Concentrations Of Amiloride Research Articles

Extracellular alpha 6 integrin cleavage by urokinase-type plasminogen activator in human prostate cancer

During human prostate cancer progression, the integrin α6β1 (laminin receptor) is expressed on the cancer cell surface during invasion and in lymph node metastases. We previously identified a novel structural variant of the α6 integrin called α6p. This variant was produced on the cell surface and was missing the β-barrel extracellular domain. Using several different concentrations of amiloride, aminobenzamidine and PAI-1 and the urokinase-type plasminogen activator (uPA) function-blocking antibody (3689), we showed that uPA, acting as a protease, is responsible for production of α6p. We also showed that addition of uPA in the culture media of cells that do not produce α6p, resulted in a dose-dependent α6p production. In contrast, the addition of uPA did not result in the cleavage of other integrins. Using α2-antiplasmin and plasmin depleted media, we observed that uPA cleaves the α6 integrin directly. Further, 12- o-tetradecanoyl-phorbol-13-acetate (TPA) induced the production of α6p, and this induction was abolished by PAI-1 but not α2-antiplasmin. Finally, the α6p integrin variant was detected in invasive human prostate carcinoma tissue indicating that this is not a tissue culture phenomenon. These data, taken together, suggest that this is a novel function of uPA, that is, to remove the β-barrel ligand-binding domain of the integrin while preserving its heterodimer association.

Demonstration of a functional apical sodium hydrogen exchanger in isolated rat gastric glands.

Previous studies have shown that gastric glands express at least sodium-hydrogen exchanger (NHE) isoforms 1-4. Our aim was to study NHE-3 localization in rat parietal cells and to investigate the functional activity of an apical membrane NHE-3 isoform in parietal cells of rats. Western blot analysis and immunohistochemistry showed expression of NHE-3 in rat stomach colocalizing the protein in parietal cells together with the beta-subunit of the H(+)-K(+)-ATPase. Functional studies in luminally perfused gastric glands demonstrated the presence of an apical NHE isoform sensitive to low concentrations of 5-ethylisopropyl amiloride (EIPA). Intracellular pH measurements in parietal cells conducted in omeprazole-pretreated superfused gastric glands showed an Na+-dependent proton extrusion pathway that was inhibited both by low concentrations of EIPA and by the NHE-3 specific inhibitor S3226. This pathway for proton extrusion had a higher activity in resting glands and was inhibited on stimulation of histamine-induced H(+)-K(+)-ATPase proton extrusion. We conclude that the NHE-3 isoform located on the apical membrane of parietal cells offers an additional pathway for proton secretion under resting conditions. Furthermore, the gastric NHE-3 appears to work under resting conditions and inactivates during periods of H(+)-K(+)-ATPase activity.

Role of basolateral membrane conductance in the regulation of transepithelial sodium transport across frog skin.

Circuit analyses of the principal cell compartment of frog skin ( Rana temporaria and R. esculenta) were made using microelectrode measurements under short-circuit conditions and with the aid of the Na(+) channel blocker amiloride. Under control conditions, intracellular potential ranged between -65 and -5 mV, and the conductances of the apical and basolateral membranes were related directly to the short-circuit current and inversely to the cellular potential. Blockade of apical Na(+) uptake by amiloride hyperpolarized the cells to nearly the same value, irrespective of the potential under transporting conditions. Under these conditions, basolateral membrane conductance increased greatly, which led to paradoxical reactions of the transepithelial Na(+) transport at lower concentrations of amiloride. The half-maximal inhibitory concentration of amiloride estimated from the response of the apical membrane conductance (99+/-10 nM) was about 5 times lower than the value derived from transepithelial current or conductance in the same tissues. The results are discussed in the context of the importance of the membrane potential for acute control of membrane conductance and transepithelial transport.

