ATP-mediated increase in H+ efflux from retinal Müller cells of the axolotl.

Previous work has shown that activation of tiger salamander retinal radial glial cells by extracellular ATP induces a pronounced extracellular acidification, which has been proposed to be a potent modulator of neurotransmitter release. This study demonstrates that low micromolar concentrations of extracellular ATP similarly induce significant H+ effluxes from Müller cells isolated from the axolotl retina. Müller cells were enzymatically isolated from axolotl retina and H+ fluxes were measured from individual cells using self-referencing H+-selective microelectrodes. The increased H+ efflux from axolotl Müller cells induced by extracellular ATP required activation of metabotropic purinergic receptors and was dependent upon calcium released from internal stores. We further found that the ATP-evoked increase in H+ efflux from Müller cells of both tiger salamander and axolotl were sensitive to pharmacological agents known to interrupt calmodulin and protein kinase C (PKC) activity: chlorpromazine (CLP), trifluoperazine (TFP), and W-7 (all calmodulin inhibitors) and chelerythrine, a PKC inhibitor, all attenuated ATP-elicited increases in H+ efflux. ATP-initiated H+ fluxes of axolotl Müller cells were also significantly reduced by amiloride, suggesting a significant contribution by sodium-hydrogen exchangers (NHEs). In addition, α-cyano-4-hydroxycinnamate (4-cin), a monocarboxylate transport (MCT) inhibitor, also reduced the ATP-induced increase in H+ efflux in both axolotl and tiger salamander Müller cells, and when combined with amiloride, abolished ATP-evoked increase in H+ efflux. These data suggest that axolotl Müller cells are likely to be an excellent model system to understand the cell-signaling pathways regulating H+ release from glia and the role this may play in modulating neuronal signaling.NEW & NOTEWORTHY Glial cells are a key structural part of the tripartite synapse and have been suggested to regulate synaptic transmission, but the regulatory mechanisms remain unclear. We show that extracellular ATP, a potent glial cell activator, induces H+ efflux from axolotl retinal Müller (glial) cells through a calcium-dependent pathway that is likely to involve calmodulin, PKC, Na+/H+ exchange, and monocarboxylate transport, and suggest that such H+ release may play a key role in modulating neuronal transmission.

Hereditary causes of hypertension due to increased sodium transport.

PURPOSE OF REVIEW: Hypertension, commonly known as high blood pressure, is a widespread health condition affecting a large number of individuals across the globe. Although lifestyle choices and environmental factors are known to have a significant impact on its development, there is growing recognition of the influence of genetic factors in the pathogenesis of hypertension. This review specifically focuses on the hereditary causes of hypertension that are associated with increased sodium transport through the thiazide-sensitive NaCl cotransporter (NCC) or amiloride-sensitive epithelial sodium channel (ENaC), crucial mechanisms involved in regulating blood pressure in the kidneys. By examining genetic mutations and signaling molecules linked to the dysregulation of sodium transport, this review aims to deepen our understanding of the hereditary causes of hypertension and shed light on potential therapeutic targets. RECENT FINDINGS: Liddle syndrome (LS) is a genetic disorder that typically manifests early in life and is characterized by hypertension, hypokalemic metabolic alkalosis, hyporeninemia, and suppressed aldosterone secretion. This condition is primarily caused by gain-of-function mutations in ENaC. In contrast, Pseudohypoaldosteronism type II (PHAII) is marked by hyperkalemia and hypertension, alongside other clinical features such as hyperchloremia, metabolic acidosis, and suppressed plasma renin levels. PHAII results from overactivations of NCC, brought about by gain-of-function mutations in its upstream signaling molecules, including WNK1 (with no lysine (K) 1), WNK4, Kelch-like 3 (KLHL3), and cullin3 (CUL3). SUMMARY: NCC and ENaC are integral components, and their malfunctions lead to disorders like LS and PHAII, hereditary causes of hypertension. Current treatments for LS involve ENaC blockers (e.g., triamterene and amiloride) in conjunction with low-sodium diets, effectively normalizing blood pressure and potassium levels. In PHAII, thiazide diuretics, which inhibit NCC, are the mainstay treatment, albeit with some limitations and potential side effects. Ongoing research in developing alternative treatments, including small molecules targeting key regulators, holds promise for more effective and tailored hypertension solutions.

