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Journal of Bacteriology Oct 2021The bacterial flagellar motor (BFM) is a protein complex that confers motility to cells and contributes to survival and virulence. The BFM consists of stators that are...
The bacterial flagellar motor (BFM) is a protein complex that confers motility to cells and contributes to survival and virulence. The BFM consists of stators that are ion-selective membrane protein complexes and a rotor that directly connects to a large filament, acting as a propeller. The stator complexes couple ion transit across the membrane to torque that drives rotation of the motor. The most common ion gradients that drive BFM rotation are protons (H) and sodium ions (Na). The sodium-powered stators, like those in the PomA/PomB stator complex of spp., can be inhibited by sodium channel inhibitors, in particular, by phenamil, a potent and widely used inhibitor. However, relatively few new sodium motility inhibitors have been described since the discovery of phenamil. In this study, we characterized two possible motility inhibitors, HM2-16F and BB2-50F, from a small library of previously reported amiloride derivatives. We used three approaches: effect on rotation of tethered cells, effect on free-swimming bacteria, and effect on rotation of marker beads. We showed that both HM2-16F and BB2-50F stopped rotation of tethered cells driven by Na motors comparable to phenamil at matching concentrations and could also stop rotation of tethered cells driven by H motors. Bead measurements in the presence and absence of stators confirmed that the compounds did not inhibit rotation via direct association with the stator, in contrast to the established mode of action of phenamil. Overall, HM2-16F and BB2-50F stopped swimming in both Na and H stator types and in pathogenic and nonpathogenic strains. Here, we characterized two novel amiloride derivatives in the search for antimicrobial compounds that target bacterial motility. These compounds were shown to inhibit flagellar motility at 10 μM across multiple strains: from nonpathogenic Escherichia coli with flagellar rotation driven by proton or chimeric sodium-powered stators, to proton-powered pathogenic E. coli (enterohemorrhagic E. coli or uropathogenic E. coli [EHEC or UPEC, respectively]), and finally, sodium-powered Vibrio alginolyticus. Broad antimotility compounds such as these are important tools in our efforts to control virulence of pathogens in health and agricultural settings.
Topics: Acid Sensing Ion Channel Blockers; Amiloride; Escherichia coli; Movement; Vibrio alginolyticus
PubMed: 34516280
DOI: 10.1128/JB.00367-21 -
Biomolecules Sep 2022P2X7 is an extracellular adenosine 5'-triphopshate (ATP)-gated cation channel present on leukocytes, where its activation induces pro-inflammatory cytokine release and...
P2X7 is an extracellular adenosine 5'-triphopshate (ATP)-gated cation channel present on leukocytes, where its activation induces pro-inflammatory cytokine release and ectodomain shedding of cell surface molecules. Human P2X7 can be partially inhibited by amiloride and its derivatives at micromolar concentrations. This study aimed to screen a library of compounds derived from amiloride or its derivative 5-(,-hexamethylene) amiloride (HMA) to identify a potential P2X7 antagonist. 6-Furopyridine HMA (6-FPHMA) was identified as a novel P2X7 antagonist and was characterized further. 6-FPHMA impaired ATP-induced dye uptake into human RPMI8226 multiple myeloma cells and human P2X7-HEK293 cells, in a concentration-dependent, non-competitive manner. Likewise, 6-FPHMA blocked ATP-induced Ca fluxes in human P2X7-HEK293 cells in a concentration-dependent, non-competitive manner. 6-FPHMA inhibited ATP-induced dye uptake into human T cells, and interleukin-1β release within human blood and CD23 shedding from RPMI8226 cells. 6-FPHMA also impaired ATP-induced dye uptake into murine P2X7- and canine P2X7-HEK293 cells. However, 6-FPHMA impaired ATP-induced Ca fluxes in human P2X4-HEK293 cells and non-transfected HEK293 cells, which express native P2Y, P2Y and P2Y. In conclusion, 6-FPHMA inhibits P2X7 from multiple species. Its poor selectivity excludes its use as a specific P2X7 antagonist, but further study of amiloride derivatives as P2 receptor antagonists is warranted.