Rat ENaC expressed in Xenopus laevis oocytes is activated by cAMP and blocked by Ni 2+

We used oocytes of the South African clawed toad Xenopus laevis to express the three subunits of the epithelial Na + channel from rat distal colon (rENaC). We combined conventional dual-microelectrode voltage-clamp with continuous capacitance ( C m) measurements and noise analysis to evaluate the effects of cAMP and Ni 2+ on rENaC. Control oocytes or rENaC-expressing oocytes exhibited no spontaneous fluctuations in current. However, in rENaC-expressing oocytes amiloride induced a marked plateau-shaped rise of the power density spectra. Recordings using four different concentrations of amiloride revealed that the blocker–channel interactions were of the first order. A cocktail of the membrane permeant cAMP analogue chlorophenylthio-cAMP and IBMX (cAMP cocktail) increased amiloride-sensitive current ( I ami) and conductance ( G ami). Furthermore, C m was also increased following cAMP application, indicating an increase in plasma membrane surface area. Noise analysis showed that cAMP increased the number of active channels in the oocyte membrane while single-channel current decreased. From these data we conclude that cAMP triggered exocytotic delivery of preformed rENaCs to the plasma membrane. Ni 2+ (2.5 mM) inhibited about 60% of the rENaC current and conductance while C m remained unaffected. Noise analysis revealed that this inhibition could be attributed to a decrease in the apparent channel density, while single-channel current did not change significantly. These observations argue for direct effects of Ni 2+ on channel activity rather than induction of endocytotic removal of active channels from the plasma membrane.

The effect of amiloride on the in vivo effective permeability of amoxicillin in human jejunum: experience from a regional perfusion technique

The purpose of this human intestinal perfusion study (in vivo) was twofold. Firstly, we aimed to determine the effective in vivo jejunal permeability ( P eff) of amoxicillin and to classify it according to the Biopharmaceutics Classification System (BCS). Secondly, we investigated the acute effect of amiloride on the jejunal P eff of amoxicillin. Amoxicillin, a β-lactam antibiotic, has been reported to be absorbed across the intestinal mucosa by both passive diffusion and active transport. A regional single-pass perfusion of the jejunum was performed using a Loc-I-Gut ® perfusion tube in 14 healthy volunteers. Each perfusion lasted for 200 min and was divided into two periods of 100 min each. The concentration of amoxicillin entering the jejunal segment was 300 mg/l in both periods, and amiloride, an inhibitor of the Na +/H + exchanger, was added at 25 mg/l in period 2. The concentrations of amoxicillin and amiloride in the outlet jejunal perfusate were measured with two different HPLC-methods. Antipyrine and [ 14C]PEG 4000 were added as internal standards to the perfusion solution. Amiloride had no significant effect on the jejunal P eff of amoxicillin. The human in vivo jejunal P eff for amoxicillin was 0.34±0.11×10 −4 and 0.46±0.12×10 −4 cm/s in periods 1 and 2, respectively. The high jejunal P eff for amiloride was 1.63±0.51×10 −4 cm/s which predicts an intestinal absorption of more than 90%. Following the BCS amoxicillin was classified as a low P eff drug, and amiloride had no acute effect on the in vivo jejunal P eff of amoxicillin.

Influence of voltage and extracellular Na(+) on amiloride block and transport kinetics of rat epithelial Na(+) channel expressed in Xenopus oocytes.

We expressed the three subunits of the epithelial amiloride-sensitive Na(+) channel (ENaC) from rat distal colon heterologously in oocytes of Xenopus laevis and analysed blocker-induced fluctuations in current using conventional dual-microelectrode voltage-clamp. To minimize Na(+) accumulation we performed all experiments in low-Na(+) solutions (15 mM). Noise analysis revealed that control or ENaC-injected oocytes did not exhibit spontaneous relaxation noise. However, in ENaC-expressing oocytes, amiloride induced a distinct Lorentzian component in the power density spectra. With three amiloride concentrations and a linear analysis of the respective changes in the corner frequency f(c) (2 pi f(c) plot) we determined the rate constants k(on) and k(off) for the amiloride-ENaC interaction. At a clamp potential (V(m)) of -60 mV k(on) was 80.8 +/- 5.1 microM(-1) s(-1) and k(off) 15.4 +/- 4.2 s(-1). The half-maximal blocker concentration (K(mic,ami)) was 0.19 microM (V(m)=-60 mV). While k(on) was voltage-independent in the range -50 to -100 mV, k(off) and K(mic,ami) decreased significantly with increasing membrane hyperpolarization, resulting in an increased affinity of amiloride for its binding site on ENaC. Increasing extracellular [Na(+)] ([Na(+)](o)) led to saturation of ENaC. Subsequent noise analysis revealed that single-channel current increased non-linearly with [Na(+)](o) and that saturation was not due to a reduction in the number of open channels. The apparent affinity of Na(+) for its binding site on the channel was voltage dependent and increased with hyperpolarization. Noise analysis revealed that k(on) and k(off) for amiloride decreased with increasing [Na(+)](o), while the affinity of the amiloride-binding site did not change. These findings show that the affinity of rat intestinal ENaC for amiloride is voltage dependent and is influenced non-competitively by [Na(+)](o), indicating that Na(+) and amiloride do not compete for the same binding site at the channel.

