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The Journal of General Physiology Jul 1976The inhibition of short-circuit current (Isc) in isolated frog skin and the induction of surface potentials in lipid bilayer membranes produced by the diuretic drug...
The inhibition of short-circuit current (Isc) in isolated frog skin and the induction of surface potentials in lipid bilayer membranes produced by the diuretic drug amiloride and a number of its chemical analogues was studied. The major conclusions of our study are: (a) The charged form of amiloride is the biologically active species. (b) Both the magnitude of Isc and the amiloride inhibitory effect are sensitive to the ionic milieu bathing the isolated skin, and these two features are modulated at separate and distinct regions on the transport site. (c) Amiloride is very specific in its inhibitory interaction with the Na+ transport site since slight structural modifications can result in significant changes in drug effectiveness. We found that substitutions at pyrazine ring position 5 greatly diminish drug activity, while changes at position 6 are less drastic. Alterations in the guanidinium moiety only diminish activity if the result is a change in the spatial orientation of the amino group carrying the positive charge. (d) Amiloride can bind to and alter the charge on membrane surfaces, but this action cannot explain its highly specific effects in biological systems.
Topics: Amiloride; Animals; Biological Transport; Dose-Response Relationship, Drug; Hydrogen-Ion Concentration; Membrane Potentials; Membranes; Membranes, Artificial; Pyrazines; Skin; Sodium
PubMed: 7635
DOI: 10.1085/jgp.68.1.43 -
Neuroscience Research Apr 2013The molecular mechanisms of sodium taste transduction are not completely understood, especially those responsible for the portion of NaCl's taste in rodents that is not...
The molecular mechanisms of sodium taste transduction are not completely understood, especially those responsible for the portion of NaCl's taste in rodents that is not blocked by amiloride. As a prelude to conducting genetic analyses of peripheral NaCl taste responsiveness, we performed multiunit electrophysiological recordings from the chorda tympani (CT) nerve in C57BL/6J (B6) and A/J mice. Mice were anesthetized, the CT was accessed, and taste solutions were flowed over the tongue in order to measure the integrated whole-nerve response. NaCl was delivered before and during application of 100μM amiloride. Pre-amiloride responses were significantly larger in A/J than B6 mice for 1-8mM NaCl. Responses to NaCl were suppressed significantly by amiloride in both strains and to similar degrees. However, the size of the amiloride-insensitive NaCl response component was significantly larger in A/J mice than in B6 mice for NaCl at 2-16mM. These data help to explain the prior observation that the strains differ in behavioral taste thresholds for NaCl. Specifically, the results suggest that perception of sodium-specific taste by mice depends on the ratio of amiloride-sensitive and -insensitive responses in the CT, rather than on the absolute level of the whole-nerve response to NaCl or on the size of the amiloride-sensitive component alone. Because the B6 and A/J mice differed in the size of their amiloride-insensitive components, they may prove useful in future genetic work designed to characterize the underlying transduction mechanisms.
Topics: Action Potentials; Amiloride; Animals; Chorda Tympani Nerve; Dose-Response Relationship, Drug; Drug Administration Schedule; Drug Interactions; Epithelial Sodium Channel Blockers; Male; Mice; Mice, Inbred C57BL; Sodium Chloride
PubMed: 23458904
DOI: 10.1016/j.neures.2013.02.003 -
The Journal of Physiology May 2023Acid-sensing ion channels (ASICs) are members of the diverse family of degenerin/epithelial sodium channels (DEG/ENaCs). They perform a wide range of physiological roles...
