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The Journal of Biological Chemistry Jul 2005The epithelial Na+ channel (ENaC) belongs to the structurally conserved ENaC/Degenerin superfamily. These channels are blocked by amiloride and its analogues. Several...
The epithelial Na+ channel (ENaC) belongs to the structurally conserved ENaC/Degenerin superfamily. These channels are blocked by amiloride and its analogues. Several amino acid residues have been implicated in amiloride binding. Primary among these are alphaSer-583, betaGly-525, and gammaGly-542, which are present at a homologous site within the three subunits of ENaC. Mutations of the beta and gamma glycines greatly weakened amiloride block, but, surprisingly, mutation of the serine of the alpha subunit resulted in moderate (<5-fold) weakening of amiloride K(i). We investigated the role of alphaSer-583 in amiloride binding by systematically mutating alphaSer-583 and analyzing the mutant channels with two-electrode voltage clamp. We observed that most mutations had moderate effects on amiloride block, whereas those introducing rings showed dramatic effects on amiloride block. In addition, mutations introducing a beta-methyl group at this site altered the electric field of ENaC, affecting both amiloride binding and the voltage dependence of channel gating. We also found that the His mutation, in addition to greatly weakening amiloride binding, appends a voltage-sensitive gate within the pore of ENaC at low pH. Because diverse residues at alpha583, such as Asn, Gln, Ser, Gly, Thr, and Ala, have similar amiloride binding affinities, our results suggest that the wild type Ser side chain is not important for amiloride binding. However, given that some alphaSer-583 mutations affect the electrical properties of the channel whereas those introducing rings greatly weaken amiloride block, we conclude that amiloride binds at or near this site and that alphaSer-583 may have a role in ion permeation through ENaC.
Topics: Amiloride; Animals; Binding Sites; Diuretics; Dose-Response Relationship, Drug; Epithelial Sodium Channels; Glycine; Histidine; Hydrogen-Ion Concentration; Ions; Kinetics; Models, Chemical; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Oocytes; Patch-Clamp Techniques; Protein Binding; Protein Conformation; Protein Structure, Quaternary; Protein Structure, Tertiary; RNA, Complementary; Serine; Sodium; Sodium Channels; Xenopus laevis
PubMed: 15908426
DOI: 10.1074/jbc.M503500200 -
Journal of Korean Medical Science Aug 2013Amiloride and benzamil showed antinocicepitve effects in several pain models through the inhibition of acid sensing ion channels (ASICs). However, their role in...
Amiloride and benzamil showed antinocicepitve effects in several pain models through the inhibition of acid sensing ion channels (ASICs). However, their role in neuropathic pain has not been investigated. In this study, we investigated the effect of the intrathecal amiloride and benzamil in neuropathic pain model, and also examined the role of ASICs on modulation of neuropathic pain. Neuropathic pain was induced by L4-5 spinal nerve ligation in male Sprague-Dawley rats weighing 100-120 g, and intrathecal catheterization was performed for drug administration. The effects of amiloride and benzamil were measured by the paw-withdrawal threshold to a mechanical stimulus using the up and down method. The expression of ASICs in the spinal cord dorsal horn was also analyzed by RT-PCR. Intrathecal amiloride and benzamil significantly increased the paw withdrawal threshold in spinal nerve-ligated rats (87%±12% and 76%±14%, P=0.007 and 0.012 vs vehicle, respectively). Spinal nerve ligation increased the expression of ASIC3 in the spinal cord dorsal horn (P=0.01), and this increase was inhibited by both amiloride and benzamil (P<0.001 in both). In conclusion, intrathecal amiloride and benzamil display antinociceptive effects in the rat spinal nerve ligation model suggesting they may present an alternative pharmacological tool in the management of neuropathic pain at the spinal level.