Direct analysis of amiloride and triamterene mixtures by fluorescence spectrometry using partial-least squares calibration

The simultaneous determination of amiloride and triamterene in urine samples using fluorimetry in combination with partial-least squares (PLS) multivariate calibration is proposed. Triamterene and amiloride exhibit overlapped spectra and urine produces background fluorescence that precludes the direct determination of these diuretics by conventional fluorimetry. Although, the qualitative composition of the fluorescent metabolites in urine from healthy people is virtually invariable, their quantitative composition exhibits some differences. Thus, the shape of the spectrum and the position of the fluorescence maxima change as the urine is diluted.The determination was performed in a 1:1 (v/v) ethanol /water medium at an apparent pH of 6.3 provided by 0.01M sodium citrate/citric acid buffer. An excitation wavelength of 365nm was used for both amiloride and triamterene. The corresponding emission maxima were located at 413 and 437nm for amiloride and triamterene, respectively.A calibration set consisting of samples standards was used in combination with a factor design; two levels per factor and a central star design (i.e. a central composite design) were used. In order to ensure accurate results, the calibration matrix was implement an urine sample containing no triamterene or amiloride (i.e. urine blank). The components of the calibration matrix were triamterene, amiloride and urine. The concentration of amiloride was varied from 64 to 320ng/ml and that of triamterene from 20 to 100ng/ml. Urine dilution was varied from 1:35 to 1:65. Urine was used as the third component of the calibration matrix in order to include the information inherent in changes in the fluorescence spectrum for urine upon dilution.

Identification of sites in the second exomembrane loop and ninth transmembrane helix of the mammalian Na+/H+ exchanger important for drug recognition and cation translocation.

Mammalian Na(+)/H(+) exchanger (NHE) isoforms are differentially sensitive to inhibition by several distinct classes of pharmacological agents, including amiloride- and benzoyl guanidinium-based derivatives. The determinants of drug sensitivity, however, are only partially understood. Earlier studies of the drug-sensitive NHE1 isoform have shown that residues within the fourth membrane-spanning helix (M4) (Phe(165), Phe(166), Leu(167), and Gly(178)) and a 66-amino acid segment encompassing M9 contribute significantly to drug recognition. In this report, we have identified two residues within M9, one highly conserved (Glu(350)) and the other non-conserved (Gly(356)), that are major determinants of drug sensitivity. In addition, residues in the second exomembrane loop between M3 and M4 (Gly(152), Phe(157), and Pro(158)) were also found to modestly influence drug sensitivity. A double substitution of crucial sites within M4 and M9 of NHE1 with the corresponding residues present in the drug-resistant NHE3 isoform (i.e. L167F/G356A) greatly reduced drug sensitivity in a cooperative manner to levels nearing that of wild type NHE3. The above mutations did not appreciably affect Na(o)(+) affinity but did markedly decrease the catalytic turnover of the transporter. These data suggest that specific sites encompassing M4 and M9 are critical determinants of both drug recognition and cation translocation.