Acid-sensing ion channels (ASICs) are members of the diverse family of degenerin/epithelial sodium channels (DEG/ENaCs). They perform a wide range of physiological roles in healthy organisms, including in gut function and synaptic transmission, but also play important roles in disease, as acidosis is a hallmark of painful inflammatory and ischaemic conditions. We performed a screen for acid sensitivity on all 30 subunits of the Caenorhabditis elegans DEG/ENaC family using two-electrode voltage clamp in Xenopus oocytes. We found two groups of acid-sensitive DEG/ENaCs characterised by being either inhibited or activated by increasing proton concentrations. Three of these acid-sensitive C. elegans DEG/ENaCs were activated by acidic pH, making them functionally similar to the vertebrate ASICs. We also identified three new members of the acid-inhibited DEG/ENaC group, giving a total of seven additional acid-sensitive channels. We observed sensitivity to the anti-hypertensive drug amiloride as well as modulation by the trace element zinc. Acid-sensitive DEG/ENaCs were found to be expressed in both neurons and non-neuronal tissue, highlighting the likely functional diversity of these channels. Our findings provide a framework to exploit the C. elegans channels as models to study the function of these acid-sensing channels in vivo, as well as to study them as potential targets for anti-helminthic drugs. KEY POINTS: Acidosis plays many roles in healthy physiology, including synaptic transmission and gut function, but is also a key feature of inflammatory pain, ischaemia and many other conditions. Cells monitor acidosis of their surroundings via pH-sensing channels, including the acid-sensing ion channels (ASICs). These are members of the degenerin/epithelial sodium channel (DEG/ENaC) family, along with, as the name suggests, vertebrate ENaCs and degenerins of the roundworm Caenorhabditis elegans. By screening all 30 C. elegans DEG/ENaCs for pH dependence, we describe, for the first time, three acid-activated members, as well as three additional acid-inhibited channels. We surveyed both groups for sensitivity to amiloride and zinc; like their mammalian counterparts, their currents can be blocked, enhanced or unaffected by these modulators. Likewise, they exhibit diverse ion selectivity. Our findings underline the diversity of acid-sensitive DEG/ENaCs across species and provide a comparative resource for better understanding the molecular basis of their function.
Topics: Animals; Caenorhabditis elegans; Epithelial Sodium Channels; Degenerin Sodium Channels; Acid Sensing Ion Channels; Amiloride; Mammals
PubMed: 36200489
DOI: 10.1113/JP283238 -
Journal of Cystic Fibrosis : Official... Jan 2013Burkholderia dolosa can result in chronic airway infection and rapid decline in lung function in patients with cystic fibrosis (CF). Amiloride has antibacterial... (Clinical Trial)
Clinical Trial
BACKGROUND
Burkholderia dolosa can result in chronic airway infection and rapid decline in lung function in patients with cystic fibrosis (CF). Amiloride has antibacterial properties that may be synergistic with aminoglycosides against other species belonging to the Burkholderia cepacia complex (Bcc). We attempted to eradicate B. dolosa using a combination of nebulized tobramycin and nebulized amiloride in infected CF patients.
METHODS
A 6-month, open-label trial of continuous inhaled amiloride, delivered via nebulization four times daily, and continuous inhaled tobramycin (TIS or TOBI®) nebulized twice daily, was offered to all CF patients at our institution who are chronically infected with B. dolosa.
RESULTS
Twenty two of 27 patients with B. dolosa were eligible and twelve elected to participate. Eradication of B. dolosa was not noted in any study subject. While patients tolerated treatment with no adverse effects, there was also no apparent impact on other secondary outcome measures.
CONCLUSIONS
Concurrent, continuous inhalation of amiloride and tobramycin for 6 months was not effective for the eradication of chronic B. dolosa airway infection in CF patients.
Topics: Administration, Inhalation; Amiloride; Anti-Bacterial Agents; Burkholderia Infections; Chronic Disease; Drug Synergism; Drug Therapy, Combination; Endpoint Determination; Humans; Tobramycin; Treatment Failure
PubMed: 23046902
DOI: 10.1016/j.jcf.2012.06.006 -
Journal of Virology Oct 2011Amiloride and its derivative 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were previously shown to inhibit coxsackievirus B3 (CVB3) RNA replication in cell culture, with two...