Topics: Acid Sensing Ion Channels; Amiloride; Analgesics; Animals; Disease Models, Animal; Male; Neuralgia; RNA, Messenger; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Spinal Cord; Transcription, Genetic
PubMed: 23960454
DOI: 10.3346/jkms.2013.28.8.1238 -
British Medical Journal (Clinical... Jun 1983The biochemical disturbance produced by thiazide diuretics and by amiloride during treatment of moderate hypertension were compared. Two parallel studies were initiated.... (Clinical Trial)
Clinical Trial Comparative Study Randomized Controlled Trial
The biochemical disturbance produced by thiazide diuretics and by amiloride during treatment of moderate hypertension were compared. Two parallel studies were initiated. In one 40 patients with newly diagnosed hypertension were treated with metoprolol and a diuretic, either hydrochlorothiazide or amiloride. In a second study 38 patients receiving longstanding treatment with hypotensives and thiazides either continued the treatment or replaced the thiazide with amiloride. Initial biochemical assessments were compared with those after two years in the study. In previously untreated patients, thiazide produced a significant fall in plasma potassium and hyperuricaemia that did not occur with amiloride (p less than 0.001). Those patients receiving long term treatment for their hypertension who continued to take thiazides had persistent hypokalaemia and hyperuricaemia. Substitution with amiloride corrected the hypokalaemia and serum uric acid returned toward normal ranges, but this change was not statistically significant. Patients receiving long term treatment also had impaired glucose tolerance, this remained unchanged in those receiving thiazide but was corrected in those receiving amiloride. Compared with amiloride thiazides produced undesirable but reversible biochemical changes. As control of hypertension was equally effective with both preparations, we suggest that a combination of amiloride with a beta blocker in treatment of moderate hypertension in preferred.
Topics: Adult; Aged; Amiloride; Drug Combinations; Drug Therapy, Combination; Humans; Hydrochlorothiazide; Hypertension; Metoprolol; Middle Aged; Potassium; Pyrazines
PubMed: 6409208
DOI: 10.1136/bmj.286.6383.2015 -
Physiological Reports Jan 2021In the aldosterone-sensitive distal nephron (ASDN), epithelial sodium channel (ENaC)-mediated Na absorption drives K excretion. K excretion depends on the delivery of Na...
BACKGROUND
In the aldosterone-sensitive distal nephron (ASDN), epithelial sodium channel (ENaC)-mediated Na absorption drives K excretion. K excretion depends on the delivery of Na to the ASDN and molecularly activated ENaC. Furosemide is known as a K wasting diuretic as it greatly enhances Na delivery to the ASDN. Here, we studied the magnitude of acute furosemide-induced kaliuresis under various states of basal molecular ENaC activity.
METHODS
C57/Bl6J mice were subjected to different dietary regimens that regulate molecular ENaC expression and activity levels. The animals were anesthetized and bladder-catheterized. Diuresis was continuously measured before and after administration of furosemide (2 µg/g BW) or benzamil (0.2 µg/g BW). Flame photometry was used to measure urinary [Na ] and [K ]. The kidneys were harvested and, subsequently, ENaC expression and cleavage activation were determined by semiquantitative western blotting.
RESULTS
A low K and a high Na diet markedly suppressed ENaC protein expression, cleavage activation, and furosemide-induced kaliuresis. In contrast, furosemide-induced kaliuresis was greatly enhanced in animals fed a high K or low Na diet, conditions with increased ENaC expression. The furosemide-induced diuresis was similar in all dietary groups.
CONCLUSION
Acute furosemide-induced kaliuresis differs greatly and depends on the a priori molecular expression level of ENaC. Remarkably, it can be even absent in animals fed a high Na diet, despite a marked increase of tubular flow and urinary Na excretion. This study provides auxiliary evidence that acute ENaC-dependent K excretion requires both Na as substrate and molecular activation of ENaC.
Topics: Amiloride; Animals; Epithelial Sodium Channels; Furosemide; Ion Transport; Kidney; Mice; Mice, Inbred C57BL; Natriuresis; Potassium; Sodium, Dietary
PubMed: 33410279
DOI: 10.14814/phy2.14668 -
Japanese Journal of Pharmacology Jul 1995The effects of amiloride and its analogues (3',4'-dichlorobenzamil (DCB), 2',4'-dimethylbenzamil (DMB), 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and...