Advances in the diagnosis of cystic fibrosis in infants

Advances in the diagnosis of cystic fibrosis in infants

A modified technique for measurement of nasal transepithelial potential difference in infants

Objective: To establish a method for measuring nasal transepithelial potential difference (PD) in infants. Study design: A modified infant method (smaller catheter size, reduced flow rates, and shorter protocol time) was compared with an established adult nasal PD method in 10 adult volunteers (4 with cystic fibrosis [CF]). Nasal PD was measured in 13 infants with a possible diagnosis of CF. Results: Recordings were similar for the established and the modified methods in adult volunteers. An amiloride concentration of 10–4 mol/L was necessary for full inhibition of amiloride-sensitive sodium ion (Na+) transport. Of the 13 infants, 2 had PD values suggestive of CF (mean baseline PD, –50.1 mV and –31.4 mV; maximum baseline PD, –61 mV and –49 mV; change in PD after perfusion with zero chloride solution with isoprenaline and amiloride [ΔzeroCl–/Iso], –1 mV and +3.5 mV), and 11 had normal values (mean ± SEM baseline PD, –13.2 ± 1.0 mV; maximum baseline PD, –21.4 ± 2.0; ΔzeroCl–/Iso, –15.3 ± 1.9 mV). These results correlated with subsequent sweat test data, mutation analysis, and clinical outcome. Conclusion: Nasal PD measured with this modified method is comparable to that measured with an established adult method. The measurements were well tolerated in 13 infants and discriminated bioelectric profiles characteristic of normal and CF respiratory epithelium. This study supports the use of this modified nasal PD technique as a diagnostic test for CF in newborn infants. (J Pediatr 2001;139:353-8)

Expression of highly selective sodium channels in alveolar type II cells is determined by culture conditions.

Alveolar fluid clearance in the developing and mature lungs is believed to be mediated by some form of epithelial Na channels (ENaC). However, single-channel studies using isolated alveolar type II (ATII) cells have failed to demonstrate consistently the presence of highly selective Na+ channels that would be expected from ENaC expression. We postulated that in vitro culture conditions might be responsible for alterations in the biophysical properties of Na+ conductances observed in cultured ATII cells. When ATII cells were grown on glass plates submerged in media that lacked steroids, the predominant channel was a 21-pS nonselective cation channel (NSC) with a Na+-to-K+ selectivity of 1; however, when grown on permeable supports in the presence of steroids and air interface, the predominant channel was a low-conductance (6.6 +/- 3.4 pS, n = 94), highly Na+-selective channel (HSC) with a P(Na)/P(K) >80 that is inhibited by submicromolar concentrations of amiloride (K(0.5) = 37 nM) and is similar in biophysical properties to ENaC channels described in other epithelia. To establish the relationship of this HSC channel to the cloned ENaC, we employed antisense oligonucleotide methods to inhibit the individual subunit proteins of ENaC (alpha, beta, and gamma) and used patch-clamp techniques to determine the density of this channel in apical membrane patches of ATII cells. Overnight treatment of cells with antisense oligonucleotides to any of the three subunits of ENaC resulted in a significant decrease in the density of HSC channels in the apical membrane cell-attached patches. Taken together, these results show that when grown on permeable supports in the presence of steroids and air interface, the predominant channels expressed in ATII cells have single-channel characteristics resembling channels that are associated with the coexpression of the three cloned ENaC subunits alpha-, beta-, and gamma-ENaC.

Na+-dependent recovery of intracellular pH from acid loading in mouse colonic crypt cells.

The membrane transport mechanism for regulating the intracellular pH value (pHi) was investigated in mouse distal colon crypt cells. pHi was measured by microfluorometry in an isolated crypt fragment loaded with the pH-sensitive fluoroprobe, 2',7'-bis-(2-carboxyethyl)-5-(6) carboxyfluorescein. The pHi recovery process after acid loading induced by a 40 mM NH4Cl prepulse was almost totally dependent on Na+ in both the presence and absence of CO2/HCO3- in the perfusion solution. In the CO2/HCO3(-)-free, HEPES-buffered solution, amiloride partially inhibited the pHi recovery rate from acid loading with an ED50 value of 15 microM and maximum inhibition of 83%. In a CO2/HCO3- solution, amiloride inhibited the pHi recovery rate with an ED50 value of 18 microM, which was similar to that in the HEPES-buffered solution, while the rate of pHi recovery remaining in the presence of the maximum effective concentration of amiloride was significantly larger than that in the HEPES-buffered solution. The Na+-dependent pHi recovery from the acid loading was significantly less (by 18%) in the presence of forskolin. These results suggest that the pHi recovery from acid loading was mediated by 1) amiloride-sensitive Na+/H+ exchanger, 2) the amiloride-insensitive Na+/H+ exchanger, and 3) the Na+- and HCO3(-)-dependent acid extruder. The pHi recovery could be inhibited by cAMP.