Topics: Adenosine; Adenosine Triphosphate; Amiloride; Animals; Dogs; HEK293 Cells; Humans; Interleukin-1beta; Mice; Purinergic P2X Receptor Antagonists; Receptors, Purinergic P2X7
PubMed: 36139148
DOI: 10.3390/biom12091309 -
Molecules (Basel, Switzerland) May 2022THz 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 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 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.
Topics: Amiloride; Animals; Epithelial Sodium Channels; Oocytes; Spectrum Analysis; Xenopus laevis
PubMed: 35630748
DOI: 10.3390/molecules27103271 -
Biochimica Et Biophysica Acta Nov 2002Recently, three proton pump inhibitors were shown to have no effect on proton excretion and little on Na uptake in tapwater-adapted (TW) crayfish, while all three... (Review)
Review
Recently, three proton pump inhibitors were shown to have no effect on proton excretion and little on Na uptake in tapwater-adapted (TW) crayfish, while all three reduced Na-H exchange in salt-depleted (SD) animals. It appeared that the exchange was mediated by the Na channel-H pump in SD crayfish but not in TW animals. An alternative, a 2Na-1H exchanger, might function in the latter. To test this, the effects of amiloride (AM) and ethylisopropyl AM (EIPA) on Na fluxes were observed. AM inhibits the Na channel but is a much weaker blocker of Na-H exchangers. In contrast, EIPA inhibits exchangers but acts weakly on the Na channel. If an exchanger functions in TW crayfish, we should expect EIPA to block Na influx in them with AM having a weaker action. The reverse should be true in SD animals. Experimental data showed that EIPA was a potent inhibitor of Na influx in TW crayfish with half-maximal inhibition at about 0.2 microM. However, AM proved to be equipotent. In SD crayfish, EIPA was as effective as in TW animals, and again AM was equally potent. The data fail to show the expected differential action. Therefore, AM and its analogues cannot be used to distinguish between the two models of Na-H exchange in crayfish.
Topics: Adaptation, Physiological; Amiloride; Animals; Astacoidea; Models, Animal; Sodium; Sodium Channels; Sodium-Hydrogen Exchangers; Vacuolar Proton-Translocating ATPases
PubMed: 12421538
DOI: 10.1016/s0005-2736(02)00585-0 -
PloS One 2020Anxiety disorders (AD) are the most common mental conditions affecting an estimated 40 million adults in the United States. Amiloride, a diuretic agent, has shown...
Anxiety disorders (AD) are the most common mental conditions affecting an estimated 40 million adults in the United States. Amiloride, a diuretic agent, has shown efficacy in reducing anxious responses in preclinical models by inhibiting the acid-sensing ion channels (ASIC). By delivering amiloride via nasal route, rapid onset of action can be achieved due to direct "nose-to-brain" access. Therefore, this study reports the formulation, physical, chemical, and microbiological stability of an extemporaneously prepared amiloride 2 mg/mL nasal spray. The amiloride nasal spray was prepared by adding 100 mg of amiloride hydrochloride to 50 mL of sterile water for injection in a sterile reagent bottle. A stability-indicating high-performance liquid chromatography (HPLC) method was developed and validated. Forced-degradation studies were performed to confirm the ability of the HPLC method to identify the degradation products from amiloride distinctively. The physical stability of the amiloride nasal spray was assessed by pH, clarity, and viscosity assessments. For chemical stability studies, samples of nasal sprays stored at room temperature were collected at time-points 0, 3 hr., 24 hr., and 7 days and were assayed in triplicate using the stability-indicating HPLC method. Microbiological stability of the nasal spray solution was evaluated for up to 7 days based on the sterility test outlined in United States Pharmacopoeia (USP) chapter 71. The stability-indicating HPLC method identified the degradation products of amiloride without interference from amiloride. All tested solutions retained over 90% of the initial amiloride concentration for the 7-day study period. There were no changes in color, pH, and viscosity in any sample. The nasal spray solutions were sterile for up to 7 days in all samples tested. An extemporaneously prepared nasal spray solution of amiloride hydrochloride (2 mg/mL) was physically, chemically, and microbiologically stable for 7 days when stored at room temperature.
Topics: Amiloride; Drug Compounding; Drug Stability; Drug Storage; Nasal Sprays
PubMed: 32649677
DOI: 10.1371/journal.pone.0232435 -
International Journal of Molecular... Mar 2021The K-sparing diuretic amiloride shows off-target anti-cancer effects in multiple rodent models. These effects arise from the inhibition of two distinct cancer targets:...