Capsazepine activates amiloride-insensitive ENaC-like channels in human leukemia cells.

Sodium influx carried out by ion channels is one of the main regulators of water-salt and volume balance in cells of blood origin. Previously, we described amiloride-insensitive ENaC-like channels in human myeloid leukemia K562 cells; the intracellular regulatory mechanisms of the channels are associated with actin cytoskeleton dynamics. Recently, an extracellular mechanism of ENaC-like channels activation in K562 cells by the action of serine protease trypsin has been revealed. The other extracellular pathways that modulate ENaC (epithelial Na+ channel) activity and sodium permeability in transformed blood cells are not yet fully investigated. Here, we study the action of capsazepine (CPZ), as δ-ENaC activator, on single channel activity in K562 cells in whole-cell patch clamp experiments. Addition of CPZ (2 µM) to the extracellular solution caused an activation of sodium channels with typical features; unitary conductance was 15.1 ± 0.8 pS. Amiloride derivative benzamil (50 µM) did not inhibit their activity. Unitary currents and conductance of CPZ-activated channels were higher in Na+-containing extracellular solution than in Li+, that is one of the main fingerprints of δ-ENaC. The results of RT-PCR analysis and immunofluorescence staining also confirmed the expression of δ-hENaC (as well as α-, ß-, γ-ENaC) at the mRNA and protein level. These findings allow us to speculate that CPZ activates amiloride-insensitive ENaC-like channels that contain δ-ENaC in К562 cells. Our data reveal a novel extracellular mechanism for ENaC-like activation in human leukemia cells.

Effect of irinotecan administration on amiloride-sensitive sodium taste responses in mice.

Taste alteration is a frequently reported side effect in patients receiving the chemotherapeutic agent, irinotecan. However, the way in which irinotecan causes taste disturbance and the type of taste impairment that is affected remain elusive. Here, we used the two-bottle preference test to characterize behavioral taste responses and employed immunohistochemical analyses to clarify the types and mechanisms of taste alteration induced, in mice, by irinotecan administration. Irinotecan administration resulted in a reduced intake of sodium taste solution but had no effect on sweet taste responses, as determined in the two-bottle preference test. In the presence of amiloride, which inhibits the function of the epithelial sodium channel (ENaC) in the periphery, the intake of sodium taste solution was comparable between the irinotecan-treated and control groups. Immunohistochemical analyses revealed that α-ENaC immunoreactivity detected in taste bud cells decreased slowly after irinotecan administration, and that administration of irinotecan had little effect on the number of cells expressing the cellular proliferation marker, Ki67, within or around taste buds. Our results imply that irinotecan administration may be responsible for altered behavioral sodium taste responses originating from ENaC function in the periphery, while being accompanied by the reduction of α-ENaC expression at the apical membrane of taste receptor cells without disturbing taste cell renewal.

Amiloride is a suitable fluorescent substrate for the study of the drug transporter human multidrug and toxin extrusion 1 (MATE1).

Human multidrug and toxin extrusion 1 (MATE1; SLC47A1) is highly expressed in the kidneys and the liver. It plays a significant role in drug and endogenous compound disposition, and therefore, a rapid evaluation of its inhibition is important for drug development and for the understanding of renal and hepatic physiology. Amiloride is a potassium-sparing diuretic used for treating hypertension; it also demonstrates strong fluorescence in organic solvent or detergent solutions. In this study, we investigated the transport characteristics of amiloride by human MATE1. Cellular accumulation of amiloride was evaluated in control vector- or MATE1-transfected HEK293 cells. Cells were lysed with 1% sodium dodecyl sulfate, and fluorescence was measured using a microplate reader at wavelengths of 364ex and 409em. With ammonium prepulse-induced intracellular acidification, MATE1 transported amiloride at an extracellular pH of 7.4. The uptake demonstrated an overshoot phenomenon and saturated, with the Km and Vmax being 23.5 µM and 1.01 nmol/mg/min, respectively. MATE1-mediated amiloride transport also presented with a bell-shaped pH profile that reached a maximum pH value of 7.4. The inhibitor sensitivity of MATE1-facilitated amiloride transport was similar to those of known substrates, such as tetraethylammonium and metformin. Among the tested inhibitors, pyrimethamine demonstrated the most potent inhibition with an IC50 value of 0.266 µM. Furthermore, MATE1 was found to be inhibited by fampridine, which was previously considered to be a non-inhibitor of MATE1. This study demonstrates that amiloride is a suitable fluorescent substrate for the in vitro study of the transport activity of MATE1.

