presence and absence of various K+ channel blockers. Barium and cesium showed pronounced voltage-dependent blockage, with interaction at about 35% inside the pores. Lidocain, as well as quinine and quinidine also blocked, but with different kinetics and binding charactertics. Both TEA and verapamil were ineffective. We also explored the effects of extra- (pHo) and intracellular pH (pHi) on the K+ conductance. An increase of pHo, as well as pHi, caused membrane hyperpolarization, and the shift of the current-voltage relationship indicates a stimulation of K+ channels by decreasing external and/or internal H+ concentration. The results provide the first whole-cell measurements on isolated amphibian colonic epithelial cells and demonstrate the presence of various K+ channel types in this preparation.

Characterization of a voltage-dependent conductance in the basolateral membrane of leech skin epithelium.

Voltage clamp studies were performed on the dorsal integument of Hirudo medicinalis. Under apical calcium-free conditions an inward-directed component of transepithelial current was activated by changes of transepithelial voltage. Depolarization caused up to 50% increase of the transepithelial sodium current. Hyperpolarization had no comparable effects. With calcium (1.8 mM) or amiloride (100 microM) in the apical solution and in sodium-free solutions the inward-directed current failed to increase after depolarization. Activation also occurred under chloride-free conditions. Permeabilization of the apical membrane by nystatin (5 microM) increased the current activation significantly. After nystatin, calcium as well as amiloride lost their inhibitory effects. This indicates a basolateral localization of the voltage-dependent conductance. Vesicle insertion or cytoskeletal structures are probably not involved in regulation, as seen by the lack of effects of brefeldin A and the cytochalasins B and D. However, serosal hyposmolar solutions (170 mosmol.1(-1)) caused a reinforced activation of the current. Our results indicate a voltage-dependent conductance in a tight sodium-absorbing epithelium.

Delta-aminolevulinic acid transport by intestinal and renal peptide transporters and its physiological and clinical implications.

Delta-aminolevulinic acid (ALA) is the precursor of porphyrin synthesis and has been recently used in vitro and in clinical studies as an endogenous photosensitizer for photodynamic therapy in the treatment of various tumors. For this purpose, ALA is given topically, systemically, or orally. When administered by the oral route, it shows excellent intestinal absorption. ALA is also efficiently reabsorbed in the renal proximal tubule after glomerular filtration. However, the pathways and mechanisms for its transmembrane transport into epithelial cells of intestine and kidney are unknown. Here we demonstrate that ALA uses the intestinal and renal apical peptide transporters for entering into epithelial cells. Kinetics and characteristics of ALA transport were determined in Xenopus laevis ooyctes and Pichia pastoris yeast cells expressing either the cloned intestinal peptide transporter PEPT1 or the renal form PEPT2. By using radiolabeled ALA and electrophysiological techniques in these heterologous expression systems, we established that: (a) PEPT1 and PEPT2 translocate 3H-ALA by saturable and pH-dependent transport mechanisms, (b) that ALA and di-/tripeptides, but not GABA or related amino acids, compete at the same substrate-binding site of the carriers, and (c) that ALA transport is electrogenic in nature as a consequence of H+/ALA cotransport. Reverse transcriptase-PCR analysis performed with specific primers for PEPT1 and PEPT2 in rabbit tissues demonstrates that, in particular, the PEPT2 mRNA is expressed in a variety of other tissues including lung, brain, and mammary gland, which have been shown to accumulate ALA. This suggests that these tissues could take up the porphyrin precusor via expressed peptide transporters, providing the endogenous photosensitizers for efficient photodynamic therapy.

Effects of nicotine on human nasal epithelium: evidence for nicotinic receptors in non-excitable cells.

We investigated the effects of nicotine and its derivate nicotine di-d-tartrate on primary cultured human nasal epithelial cells. Both substances evoked an increase in the intracellular free calcium concentration. In the presence of extracellular Ca2+ the cytosolic Ca2+ ([Ca2+]i) increase was long lasting, whereas in the absence of external Ca2+ there was a transient increase of [Ca2+]i indicating that nicotine has an influence on Ca2+ conductances across the membranes and on intracellular Ca2+ stores. Both effects could be blocked by the nicotinic receptor antagonist methyllycaconitine (MLA). Apical or basolateral application of nicotine during transepithelial transport measurements with confluent monolayers of cultured human nasal cells resulted in a significant, reversible decrease of amiloride-sensitive sodium absorption with an apparent half-maximal blocker concentration of about 950 microM. To exclude the possibility that remnant neuronal components were responsible for the observed effects we used tetrodotoxin and verapamil to block putative neuronal channels and 4-(4-diethylamino)styryl-N-methylpyridinium iodide (4-di-2-Asp) to stain neuronal tissue. Both experimental approaches demonstrated that there were no neuronal-mediated effects. These results indicate the direct effects of nicotine on human nasal epithelium, giving the first evidence of the existence of nicotinic receptors in non-excitable cells.

Amiloride-sensitive Na+ channels in human nasal epithelium are different from classical epithelial Na+ channels.

We characterized Na+ absorption in confluent monolayers of primary cultured epithelia derived from human nasal cystic fibrosis (CF) and non-CF epithelium in modified Ussing chambers. Amiloride-sensitive Na+ channels in cells obtained from CF as well as from non-CF patients showed properties different from all previously described epithelial Na+ channels (ENaC). DPC, a potent Cl- channel blocker, which has never been described to block ENaC, inhibited a considerable portion of the amiloride-sensitive Na+ absorption. In contrast to classical ENaC, cAMP induced no activation of amiloride-sensitive short-circuit current. Aldosterone failed to induce any functional stimulation of Na+ absorption in vitro when applied to the cell culture medium prior to measurements. Together with the reportedly reversible inhibition by phenamil we propose that Na+ absorption in human nasal epithelia is either regulated differently or is mediated by a yet still unknown member of the ENaC superfamily.