The K-sparing diuretic amiloride shows off-target anti-cancer effects in multiple rodent models. These effects arise from the inhibition of two distinct cancer targets: the trypsin-like serine protease urokinase-type plasminogen activator (uPA), a cell-surface mediator of matrix degradation and tumor cell invasiveness, and the sodium-hydrogen exchanger isoform-1 (NHE1), a central regulator of transmembrane H that supports carcinogenic progression. In this study, we co-screened our library of 5- and 6-substituted amilorides against these two targets, aiming to identify single-target selective and dual-targeting inhibitors for use as complementary pharmacological probes. Closely related analogs substituted at the 6-position with pyrimidines were identified as dual-targeting (pyrimidine uPA IC = 175 nM, NHE1 IC = 266 nM, uPA selectivity ratio = 1.5) and uPA-selective (methoxypyrimidine uPA IC = 86 nM, NHE1 IC = 12,290 nM, uPA selectivity ratio = 143) inhibitors, while high NHE1 potency and selectivity was seen with 5-morpholino ( NHE1 IC = 129 nM, uPA IC = 10,949 nM; NHE1 selectivity ratio = 85) and 5-(1,4-oxazepine) ( NHE1 IC = 85 nM, uPA IC = 5715 nM; NHE1 selectivity ratio = 67) analogs. Together, these amilorides comprise a new toolkit of chemotype-matched, non-cytotoxic probes for dissecting the pharmacological effects of selective uPA and NHE1 inhibition versus dual-uPA/NHE1 inhibition.
Topics: Amiloride; Breast Neoplasms; Cell Line, Tumor; Diuretics; Female; Humans; Models, Molecular; Neoplasm Invasiveness; Sodium-Hydrogen Exchanger 1; Structure-Activity Relationship; Urokinase-Type Plasminogen Activator
PubMed: 33804289
DOI: 10.3390/ijms22062999 -
European Journal of Pharmacology Jan 2024The use of morphine in clinical medicine is severely constrained by tolerance. Therefore, it is essential to examine pharmacological therapies that suppress the...
BACKGROUND
The use of morphine in clinical medicine is severely constrained by tolerance. Therefore, it is essential to examine pharmacological therapies that suppress the development of morphine tolerance. Amiloride suppressed the expression of inflammatory cytokines by inhibiting microglial activation. Microglia play a crucial role in the establishment of morphine tolerance. Thus, we anticipated that amiloride might suppress the development of morphine tolerance. During this investigation, we assessed the impact of amiloride on mouse morphine tolerance.
METHODS
Mice received morphine (10 mg/kg, s.c.) twice daily with intrathecally injected amiloride (0.3 μg/5 μl, 1 μg/5 μl, and 3 μg/5 μl) for nine continuous days. To assess morphine tolerance, mice underwent the tail-flick and hot plate tests. BV-2 cells were used to investigate the mechanism of amiloride. By using Western blotting, real-time PCR, and immunofluorescence labeling methods, the levels of acid-sensing ion channels (ASICs), nuclear factor kappa B (NF-kB) p65, p38 mitogen-activated protein kinase (MAPK) proteins, and neuroinflammation-related cytokines were determined.
RESULTS
The levels of ASIC3 in the spinal cord were considerably increased after long-term morphine administration. Amiloride was found to delay the development of tolerance to chronic morphine assessed via tail-flick and hot plate tests. Amiloride reduced microglial activation and downregulated the cytokines IL-1β and TNF-a by inhibiting ASIC3 in response to morphine. Furthermore, amiloride reduced p38 MAPK phosphorylation and inhibited NF-κB expression.
CONCLUSIONS
Amiloride effectively reduces chronic morphine tolerance by suppressing microglial activation caused by morphine by inhibiting ASIC3.
Topics: Mice; Animals; Morphine; Analgesics, Opioid; Amiloride; Neuroinflammatory Diseases; NF-kappa B; Microglia; Cytokines; Spinal Cord
PubMed: 37918499
DOI: 10.1016/j.ejphar.2023.176173 -
The Journal of Biological Chemistry Apr 1994Diamine oxidase (histaminase), an enzyme that oxidatively deaminates putrescine and histamine, was purified from human placenta and from pig kidney. Both NH2-terminal...