Neural representation of salts in the rat solitary nucleus: brain stem correlates of taste discrimination.

One mechanism of salt taste transduction by gustatory receptor cells involves the influx of cations through epithelial sodium channels that can be blocked by oral application of amiloride. A second mechanism is less clearly defined but seems to depend on electroneutral diffusion of the salt through the tight junctions between receptor cells; this paracellular pathway is insensitive to amiloride. Because the first mechanism is more sensitive to sodium salts and the second to nonsodium salts, these peripheral events could underlie the ability of rats to discriminate sodium from nonsodium salts on the basis of taste. Behavioral experiments indicate that amiloride, at concentrations that are tasteless to rats, impairs a rat's ability to discriminate NaCl from KCl and may do so by making both salts taste like KCl. In the present study, we examined the neural representation of NaCl and KCl (0.05-0.2 M), and mixtures of these salts with amiloride (0, 3, and 30 microM), to explore the neural correlates of this behavioral result. NaCl and KCl were represented by distinct patterns of activity in the nucleus of the solitary tract. Amiloride, in a concentration-dependent manner, changed the pattern for NaCl to one more characteristic of KCl, primarily by reducing activity in neurons responding best to NaCl and sucrose. The effect of amiloride concentration on the response to 0.1 M NaCl in NaCl-best neurons was virtually identical to its effect on behavioral discrimination performance. Modeling the effects of blocking the amiloride-insensitive pathway also resulted in highly similar patterns of activity for NaCl and KCl. These results suggest that activity in both the amiloride-sensitive and -insensitive pathways is required for the behavioral discrimination between NaCl and KCl. In the context of published behavioral data, the present results suggest that amiloride-sensitive activity alone is not sufficient to impart a unique signal for the taste of sodium salts.

Intracellular pH regulatory mechanism in a human renal proximal cell line (HKC-8): evidence for Na+/H+ exchanger, CI-/HCO3- exchanger and Na+-HCO3- cotransporter.

In the present study we investigated whether an immortalized human renal proximal cell line, HKC-8, expresses a recently cloned Na+-HCO3- cotransporter (NBC-1) and, if so, which isoform (kNBC-1 from kidney or pNBC-1 from pancreas) is expressed in this cell line. Cell pH (pHi) measurements using a pH-sensitive fluorescence probe in the absence of HCO3-/CO2 revealed the presence of a Na+/H+ exchanger that required high concentrations of amiloride for full inhibition. In the presence of HCO3-/CO2 another pHi recovery process, dependent on Na+ but independent of Cl-, was identified. This process was electrogenic and was inhibited by 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulphonic acid (DIDS), being consistent with the Na+-HCO3- cotransporter. In addition, the pHi responses to Cl- removal were compatible with the presence of a Na+-independent Cl-/HCO3- exchanger that was also inhibited by DIDS. Reverse transcriptase polymerase chain reaction (RT-PCR) using primers designed for specific and common regions detected mRNAs of both kNBC-1 and pNBC-1 and Western blot analysis confirmed the expression of NBC-1 protein. These results indicate that HKC-8 has transport activities similar to intact proximal tubules and also suggest that both kNBC-1 and pNBC-1 may contribute to the Na+-HCO3- cotransport activity in this cell line.