Sodium Energetic Cycle in the Natronophilic Bacterium Thioalkalivibrio versutus.

As inhabitants of soda lakes, Thioalkalivibrio versutus are halo- and alkaliphilic bacteria that have previously been shown to respire with the first demonstrated Na+-translocating cytochrome-c oxidase (CO). The enzyme generates a sodium-motive force (Δs) as high as -270 mV across the bacterial plasma membrane. However, in these bacteria, operation of the possible Δs consumers has not been proven. We obtained motile cells and used them to study the supposed Na+ energetic cycle in these bacteria. The resulting motility was activated in the presence of the protonophore 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), in line with the same effect on cell respiration, and was fully blocked by amiloride-an inhibitor of Na+-motive flagella. In immotile starving bacteria, ascorbate triggered CO-mediated respiration and motility, both showing the same dependence on sodium concentration. We concluded that, in T. versutus, Na+-translocating CO and Na+-motive flagella operate in the Na+ energetic cycle mode. Our research may shed light on the energetic reason for how these bacteria are confined to a narrow chemocline zone and thrive in the extreme conditions of soda lakes.

Epithelial Sodium Channel Inhibition by Amiloride Addressed with THz Spectroscopy and Molecular Modeling.

THz spectroscopy is important for the study of ion channels because it directly addresses the low frequency collective motions relevant for their function. Here we used THz spectroscopy to investigate the inhibition of the epithelial sodium channel (ENaC) by its specific blocker, amiloride. Experiments were performed on A6 cells' suspensions, which are cells overexpressing ENaC derived from Xenopus laevis kidney. THz spectra were investigated with or without amiloride. When ENaC was inhibited by amiloride, a substantial increase in THz absorption was noticed. Molecular modeling methods were used to explain the observed spectroscopic differences. THz spectra were simulated using the structural models of ENaC and ENaC-amiloride complexes built here. The agreement between the experiment and the simulations allowed us to validate the structural models and to describe the amiloride dynamics inside the channel pore. The amiloride binding site validated using THz spectroscopy agrees with previous mutagenesis studies. Altogether, our results show that THz spectroscopy can be successfully used to discriminate between native and inhibited ENaC channels and to characterize the dynamics of channels in the presence of their specific antagonist.

Ion Selectivity in the ENaC/DEG Family: A Systematic Review with Supporting Analysis.

The Epithelial Sodium Channel/Degenerin (ENaC/DEG) family is a superfamily of sodium-selective channels that play diverse and important physiological roles in a wide variety of animal species. Despite their differences, they share a high homology in the pore region in which the ion discrimination takes place. Although ion selectivity has been studied for decades, the mechanisms underlying this selectivity for trimeric channels, and particularly for the ENaC/DEG family, are still poorly understood. This systematic review follows PRISMA guidelines and aims to determine the main components that govern ion selectivity in the ENaC/DEG family. In total, 27 papers from three online databases were included according to specific exclusion and inclusion criteria. It was found that the G/SxS selectivity filter (glycine/serine, non-conserved residue, serine) and other well conserved residues play a crucial role in ion selectivity. Depending on the ion type, residues with different properties are involved in ion permeability. For lithium against sodium, aromatic residues upstream of the selectivity filter seem to be important, whereas for sodium against potassium, negatively charged residues downstream of the selectivity filter seem to be important. This review provides new perspectives for further studies to unravel the mechanisms of ion selectivity.

Exploring the quinone/inhibitor-binding pocket in mitochondrial respiratory complex I by chemical biology approaches.