Diamine oxidase (histaminase), an enzyme that oxidatively deaminates putrescine and histamine, was purified from human placenta and from pig kidney. Both NH2-terminal sequences are highly homologous to the human kidney amiloride-binding protein, previously thought to be a component of the amiloride-sensitive Na+ channel. Monoclonal antibodies raised against the pig kidney amiloride-binding protein immunoprecipitate a polypeptide with the same M(r) (105,000) as that of pig kidney diamine oxidase. That polypeptide has both diamine oxidase activity and the capacity to bind [3H]phenamil, a tritiated amiloride derivative. Cells stably transfected with human kidney amiloride-binding protein cDNA express a high diamine oxidase activity. In transfected cells as well as with the purified enzyme, this activity was inhibited by amiloride and by some of its derivatives, such as phenamil and ethylpropylamiloride. Amiloride inhibition seems to be due to drug binding at the active site of the enzyme. These data indicate that human placental diamine oxidase is identical to the human kidney amiloride-binding protein and that amiloride analogues may have wider physiological effects besides those on epithelial ion transport.
Topics: Amiloride; Amine Oxidase (Copper-Containing); Amino Acid Sequence; Animals; Antibodies, Monoclonal; Carrier Proteins; Colon; DNA, Complementary; Female; Humans; Kidney; Kinetics; Molecular Sequence Data; Molecular Weight; Placenta; Pregnancy; Sequence Homology, Amino Acid; Swine; Transfection
PubMed: 8144586
DOI: No ID Found -
Scientific Reports Aug 2022Long-term administration of lithium is associated with chronic interstitial fibrosis that is partially reduced with exposure to amiloride. We examined potential pathways...
Long-term administration of lithium is associated with chronic interstitial fibrosis that is partially reduced with exposure to amiloride. We examined potential pathways of how amiloride may reduce interstitial fibrosis. Amiloride was administered to a rat model of lithium induced interstitial fibrosis over a long term (6 months), as well as for short terms of 14 and 28 days. Kidney cortical tissue was subjected to RNA sequencing and microRNA expression analysis. Gene expression changes of interest were confirmed using immunohistochemistry on kidney tissue. Pathways identified by RNA sequencing of kidney tissue were related to 'promoting inflammation' for lithium and 'reducing inflammation' for amiloride. Validation of candidate genes found amiloride reduced inflammatory components induced by lithium including NF-κB/p65 and activated pAKT, and increased p53 mediated regulatory function through increased p21 in damaged tubular epithelial cells. Amiloride also reduced the amount of Notch1 positive PDGFrβ pericytes and infiltrating CD3 cells in the interstitium. Thus, amiloride attenuates a multitude of pro-inflammatory components induced by lithium. This suggests amiloride could be repurposed as a possible anti-inflammatory, anti-fibrotic agent to prevent or reduce the development of chronic interstitial fibrosis.
Topics: Amiloride; Animals; Fibrosis; Inflammation; Kidney; Lithium; Lung Diseases, Interstitial; Rats
PubMed: 36028651
DOI: 10.1038/s41598-022-18825-1 -
Mediators of Inflammation 2017Acid-sensing ion channels (ASICs) are a family of proton-sensing channels that are voltage insensitive, cation selective (mostly permeable to Na), and nonspecifically... (Review)
Review
Acid-sensing ion channels (ASICs) are a family of proton-sensing channels that are voltage insensitive, cation selective (mostly permeable to Na), and nonspecifically blocked by amiloride. Derived from 5 genes (), 7 subunits have been identified, 1a, 1b, 2a, 2b, 3, 4, and 5, that are widely expressed in the peripheral and central nervous system as well as other tissues. Over the years, different studies have shown that activation of these channels is linked to various physiological and pathological processes, such as memory, learning, fear, anxiety, ischemia, and multiple sclerosis to name a few, so their potential as therapeutic targets is increasing. This review focuses on recent advances that have helped us to better understand the role played by ASICs in different pathologies related to neurodegenerative diseases, inflammatory processes, and pain.
Topics: Acid Sensing Ion Channels; Amiloride; Animals; Central Nervous System; Humans; Neurodegenerative Diseases
PubMed: 29056828
DOI: 10.1155/2017/3728096