Cystic fibrosis and non-cystic-fibrosis human nasal epithelium show analogous Na+ absorption and reversible block by phenamil.

Transepithelial short-circuit current (ISC), potential (VT) and resistance (RT) of confluent monolayers of human nasal epithelium cultured from patients with and without cystic fibrosis (CF) were measured. In our Ussing chamber experiments with monolayers derived from non-CF and CF patients neither ISC (non-CF: 14.1 +/- 1.0 microA/cm2, n = 77; CF: 16.7 +/- 1.5 microA/cm2, n = 42), nor RT (non-CF: 288 +/- 15 Omega . cm2; CF: 325 +/- 20 Omega . cm2) showed any significant differences, only VT showed moderate but significant different values (non-CF: -3.6 +/- 0.4 mV; CF: -5.6 +/- 0.7 mV, respectively). Total ISC in CF cells was nearly completely inhibited by amiloride (92 +/- 9.6%), while in non-CF tissue amiloride-insensitive conductances mediated a considerable amount of the ISC (36.3 +/- 6.1%), indicating a lower activity of amiloride-sensitive Na+ conductances in non-CF cells. In both tissues the amiloride-sensitive ISC could also be blocked by the amiloride analogues benzamil, phenamil and 5-(N-ethyl-N-isopropyl)2', 4'-amiloride (EIPA) with different affinities. However, amiloride had a significant lower affinity in CF tissue (half-maximal blocker concentration, K1/2 = 586 +/- 59 nM) compared with non-CF tissue (K1/2 = 294 +/- 22 nM). Astonishingly, phenamil, a blocker which irreversibly blocks all epithelial Na+ channels hitherto described, inhibited the Na+ conductances of human nasal epithelium in a completely reversible way, but nevertheless with high affinity (non-CF: K1/2 = 12.5 +/- 1.2 nM; CF: K1/2 = 17.1 +/- 1.1 nM). Even in high doses none of these blockers had any effect on intracellular Ca2+ concentration as measured with Fura-2. From these findings, we conclude that the epithelial Na+ conductances of human CF nasal epithelium show modified regulation or are functionally different from those of other tissues.

[Benzamil and mucoviscidosis. Primary culture of nasal mucosa as an electrophysiologic in vitro model]. / Benzamil und Mukoviszidose. Primärkultur der Nasenschleimhaut als elektrophysiologisches In-vitro-Modell.

The genetic disease cystic fibrosis (CF) is characterized by a defect in the gene encoding for an epithelial chloride channel. This gene product, cystic fibrosis transmembrane conductance regulator (CFTR), in turn leads to a decreased chloride secretion. When this occurs sodium absorption is increased drastically because of an up-regulation of the epithelial sodium channel. In airway epithelia these changes in ion permeabilities result in dehydration of the mucus blanket and impaired mucociliary clearance, and subsequently lead to chronic infections of the airways. In this study we established a primary cell culture of human nasal epithelium from CF and non-CF patients. The increased transepithelial potential in CF exclusively was due to the greater sodium absorption. In previous studies, amiloride was found to specifically block the epithelial sodium channel. Inhalation of nebulized amiloride has been used clinically in the treatment of CF to inhibit sodium absorption and exert a secondary effect on water withdrawal. In the present cell culture model benzamil, an amiloride analogue, was found to inhibit sodium absorption completely, but at strikingly lower concentrations than amiloride. This raises the possibility of using benzamil for inhalation and to provide better efficacy in the symptomatic therapy of patients with CF.

cAMP-activation of amiloride-sensitive Na+ channels from guinea-pig colon expressed in Xenopus oocytes.

Guinea-pig distal colonic mRNA injection into Xenopus laevis oocytes resulted in expression of functional active epithelial Na+ channels in the oocyte plasma membrane. Poly(A)+ RNA was extracted from distal colonic mucosa of animals fed either a high-salt (HS) or a low-salt (LS) diet. The electrophysiological properties of the expressed amiloride-sensitive Na+ conductances were investigated by conventional two-electrode voltage-clamp and patch-clamp measurements. Injection of poly(A)+ RNA from HS-fed animals [from hereon referred to as HS-poly(A)+ RNA] into oocytes induced the expression of amiloride-sensitive Na+ conductances. On the other hand, oocytes injected with poly(A)+ RNA from LS-fed animals [LS-poly(A)+ RNA] expressed a markedly larger amount of amiloride-blockable Na+ conductances. LS-poly(A)+ RNA-induced conductances were completely inhibitable by amiloride with a Ki of 77 nM, and were also blocked by benzamil with a Ki of 1.8 nM. 5-(N-Ethyl-N-isopropyl)-amiloride (EIPA), even in high doses (25 "mu"M), had no detectable effect on the Na+ conductances. Expressed amiloride-sensitive Na+ channels could be further activated by cAMP leading to nearly doubled clamp currents. When Na+ was replaced by K+, amiloride (1 "mu"M) showed no effect on the clamp current. Single-channel analysis revealed slow gating behaviour, open probabilities (Po) between 0.4 and 0.9, and slope conductances of 3. 8 pS for Na+ and 5.6 pS for Li+. The expressed channels showed to be highly selective for Na+ over K+ with a permeability ratio PNa/PK > 20. Amiloride (500 nM) reduced channel Po to values < 0.05. All these features make the guinea-pig distal colon of LS-fed animals an interesting mRNA source for the expression of highly amiloride-sensitive Na+ channels in Xenopus oocytes, which could provide new insights in the regulatory mechanism of these channels.