Amiloride and its derivative 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were previously shown to inhibit coxsackievirus B3 (CVB3) RNA replication in cell culture, with two amino acid substitutions in the viral RNA-dependent RNA polymerase 3D(pol) conferring partial resistance of CVB3 to these compounds (D. N. Harrison, E. V. Gazina, D. F. Purcell, D. A. Anderson, and S. Petrou, J. Virol. 82:1465-1473, 2008). Here we demonstrate that amiloride and EIPA inhibit the enzymatic activity of CVB3 3D(pol) in vitro, affecting both VPg uridylylation and RNA elongation. Examination of the mechanism of inhibition of 3D(pol) by amiloride showed that the compound acts as a competitive inhibitor, competing with incoming nucleoside triphosphates (NTPs) and Mg(2+). Docking analysis suggested a binding site for amiloride and EIPA in 3D(pol), located in close proximity to one of the Mg(2+) ions and overlapping the nucleotide binding site, thus explaining the observed competition. This is the first report of a molecular mechanism of action of nonnucleoside inhibitors against a picornaviral RNA-dependent RNA polymerase.
Topics: Amiloride; Antiviral Agents; Binding Sites; DNA-Directed RNA Polymerases; Enterovirus B, Human; Enzyme Inhibitors; Magnesium; Models, Molecular; Nucleotides; Protein Binding
PubMed: 21795353
DOI: 10.1128/JVI.05022-11 -
Channels (Austin, Tex.) Nov 2016Guanidine compounds act as ion channel modulators. In the case of Cys-loop receptors, the guanidine compound amiloride antagonized the heteromeric GABA-A, glycine, and...
Guanidine compounds act as ion channel modulators. In the case of Cys-loop receptors, the guanidine compound amiloride antagonized the heteromeric GABA-A, glycine, and nicotinic acetylcholine receptors. However, amiloride exhibits characteristics consistent with a positive allosteric modulator for the human GABA-A (hGABA-A) ρ1 receptor. Site-directed mutagenesis revealed that the positive allosteric modulation was influenced by the GABA-A ρ1 second transmembrane domain 15' position, a site implicated in ligand allosteric modulation of Cys-loop receptors. There are a variety of amiloride derivatives that provide opportunities to assess the significance of amiloride functional groups (e.g., the guanidine group, the pyrazine ring, etc.) in the modulation of the GABA-A ρ1 receptor activity. We utilized 3 amiloride derivatives (benzamil, phenamil, and 5-(N, N-Hexamethylene) amiloride) to assess the contribution of these groups toward the potentiation of the GABA-A ρ1 receptor. Benzamil and phenamil failed to potentiate on the wild type GABA-A ρ1 GABA-mediated current while HMA demonstrated efficacy only at the highest concentration studied. The hGABA-A ρ1 (I15'N) mutant receptor activity was potentiated by lower HMA concentrations compared to the wild type receptor. Our findings suggest that an exposed guanidine group on amiloride and amiloride derivatives is critical for modulating the GABA-A ρ1 receptor. The present study provides a conceptual framework for predicting which amiloride derivatives will demonstrate positive allosteric modulation of the GABA-A ρ1 receptor.
Topics: Allosteric Regulation; Amiloride; Binding Sites; Electrophysiology; Guanidine; HEK293 Cells; Humans; Mutagenesis, Site-Directed; Receptors, GABA-B; gamma-Aminobutyric Acid
PubMed: 27367557
DOI: 10.1080/19336950.2016.1207021 -
Proceedings of the National Academy of... Oct 1990Phenamil, an analog of amiloride, is a potent blocker of the epithelial Na+ channel. It has been used to purify the porcine kidney amiloride-binding protein. Synthetic... (Comparative Study)
Comparative Study
Phenamil, an analog of amiloride, is a potent blocker of the epithelial Na+ channel. It has been used to purify the porcine kidney amiloride-binding protein. Synthetic oligonucleotides derived from partial sequences have been used to screen a human kidney cDNA library and to isolate the cDNA encoding the human amiloride-binding protein. The primary structure was deduced from the DNA sequence analysis. The protein is 713 residues long, with a 19-amino acid signal peptide. The mRNA was expressed in 293-S and NIH 3T3 cells, yielding a glycoprotein (i) that binds amiloride and amiloride analogs with affinities similar to the amiloride receptor associated with the apical Na+ channel in pig kidney membranes and (ii) that is immunoprecipitated with monoclonal antibodies raised against pig kidney amiloride-binding protein.