NADH-quinone oxidoreductase (respiratory complex I) couples NADH-to-quinone electron transfer to the translocation of protons across the membrane. Even though the architecture of the quinone-access channel in the enzyme has been modeled by X-ray crystallography and cryo-EM, conflicting findings raise the question whether the models fully reflect physiologically relevant states present throughout the catalytic cycle. To gain further insights into the structural features of the binding pocket for quinone/inhibitor, we performed chemical biology experiments using bovine heart sub-mitochondrial particles. We synthesized ubiquinones that are oversized (SF-UQs) or lipid-like (PC-UQs) and are highly unlikely to enter and transit the predicted narrow channel. We found that SF-UQs and PC-UQs can be catalytically reduced by complex I, albeit only at moderate or low rates. Moreover, quinone-site inhibitors completely blocked the catalytic reduction and the membrane potential formation coupled to this reduction. Photoaffinity-labeling experiments revealed that amiloride-type inhibitors bind to the interfacial domain of multiple core subunits (49 kDa, ND1, and PSST) and the 39-kDa supernumerary subunit, although the latter does not make up the channel cavity in the current models. The binding of amilorides to the multiple target subunits was remarkably suppressed by other quinone-site inhibitors and SF-UQs. Taken together, the present results are difficult to reconcile with the current channel models. On the basis of comprehensive interpretations of the present results and of previous findings, we discuss the physiological relevance of these models.

Is the Amiloride-Sensitive Na+ Channel in Taste Cells Really ENaC?

Among the 5 taste qualities, salt is the least understood. The receptors, their expression pattern in taste cells, and the transduction mechanisms for salt taste are still unclear. Previous studies have suggested that low concentrations of NaCl are detected by the amiloride-sensitive epithelial Na+ channel (ENaC), which in other systems requires assembly of 3 homologous subunits (α, ß, and γ) to form a functional channel. However, a new study from Lossow and colleagues, published in this issue of Chemical Senses, challenges that hypothesis by examining expression levels of the 3 ENaC subunits in individual taste cells using gene-targeted mice in combination with immunohistochemistry and in situ hybridization. Results show a lack of colocalization of ENaC subunits in taste cells as well as expression of subunits in taste cells that show no amiloride sensitivity. These new results question the molecular identity of the amiloride-sensitive Na+ conductance in taste cells.

Segregated Expression of ENaC Subunits in Taste Cells.

Salt taste is one of the 5 basic taste qualities. Depending on the concentration, table salt is perceived either as appetitive or aversive, suggesting the contribution of several mechanisms to salt taste, distinguishable by their sensitivity to the epithelial sodium channel (ENaC) blocker amiloride. A taste-specific knockout of the α-subunit of the ENaC revealed the relevance of this polypeptide for low-salt transduction, whereas the response to other taste qualities remained normal. The fully functional ENaC is composed of α-, ß-, and γ-subunits. In taste tissue, however, the precise constitution of the channel and the cell population responsible for detecting table salt remain uncertain. In order to examine the cells and subunits building the ENaC, we generated mice carrying modified alleles allowing the synthesis of green and red fluorescent proteins in cells expressing the α- and ß-subunit, respectively. Fluorescence signals were detected in all types of taste papillae and in taste buds of the soft palate and naso-incisor duct. However, the lingual expression patterns of the reporters differed depending on tongue topography. Additionally, immunohistochemistry for the γ-subunit of the ENaC revealed a lack of overlap between all potential subunits. The data suggest that amiloride-sensitive recognition of table salt is unlikely to depend on the classical ENaCs formed by α-, ß-, and γ-subunits and ask for a careful investigation of the channel composition.

ENaC-Dependent Sodium Chloride Taste Responses in the Regenerated Rat Chorda Tympani Nerve After Lingual Gustatory Deafferentation Depend on the Taste Bud Field Reinnervated.

The chorda tympani (CT) nerve is exceptionally responsive to NaCl. Amiloride, an epithelial Na+ channel (ENaC) blocker, consistently and significantly decreases the NaCl responsiveness of the CT but not the glossopharyngeal (GL) nerve in the rat. Here, we examined whether amiloride would suppress the NaCl responsiveness of the CT when it cross-reinnervated the posterior tongue (PT). Whole-nerve electrophysiological recording was performed to investigate the response properties of the intact (CTsham), regenerated (CTr), and cross-regenerated (CT-PT) CT in male rats to NaCl mixed with and without amiloride and common taste stimuli. The intact (GLsham) and regenerated (GLr) GL were also examined. The CT responses of the CT-PT group did not differ from those of the GLr and GLsham groups, but did differ from those of the CTr and CTsham groups for some stimuli. Importantly, the responsiveness of the cross-regenerated CT to a series of NaCl concentrations was not suppressed by amiloride treatment, which significantly decreased the response to NaCl in the CTr and CTsham groups and had no effect in the GLr and GLsham groups. This suggests that the cross-regenerated CT adopts the taste response properties of the GL as opposed to those of the regenerated CT or intact CT. This work replicates the 5 decade-old findings of Oakley and importantly extends them by providing compelling evidence that the presence of functional ENaCs, essential for sodium taste recognition in regenerated taste receptor cells, depends on the reinnervated lingual region and not on the reinnervating gustatory nerve, at least in the rat.