Dual-stopped-flow spectrophotometric determination of amiloride hydrochloride in a multicommutated flow system

In this work a multicommutated flow system combining a binary sampling approach with a dual-stopped-flow strategy, for the spectrophotometric determination of amiloride hydrochloride in pharmaceutical preparations is proposed. The developed methodology is based on a two-stage reaction between amiloride hydrochloride and 3-methyl-2-benzothiazolinone hydrazone (MBTH) in the presence of Ce(IV) which is monitored at 545 nm. Reaction development is favoured by the intercalation of sample and reagent aliquots accomplishing a faster sample zone homogenisation with a low dispersion pattern. The sample zone is then halted for a certain period prior to detection resulting in an increased reaction time and a concomitant sensitivity enhancement. The implementation of a dual-stopped-flow manifold enabled the simultaneous processing of two samples, thus improving the sampling rate. Beer’s law was followed for amiloride concentrations of up to 120 μg ml −1. Results were reproducible (RSD < 2.2%, n = 10) and in agreement with those obtained by the reference procedure with relative deviations from −1.2 to 2.4%. A sampling rate of about 30 samples per hour was attained.

Maitotoxin induces insertion of different ion channels into the Xenopus oocyte plasma membrane via Ca(2+)-stimulated exocytosis.

The activation of cation channels in oocytes of Xenopus laevis by the marine poison maitotoxin (MTX) was monitored as membrane current (I(m)), conductance (Gm) and membrane surface area determined by continuous measurements of membrane capacitance (Cm). When MTX (25 pM) was added to the bathing solution there was an abrupt, large increase in inward membrane currents. Current/voltage relationships (I/V curves) were linear and suggested activation of voltage-independent non-selective cation channels (NSCC). MTX-induced Ca(2+)-sensitive currents were mainly carried by Na+ and were suppressed by low (0 mM) or high (40 mM) external Ca2+ concentrations and removal of Na+. Gadolinium (Gd3+, 10-500 microM) also had inhibitory effects, demonstrating the possible involvement of stretch-activated cation channels (SACC). In a high concentration (500 microM), amiloride substantially reduced the MTX-activated current while lower amiloride concentrations (50-100 microM) stimulated the current further. Continuous measurements of Cm revealed that MTX induced exocytotic delivery and functional insertion of new channel proteins into the plasma membrane, indicated by a Ca(2+)-dependent increase in membrane surface area by around 28%. From these data we conclude that MTX activates NSCC that require relatively high concentrations of amiloride to be blocked. Furthermore, MTX possibly stimulates activation of Gd(3+)- and Ca(2+)-sensitive mechanosensitive cation channels. Stimulation of these channels is achieved by exocytotic delivery and functional insertion of new channels into the plasma membrane in a pathway that depends on the presence of extracellular Ca2+.

Muscarinic Receptor Activity Induces an Afterdepolarization in a Subpopulation of Hippocampal CA1 Interneurons

Cholinergic input to the hippocampus may be involved in important behavioral functions and the pathophysiology of neurodegenerative diseases. Muscarinic receptor activity in interneurons of the hippocampus may play a role in these actions. In this study, we investigated the effects of muscarinic receptor activity on the excitability of different subtypes of interneurons in rat hippocampal CA1. Most interneurons displayed an afterhyperpolarizing potential (AHP) after depolarization by injected current or synaptic stimulation. In the presence of a muscarinic agonist, the AHP of a subset of these interneurons was replaced by an afterdepolarization (ADP), often of sufficient magnitude to evoke action potentials in the absence of further stimulation. The ADP was insensitive to cadmium and low extracellular calcium. It was blocked by low extracellular sodium but not by tetrodotoxin or low concentrations of amiloride. Muscarinic ADPs were sometimes observed in isolation but were often accompanied by depolarizing, hyperpolarizing, or biphasic changes in the membrane potential. Interneurons with muscarinic ADPs were found in all strata of CA1 and did not fall into a single morphological classification. The potential functions of the prolonged action potential output of interneurons produced by the ADP could include changes in hippocampal circuit properties and facilitation of the release of peptide cotransmitters in these interneurons.

Antisense oligonucleotides against the alpha-subunit of ENaC decrease lung epithelial cation-channel activity.