The effects of amiloride and its analogues (3',4'-dichlorobenzamil (DCB), 2',4'-dimethylbenzamil (DMB), 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and 5-(N-methyl-N-isobutyl)amiloride (MIBA)) on cardiac ion transporters (Na+/Ca2+ exchanger, Na+/H+ exchanger, Na+ pump and Ca2+ pump) and their cytotoxicities were tested in cardiac myocytes. All the tested compounds showed concentration-dependent inhibitory effects on the ion transporters studied in canine cardiac sarcolemmal vesicles. The concentrations (microM) of amiloride, DCB, DMB, EIPA and MIBA required to produce 50% inhibition were > 1000, 19, 10, 83 and 84, respectively, for the Na+/Ca2+ exchanger; 130, 73, 63, 16 and 14 for the Na+/H+ exchanger; > 1000, 72, > 300, > 300 and > 300 for the Na+ pump; and > 1000, 37, 93, 90 and 70 for the Ca2+ pump, respectively. Furthermore, these agents induced cell death in isolated rat cardiac myocytes and the 50% lethal concentrations (microM) were > 1000, 9.2, 30, 16 and 17, respectively. These findings demonstrate that amiloride and its analogues have non-selective inhibitory effects on cardiac ion transporters and cytotoxicity in cardiomyocytes. When these drugs are employed as experimental tools to investigate the involvement of ion transporters in cell functions, the results must be interpreted with caution.
Topics: Amiloride; Animals; Calcium-Transporting ATPases; Dogs; Dose-Response Relationship, Drug; Heart; Ion Transport; Membranes; Rats; Sodium-Hydrogen Exchangers
PubMed: 7474551
DOI: 10.1254/jjp.68.279 -
British Journal of Pharmacology Jun 19921. Patch clamp recording techniques have been used to compare the block caused by amiloride and some of its structural analogues of the mechanosensitive (MS) cation...
1. Patch clamp recording techniques have been used to compare the block caused by amiloride and some of its structural analogues of the mechanosensitive (MS) cation selective channel in frog (Xenopus laevis) oocytes. 2. Like amiloride, the amiloride analogues dimethylamiloride (DMA), benzamil and bromohexamethyleneamiloride (BrHMA) block the MS channel in a highly voltage-dependent manner. 3. All analogues tested were more potent blockers than amiloride with IC50's of 500 microM (amiloride), 370 microM (DMA), 95 microM (benzamil) and 34 microM (BrHMA). 4. Hill plots gave Hill coefficients of 2 (amiloride), 1.8 (DMA), 1 (benzamil) and 1.2 (BrHMA) indicating that the binding of two ligand molecules may be necessary for the block caused by amiloride, DMA and possibly BrHMA whereas only a single ligand molecule may be required for the block by benzamil. 5. The potential use of BrHMA as a light-activated, covalent label of the MS channel protein is discussed. 6. The amiloride analogue 'fingerprinting' of the blocking site on the MS channel indicates it is structurally different from previously described amiloride-sensitive ion transport pathways but may be related to the amiloride binding site on outer hair cells of the ear.
Topics: Amiloride; Animals; Female; In Vitro Techniques; Ion Channels; Kinetics; Oocytes; Structure-Activity Relationship; Xenopus
PubMed: 1382778
DOI: 10.1111/j.1476-5381.1992.tb14329.x -
American Journal of Physiology. Renal... Apr 2021Epithelial Na channel (ENaC) blockers elicit acute and substantial increases of urinary pH. The underlying mechanism remains to be understood. Here, we evaluated if...