Analysis of the Heterogeneous Distribution of Amiloride and Propranolol in Dried Blood Spot by UHPLC-FLD and MALDI-IMS.

Dried blood spot (DBS) has lately experienced an increase in its use in bioanalysis due to its several advantages compared with traditional blood sampling methods. Nevertheless, the use of DBS with quantitative purposes is hindered by the heterogeneous distribution of some compounds in the supporting matrix and the dependence of the response on different factors, such as the hematocrit, blood volume, and sampling position. In this study the effect of those factors in the analytical response was investigated by ultra high performance liquid chromatography coupled to fluorescence detection, using amiloride and propranolol as model compounds. The results showed a heterogeneous and drug-dependent distribution of the compounds in the blood spot. While amiloride concentration was higher in the center, propranolol concentration was higher in the periphery of the spot. Besides, the influence of the hematocrit on the quantitative results was observed. MALDI mass spectrometry imaging (MALDI-IMS) has allowed study of the distribution of the two cardiovascular drugs when they were placed in the DBS card using water:methanol solutions, demonstrating that they followed a similar distribution pattern as in blood. This work has showed the potentiality of the MALDI-IMS technique to predict the distribution of the drugs in the DBS card.

SGK1-dependent ENaC processing and trafficking in mice with high dietary K intake and elevated aldosterone.

We examined renal Na and K transporters in mice with deletions in the gene encoding the aldosterone-induced protein SGK1. The knockout mice were hyperkalemic, and had altered expression of the subunits of the epithelial Na channel (ENaC). The kidneys showed decreased expression of the cleaved forms of the γENaC subunit, and the fully glycosylated form of the ßENaC subunits when animals were fed a high-K diet. Knockout animals treated with exogenous aldosterone also had reduced subunit processing and diminished surface expression of ßENaC and γENaC. Expression of the three upstream Na transporters NHE3, NKCC2, and NCC was reduced in both wild-type and knockout mice in response to K loading. The activity of ENaC measured as whole cell amiloride-sensitive current (INa) in principal cells of the cortical collecting duct (CCD) was minimal under control conditions but was increased by a high-K diet to a similar extent in knockout and wild-type animals. INa in the connecting tubule also increased similarly in the two genotypes in response to exogenous aldosterone administration. The activities of both ROMK channels in principal cells and BK channels in intercalated cells of the CCD were unaffected by the deletion of SGK1. Acute treatment of animals with amiloride produced similar increases in Na excretion and decreases in K excretion in the two genotypes. The absence of changes in ENaC activity suggests compensation for decreased surface expression. Altered K balance in animals lacking SGK1 may reflect defects in ENaC-independent K excretion.

Amilorides bind to the quinone binding pocket of bovine mitochondrial complex I.

Amilorides, well-known inhibitors of Na(+)/H(+) antiporters, were previously shown to inhibit bacterial and mitochondrial NADH-quinone oxidoreductase (complex I) but were markedly less active for complex I. Because membrane subunits ND2, ND4, and ND5 of bovine complex I are homologous to Na(+)/H(+) antiporters, amilorides have been thought to bind to any or all of the antiporter-like subunits; however, there is currently no direct experimental evidence that supports this notion. To identify the binding site of amilorides in bovine complex I, we synthesized two photoreactive amilorides (PRA1 and PRA2), which have a photoreactive azido (-N3) group and terminal alkyne (-C≡CH) group at the opposite ends of the molecules, respectively, and conducted photoaffinity labeling with bovine heart submitochondrial particles. The terminal alkyne group allows various molecular tags to covalently attach to it via Cu(+)-catalyzed click chemistry, thereby allowing purification and/or detection of the labeled peptides. Proteomic analyses revealed that PRA1 and PRA2 label none of the antiporter-like subunits; they specifically label the accessory subunit B14.5a and core subunit 49 kDa (N-terminal region of Thr25-Glu115), respectively. Suppressive effects of ordinary inhibitors (bullatacin, fenpyroximate, and quinazoline), which bind to the putative quinone binding pocket, on labeling were fairly different between the B14.5a and 49 kDa subunits probably because the binding positions of the three inhibitors differ within the pocket. The results of this study clearly demonstrate that amilorides inhibit complex I activity by occupying the quinone binding pocket rather than directly blocking translocation of protons through the antiporter-like subunits (ND2, ND4, and ND5). The accessory subunit B14.5a may be located adjacent to the N-terminal region of the 49 kDa subunits. The structural features of the quinone binding pocket in bovine complex I were discussed on the basis of these results.