Topics: Amiloride; Amine Oxidase (Copper-Containing); Amino Acid Sequence; Animals; Base Sequence; Carrier Proteins; Cell Line; Cell Membrane; Cloning, Molecular; DNA; Gene Library; Humans; Kinetics; Molecular Sequence Data; Oligonucleotide Probes; Peptide Mapping; RNA, Messenger; Recombinant Proteins; Sequence Homology, Nucleic Acid; Swine; Transfection
PubMed: 2217167
DOI: 10.1073/pnas.87.19.7347 -
American Journal of Physiology. Renal... Apr 2016Diuretics acting on specific nephron segments to inhibit Na reabsorption have been used clinically for decades; however, drug interactions, tolerance, and derangements...
Diuretics acting on specific nephron segments to inhibit Na reabsorption have been used clinically for decades; however, drug interactions, tolerance, and derangements in serum K complicate their use to achieve target blood pressure. ROMK is an attractive diuretic target, in part, because its inhibition is postulated to indirectly inhibit the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) and the amiloride- and benzamil-sensitive epithelial Na channel (ENaC). The development of small-molecule ROMK inhibitors has created opportunities for exploring the physiological responses to ROMK inhibition. The present study evaluated how inhibition of ROMK alone or in combination with NKCC2, ENaC, or the hydrochlorothiazide (HCTZ) target NCC alter fluid and electrolyte transport in the nephron. The ROMK inhibitor VU591 failed to induce diuresis when administered orally to rats. However, another ROMK inhibitor, termed compound A, induced a robust natriuretic diuresis without kaliuresis. Compound A produced additive effects on urine output and Na excretion when combined with HCTZ, amiloride, or benzamil, but not when coadministered with bumetanide, suggesting that the major diuretic target site is the thick ascending limb (TAL). Interestingly, compound A inhibited the kaliuretic response induced by bumetanide and HCTZ, an effect we attribute to inhibition of ROMK-mediated K secretion in the TAL and CD. Compound A had no effect on heterologously expressed flow-sensitive large-conductance Ca-activated K channels (Slo1/β1). In conclusion, compound A represents an important new pharmacological tool for investigating the renal consequences of ROMK inhibition and therapeutic potential of ROMK as a diuretic target.
Topics: Amiloride; Animals; Benzimidazoles; Bumetanide; Diuresis; Diuretics; Drug Interactions; Hydrochlorothiazide; Nephrons; Potassium; Potassium Channels, Inwardly Rectifying; Rats; Sodium
PubMed: 26661652
DOI: 10.1152/ajprenal.00423.2015 -
Journal of the American College of... Nov 1995This study sought to establish the effect of amiloride on stunned myocardium and to determine the role of hemodynamic alterations and inhibition of sodium/proton...
Intracoronary amiloride prevents contractile dysfunction of postischemic "stunned" myocardium: role of hemodynamic alterations and inhibition of Na+/H+ exchange and L-type Ca2+ channels.
OBJECTIVES
This study sought to establish the effect of amiloride on stunned myocardium and to determine the role of hemodynamic alterations and inhibition of sodium/proton (Na+/H+) exchange and L-type cytosolic calcium (Ca2+) channels.
BACKGROUND
Amiloride is a nonspecific agent that may reduce reperfusion injury, but its effect on reversible dysfunction or stunned myocardium is unclear.