Amiloride-sensitive Na+ transport by lung epithelia plays a critical role in maintaining alveolar Na+ and water balance. It has been generally assumed that Na+ transport is mediated by the amiloride-sensitive epithelial Na+ channel (ENaC) because molecular biology studies have confirmed the presence of ENaC subunits alpha, beta, and gamma in lung epithelia. However, the predominant Na+-transporting channel reported from electrophysiological studies by most laboratories is a nonselective, high-conductance channel that is very different from the highly selective, low-conductance ENaC reported in other tissues. In our laboratory, single-channel recordings from apical membrane patches from rat alveolar type II (ATII) cells in primary culture reveal a nonselective cation channel with a conductance of 20.6 +/- 1.1 pS and an Na+-to-K+ selectivity of 0.97 +/- 0.07. This channel is inhibited by submicromolar concentrations of amiloride. Thus there is some question about the relationship between the gene product observed with single-channel methods and the cloned ENaC subunits. We have employed antisense oligonucleotide methods to block the synthesis of individual ENaC subunit proteins (alpha, beta, and gamma) and determined the effect of a reduction in the subunit expression on the density of the nonselective cation channel observed in apical membrane patches on ATII cells. Treatment of ATII cells with antisense oligonucleotides inhibited the production of each subunit protein; however, single-channel recordings showed that only the antisense oligonucleotide targeting the alpha-subunit resulted in a significant decrease in the density of nonselective cation channels. Inhibition of the beta- and gamma-subunit proteins alone or together did not cause any changes in the observed channel density. There were no changes in open probability or other channel characteristics. These results support the hypothesis that the alpha-subunit of ENaC alone or in combination with some protein other than the beta- or gamma-subunit protein is the major component of lung alveolar epithelial cation channels.

ETB receptor activation leads to activation and phosphorylation of NHE3.

In OKP cells expressing ETB endothelin receptors, activation of Na+/H+ antiporter activity by endothelin-1 (ET-1) was resistant to low concentrations of ethylisopropyl amiloride, indicating regulation of Na+/H+ exchanger isoform 3 (NHE3). ET-1 increased NHE3 phosphorylation in cells expressing ETB receptors but not in cells expressing ETA receptors. Receptor specificity was not due to demonstrable differences in receptor-specific activation of tyrosine phosphorylation pathways or inhibition of adenylyl cyclase. Phosphorylation was associated with a decrease in mobility on SDS-PAGE, which was reversed by treating immunoprecipitated NHE3 with alkaline phosphatase. Phosphorylation was first seen at 5 min and was maximal at 15-30 min. Phosphorylation was maximal with 10(-9) M ET-1. Phosphorylation occurred on threonine and serine residues at multiple sites. In summary, ET-1 induces NHE3 phosphorylation in OKP cells on multiple threonine and serine residues. ETB receptor specificity, time course, and concentration dependence are all similar between ET-1-induced increases in NHE3 activity and phosphorylation, suggesting that phosphorylation plays a key role in activation.

Effects of amiloride on gustatory quality descriptions and temporal patterns produced by NaCl.

The amiloride-sensitivity of perceived taste qualities and time-intensity patterns for NaCl, and interactions between amiloride and NaCl as taste stimuli, were explored using caffeine as the control treatment. NaCl at 100, 250 and 500 mM, dissolved in 10 or 100 microM amiloride, or in caffeine concentrations matched to the amiloride taste, was flowed over 39.3 mm2 of the anterodorsal tongue for 4 s using a closed stimulus delivery system. Amiloride, caffeine and NaCl in H2O were also presented. It was found that NaCl-amiloride mixtures were most frequently described as salty, with the incidence of salty descriptions directly associated with NaCl concentration but not significantly associated with the presence or concentration of amiloride. Amiloride in H2O was called 'bitter', and the incidence of bitter descriptions was significantly associated with the presence of amiloride. The perceived temporal patterns varied with NaCl concentration but did not change with the presence of amiloride, except for an increase in perceived duration. No evidence was found for a dependence upon specific amiloride-sensitive mechanisms of human description of NaCl as salty or of gustatory temporal patterns evoked by NaCl.