Identification of the Binding Position of Amilorides in the Quinone Binding Pocket of Mitochondrial Complex I.

We previously demonstrated that amilorides bind to the quinone binding pocket of bovine mitochondrial complex I, not to the hitherto suspected Na⁺/H⁺ antiporter-like subunits (ND2, ND4, and ND5) [Murai, M., et al. (2015) Biochemistry 54, 2739-2746]. To characterize the binding position of amilorides within the pocket in more detail, we conducted specific chemical labeling [alkynylation (-C≡CH)] of complex I via ligand-directed tosyl (LDT) chemistry using a newly synthesized amide-type amiloride AAT as a LDT chemistry reagent. The inhibitory potency of AAT, in terms of its IC50 value, was markedly higher (∼1000-fold) than that of prototypical guanidine-type amilorides such as commercially available EIPA and benzamil. Detailed proteomic analyses in combination with click chemistry revealed that the chemical labeling occurred at Asp160 of the 49 kDa subunit (49 kDa Asp160). This labeling was significantly suppressed in the presence of an excess amount of other amilorides or ordinary inhibitors such as quinazoline and acetogenin. Taking into consideration the fact that 49 kDa Asp160 was also specifically labeled by LDT chemistry reagents derived from acetogenin [Masuya, T., et al. (2014) Biochemistry 53, 2307-2317, 7816-7823], we found this aspartic acid to elicit very strong nucleophilicity in the local protein environment. The structural features of the quinone binding pocket in bovine complex I are discussed on the basis of this finding.

Hydrochlorothiazide attenuates lithium-induced nephrogenic diabetes insipidus independently of the sodium-chloride cotransporter.

Lithium is the most common cause of nephrogenic diabetes insipidus (Li-NDI). Hydrochlorothiazide (HCTZ) combined with amiloride is the mainstay treatment in Li-NDI. The paradoxical antidiuretic action of HCTZ in Li-NDI is generally attributed to increased sodium and water uptake in proximal tubules as a compensation for increased volume loss due to HCTZ inhibition of the Na-Cl cotransporter (NCC), but alternative actions for HCTZ have been suggested. Here, we investigated whether HCTZ exerted an NCC-independent effect in Li-NDI. In polarized mouse cortical collecting duct (mpkCCD) cells, HCTZ treatment attenuated the Li-induced downregulation of aquaporin-2 (AQP2) water channel abundance. In these cells, amiloride reduces cellular Li influx through the epithelial sodium channel (ENaC). HCTZ also reduced Li influx, but to a lower extent. HCTZ increased AQP2 abundance on top of that of amiloride and did not affect the ENaC-mediated transcellular voltage. MpkCCD cells did not express NCC mRNA or protein. These data indicated that in mpkCCD cells, HCTZ attenuated lithium-induced downregulation of AQP2 independently of NCC and ENaC. Treatment of Li-NDI NCC knockout mice with HCTZ revealed a significantly reduced urine volume, unchanged urine osmolality, and increased cortical AQP2 abundance compared with Li-treated NCC knockout mice. HCTZ treatment further resulted in reduced blood Li levels, creatinine clearance, and alkalinized urinary pH. Our in vitro and in vivo data indicate that part of the antidiuretic effect of HCTZ in Li-NDI is NCC independent and may involve a tubuloglomerular feedback response-mediated reduction in glomerular filtration rate due to proximal tubular carbonic anhydrase inhibition.

Salty taste deficits in CALHM1 knockout mice.