METHODS
Ninety-seven open chest dogs undergoing 15 min of left anterior descending coronary artery occlusion and 3 h of reperfusion with monitoring of hemodynamic variables, systolic shortening and myocardial blood flow were randomized to seven intracoronary infusions: control dogs (5% dextrose, n = 16); low dose amiloride (1 mg/min, n = 14); high dose amiloride (5 mg/min) with (n = 12) and without (n = 16) atrial pacing; sodium nitroprusside (20 micrograms/min, n = 16); hexamethylene amiloride (a specific inhibitor of Na+/H+ exchange, 60 micrograms/min, n = 14); and nifedipine (a specific inhibitor of L-type Ca2+ channels, 5 micrograms/min, n = 9). Drug infusions were started 40 min before occlusion and stopped at 30 min after reperfusion.
RESULTS
Forty-three dogs were excluded because of ventricular fibrillation or high collateral flow. The incidence of ventricular fibrillation was similar in all groups to that in control dogs. Systolic shortening completely recovered (p = NS vs. baseline; p < 0.01 vs. control group) by 2 h after reperfusion in the low dose amiloride group and 30 min in the high dose group (p < 0.01 vs. low dose). High dose amiloride increased myocardial blood flow and had positive inotropic and negative chronotropic effects (p < 0.05 vs. control group). Atrial pacing did not attenuate recovery. The only effect of low dose amiloride was increased myocardial blood flow after reperfusion. Systolic shortening did not deteriorate after washout of drug effects. Sodium nitroprusside and nifedipine similarly increased myocardial blood flow, but systolic shortening never recovered. Hexamethylene amiloride had no hemodynamic effects, and systolic shortening never recovered.
CONCLUSIONS
Amiloride prevented the contractile dysfunction of myocardial stunning but did not prevent arrhythmias. Hemodynamic alterations, increased myocardial blood flow and inhibition of Na+/H+ exchange or L-type Ca2+ channels alone did not account for the improved function. Inhibition of Na+/Ca2+ exchange may be the mechanism of improved postischemic function.
Topics: Amiloride; Animals; Calcium Channels; Dogs; Hemodynamics; Infusions, Intra-Arterial; Myocardial Contraction; Myocardial Stunning; Sodium-Hydrogen Exchangers
PubMed: 7594055
DOI: 10.1016/0735-1097(95)00326-6 -
Journal of Virology Feb 2008Amiloride derivatives are known blockers of the cellular Na(+)/H(+) exchanger and the epithelial Na(+) channel. More recent studies demonstrate that they also inhibit...
Amiloride derivatives are known blockers of the cellular Na(+)/H(+) exchanger and the epithelial Na(+) channel. More recent studies demonstrate that they also inhibit ion channels formed by a number of viral proteins. We previously reported that 5-(N-ethyl-N-isopropyl)amiloride (EIPA) modestly inhibits intracellular replication and, to a larger extent, release of human rhinovirus 2 (HRV2) (E. V. Gazina, D. N. Harrison, M. Jefferies, H. Tan, D. Williams, D. A. Anderson and S. Petrou, Antiviral Res. 67:98-106, 2005). Here, we demonstrate that amiloride and EIPA strongly inhibit coxsackievirus B3 (CVB3) RNA replication and do not inhibit CVB3 release, in contrast to our previous findings on HRV2. Passaging of plasmid-derived CVB3 in the presence of amiloride generated mutant viruses with amino acid substitutions in position 299 or 372 of the CVB3 polymerase. Introduction of either of these mutations into the CVB3 plasmid produced resistance to amiloride and EIPA, suggesting that they act as inhibitors of CVB3 polymerase, a novel mechanism of antiviral activity for these compounds.
Topics: Amiloride; Antiviral Agents; DNA-Directed RNA Polymerases; Enterovirus B, Human; Humans; Viral Proteins; Virus Replication
PubMed: 18032495
DOI: 10.1128/JVI.01374-07