Epithelial Na channel (ENaC) blockers elicit acute and substantial increases of urinary pH. The underlying mechanism remains to be understood. Here, we evaluated if benzamil-induced urine alkalization is mediated by an acute reduction in H secretion via renal H-K-ATPases (HKAs). Experiments were performed in vivo on HKA double-knockout and wild-type mice. Alterations in dietary K intake were used to change renal HKA and ENaC activity. The acute effects of benzamil (0.2 µg/g body wt, sufficient to block ENaC) on urine flow rate and urinary electrolyte and acid excretion were monitored in anesthetized, bladder-catheterized animals. We observed that benzamil acutely increased urinary pH (ΔpH: 0.33 ± 0.07) and reduced NH and titratable acid excretion and that these effects were distinctly enhanced in animals fed a low-K diet (ΔpH: 0.74 ± 0.12), a condition when ENaC activity is low. In contrast, benzamil did not affect urine acid excretion in animals kept on a high-K diet (i.e., during high ENaC activity). Thus, urine alkalization appeared completely uncoupled from ENaC function. The absence of benzamil-induced urinary alkalization in HKA double-knockout mice confirmed the direct involvement of these enzymes. The inhibitory effect of benzamil was also shown in vitro for the pig α-isoform of HKA. These results suggest a revised explanation of the benzamil effect on renal acid-base excretion. Considering the conditions used here, we suggest that it is caused by a direct inhibition of HKAs in the collecting duct and not by inhibition of the ENaC function. Bolus application of epithelial Na channel (EnaC) blockers causes marked and acute increases of urine pH. Here, we provide evidence that the underlying mechanism involves direct inhibition of the H-K pump in the collecting duct. This could provide a fundamental revision of the previously assumed mechanism that suggested a key role of ENaC inhibition in this response.
Topics: Amiloride; Animals; Epithelial Sodium Channels; H(+)-K(+)-Exchanging ATPase; Kidney Tubules, Collecting; Mice; Natriuresis; Renal Elimination; Sodium; Sodium, Dietary
PubMed: 33554781
DOI: 10.1152/ajprenal.00444.2020 -
American Journal of Physiology.... May 2007Current evidence suggests salt taste transduction involves at least two mechanisms, one that is amiloride sensitive and appears to use apically located epithelial sodium...
Current evidence suggests salt taste transduction involves at least two mechanisms, one that is amiloride sensitive and appears to use apically located epithelial sodium channels relatively selective for Na(+) and a second that is amiloride insensitive and uses a variant of the transient receptor potential vanilloid receptor 1 (TRPV1) that serves as a nonspecific cation channel. To provide a functional context for these findings, we trained Trpv1 knockout (KO) and wild-type (WT) C57BL/6J mice (n = 9 or 10/group) in a two-response operant discrimination procedure and measured detection thresholds to NaCl and KCl with and without amiloride. The KO and WT mice had similar detection thresholds for NaCl and KCl. Amiloride shifted the NaCl sensitivity curve to the same degree in both groups and had virtually no effect on KCl thresholds. In addition, a more detailed analysis of chorda tympani nerve (CT) responses to NaCl, with and without benzamil (Bz, an amiloride analog) treatment revealed that the tonic portion of the CT response of KO mice to NaCl + Bz was absent, but both KO and WT mice displayed some degree of a phasic response to NaCl with and without Bz. Because these transients constitute the entire CT response to NaCl + Bz in Trpv1 KO mice, it is possible that these signals are sufficient to maintain normal NaCl detectabilty in the behavioral task used here. Additionally, there may be other amiloride-insensitive salt transduction mechanisms in taste receptor fields other than the anterior tongue that maintain normal salt detection performance in the KO mice.