Genetic ablation of calcium homeostasis modulator 1 (CALHM1), which releases adenosine triphosphate from Type 2 taste cells, severely compromises the behavioral and electrophysiological responses to tastes detected by G protein-coupled receptors, such as sweet and bitter. However, the contribution of CALHM1 to salty taste perception is less clear. Here, we evaluated several salty taste-related phenotypes of CALHM1 knockout (KO) mice and their wild-type (WT) controls: 1) In a conditioned aversion test, CALHM1 WT and KO mice had similar NaCl avoidance thresholds. 2) In two-bottle choice tests, CALHM1 WT mice showed the classic inverted U-shaped NaCl concentration-preference function but CALHM1 KO mice had a blunted peak response. 3) In brief-access tests, CALHM1 KO mice showed less avoidance than did WT mice of high concentrations of NaCl, KCl, NH(4)Cl, and sodium lactate (NaLac). Amiloride further ameliorated the NaCl avoidance of CALHM1 KO mice, so that lick rates to a mixture of 1000 mM NaCl + 10 µM amiloride were statistically indistinguishable from those to water. 4) Relative to WT mice, CALHM1 KO mice had reduced chorda tympani nerve activity elicited by oral application of NaCl, NaLac, and sucrose but normal responses to HCl and NH(4)Cl. Chorda tympani responses to NaCl and NaLac were amiloride sensitive in WT but not KO mice. These results reinforce others demonstrating that multiple transduction pathways make complex, concentration-dependent contributions to salty taste perception. One of these pathways depends on CALHM1 to detect hypertonic NaCl in the mouth and signal the aversive taste of concentrated salt.

Human cystic fibrosis airway epithelia have reduced Cl- conductance but not increased Na+ conductance.

Loss of cystic fibrosis transmembrane conductance regulator (CFTR) anion channel function causes cystic fibrosis (CF) lung disease. CFTR is expressed in airway epithelia, but how CF alters electrolyte transport across airway epithelia has remained uncertain. Recent studies of a porcine model showed that in vivo, excised, and cultured CFTR(-/-) and CFTR(ΔF508/ΔF508) airway epithelia lacked anion conductance, and they did not hyperabsorb Na(+). Therefore, we asked whether Cl(-) and Na(+) conductances were altered in human CF airway epithelia. We studied differentiated primary cultures of tracheal/bronchial epithelia and found that transepithelial conductance (Gt) under basal conditions and the cAMP-stimulated increase in Gt were markedly attenuated in CF epithelia compared with non-CF epithelia. These data reflect loss of the CFTR anion conductance. In CF and non-CF epithelia, the Na(+) channel inhibitor amiloride produced similar reductions in Gt and Na(+) absorption, indicating that Na(+) conductance in CF epithelia did not exceed that in non-CF epithelia. Consistent with previous reports, adding amiloride caused greater reductions in transepithelial voltage and short-circuit current in CF epithelia than in non-CF epithelia; these changes are attributed to loss of a Cl(-) conductance. These results indicate that Na(+) conductance was not increased in these cultured CF tracheal/bronchial epithelia and point to loss of anion transport as key to airway epithelial dysfunction in CF.

High-fat Western diet alters crystalline silica-induced airway epithelium ion transport but not airway smooth muscle reactivity.

OBJECTIVES: Silicosis is an irreversible occupational lung disease resulting from crystalline silica inhalation. Previously, we discovered that Western diet (HFWD)-consumption increases susceptibility to silica-induced pulmonary inflammation and fibrosis. This study investigated the potential of HFWD to alter silica-induced effects on airway epithelial ion transport and smooth muscle reactivity. METHODS: Six-week-old male F344 rats were fed a HFWD or standard rat chow (STD) and exposed to silica (Min-U-Sil 5®, 15 mg/m3, 6 h/day, 5 days/week, for 39 d) or filtered air. Experimental endpoints were measured at 0, 4, and 8 weeks post-exposure. Transepithelial potential difference (Vt), short-circuit current (ISC) and transepithelial resistance (Rt) were measured in tracheal segments and ion transport inhibitors [amiloride, Na+ channel blocker; NPPB; Cl- channel blocker; ouabain, Na+, K+-pump blocker] identified changes in ion transport pathways. Changes in airway smooth muscle reactivity to methacholine (MCh) were investigated in the isolated perfused trachea preparation. RESULTS: Silica reduced basal ISC at 4 weeks and HFWD reduced the ISC response to amiloride at 0 week compared to air control. HFWD + silica exposure induced changes in ion transport 0 and 4 weeks after treatment compared to silica or HFWD treatments alone. No effects on airway smooth muscle reactivity to MCh were observed.