Topics: Amiloride; Animals; Chorda Tympani Nerve; Electrophysiology; Genotype; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Potassium Chloride; Sodium Channel Blockers; Sodium Chloride; TRPV Cation Channels; Taste; Water Deprivation
PubMed: 17234959
DOI: 10.1152/ajpregu.00587.2006 -
British Journal of Clinical Pharmacology Apr 19931. The relative potencies of amiloride (5 and 20 mg) and spironolactone (25 and 100 mg) for plasma and erythrocyte electrolytes were investigated in a double-blind,... (Clinical Trial)
Clinical Trial Comparative Study Randomized Controlled Trial
1. The relative potencies of amiloride (5 and 20 mg) and spironolactone (25 and 100 mg) for plasma and erythrocyte electrolytes were investigated in a double-blind, randomised, balanced, crossover study in 12 normal men treated concomitantly with hydrochlorothiazide 100 mg daily for 1 week. 2. Participants satisfied an a priori requirement for a fall in plasma potassium concentration of at least 0.5 mmol l-1 after 7 days of treatment with hydrochlorothiazide alone. 3. After hydrochlorothiazide alone, plasma potassium and sodium concentrations fell (P < 0.001). There were associated reductions in erythrocyte sodium (P < 0.01). Plasma magnesium concentration did not change, although erythrocyte magnesium decreased (P < 0.001). 4. Both amiloride and spironolactone attenuated the thiazide-induced fall in plasma potassium (relative potency, amiloride:spironolactone 10:1, 95% confidence interval 6.3-16.2:1). Amiloride but not spironolactone was associated with a dose-related increase in plasma magnesium; a relative potency estimation was precluded. There was little evidence of influences of amiloride or spironolactone on erythrocyte electrolytes. 5. On a weight basis, amiloride is ten times more potent than spironolactone as a potassium-sparing agent in diuretic-treated subjects but neither agent had major effects on erythrocyte potassium. The drugs may have divergent actions on magnesium handling; hydrochlorothiazide alone had no influence on plasma magnesium.
Topics: Adult; Aldosterone; Amiloride; Double-Blind Method; Drug Interactions; Erythrocytes; Humans; Hydrochlorothiazide; Intracellular Fluid; Magnesium; Male; Potassium; Spironolactone
PubMed: 8485017
DOI: 10.1111/j.1365-2125.1993.tb04153.x -
International Journal of Molecular... Feb 2021Freshwater fishes maintain an internal osmolality of ~300 mOsm, while living in dilute environments ranging from 0 to 50 mOsm. This osmotic challenge is met at least...
Freshwater fishes maintain an internal osmolality of ~300 mOsm, while living in dilute environments ranging from 0 to 50 mOsm. This osmotic challenge is met at least partially, by Na/H exchangers (NHE) of fish gill and kidney. In this study, we cloned, expressed, and pharmacologically characterized fish-specific Nhes of the commercially important species . Trout (t) Nhe3a and Nhe3b isoforms from gill and kidney were expressed and characterized in an NHE-deficient cell line. Western blotting and immunocytochemistry confirmed stable expression of the tagged trout tNhe proteins. To measure NHE activity, a transient acid load was induced in trout tNhe expressing cells and intracellular pH was measured. Both isoforms demonstrated significant activity and recovered from an acute acid load. The effect of the NHE transport inhibitors amiloride, EIPA (5-(N-ethyl-N-isopropyl)-amiloride), phenamil, and DAPI was examined. tNhe3a was inhibited in a dose-dependent manner by amiloride and EIPA and tNhe3a was more sensitive to amiloride than EIPA, unlike mammalian NHE1. tNhe3b was inhibited by high concentrations of amiloride, while even in the presence of high concentrations of EIPA (500 µM), some activity of tNhe3b remained. Phenamil and DAPI were ineffective at inhibiting tNhe activity of either isoform. The current study aids in understanding the pharmacology of fish ion transporters. Both isoforms display inhibitory profiles uniquely different from mammalian NHEs and show resistance to inhibition. Our study allows for more direct interpretation of past, present, and future fish-specific sodium transport studies, with less reliance on mammalian NHE data for interpretation.
Topics: Amiloride; Animals; CHO Cells; Cloning, Molecular; Cricetulus; Fish Proteins; Gene Expression; Gills; Indoles; Mammals; Oncorhynchus mykiss; Organ Specificity; Sodium Channel Blockers; Sodium-Hydrogen Exchanger 3; Transfection
PubMed: 33672216
DOI: 10.3390/ijms22042205