Concentrations Of Amiloride Research Articles

Ribavirin inhibits the replication of infectious bursal disease virus predominantly through depletion of cellular guanosine pool.

The antiviral activity of different mutagens against single-stranded RNA viruses is well documented; however, their activity on the replication of double-stranded RNA viruses remains unexplored. This study aims to investigate the effect of different antivirals on the replication of a chicken embryo fibroblast-adapted Infectious Bursal Disease virus, FVSKG2. This study further explores the antiviral mechanism utilized by the most effective anti-IBDV agent. The cytotoxicity and anti-FVSKG2 activity of different antiviral agents (ribavirin, 5-fluorouracil, 5-azacytidine, and amiloride) were evaluated. The virus was serially passaged in chicken embryo fibroblasts 11 times at sub-cytotoxic concentrations of ribavirin, 5-fluorouracil or amiloride. Further, the possible mutagenic and non-mutagenic mechanisms utilized by the most effective anti-FVSKG2 agent were explored. Ribavirin was the least cytotoxic on chicken embryo fibroblasts, followed by 5-fluorouracil, amiloride and 5-azacytidine. Ribavirin inhibited the replication of FVSKG2 in chicken embryo fibroblasts significantly at concentrations as low as 0.05 mM. The extinction of FVSKG2 was achieved during serial passage of the virus in chicken embryo fibroblasts at ≥0.05 mM ribavirin; however, the emergence of a mutagen-resistant virus was not observed until the eleventh passage. Further, no mutation was observed in 1,898 nucleotides of the FVSKG2 following its five passages in chicken embryo fibroblasts in the presence of 0.025 mM ribavirin. Ribavarin inhibited the FVSKG2 replication in chicken embryo fibroblasts primarily through IMPDH-mediated depletion of the Guanosine Triphosphate pool of cells. However, other mechanisms like ribavirin-mediated cytokine induction or possible inhibition of viral RNA-dependent RNA polymerase through its interaction with the enzyme's active sites enhance the anti-IBDV effect. Ribavirin inhibits ds- RNA viruses, likely through IMPDH inhibition and not mutagenesis. The inhibitory effect may, however, be augmented by other non-mutagenic mechanisms, like induction of antiviral cytokines in chicken embryo fibroblasts or interaction of ribavirin with the active sites of RNA-dependent RNA polymerase of the virus.

TCL-318 Evaluation of the Antitumor Activity of Amiloride in Peripheral T-Cell Lymphomas Using In-Vitro Models.

Peripheral T-cell lymphomas (PTCL) have an aggressive clinical behavior and poor response to treatment; therefore, the search for new drugs is required. We have previously demonstrated the antitumor activity of amiloride in multiple myeloma. This prompted us to investigate the possibility of a cytotoxic effect of amiloride in this kind of T-cell lymphoma. To evaluate the antitumor activity of amiloride in different PTCL using in-vitro assays and identify the possible mechanisms involved. Two anaplastic large-cell lymphoma (ALCL) cell lines, KARPAS-299 and SR-786, and two cutaneous T-cell lymphoma cell lines, HH and HuT-78 (Sézary syndrome), were used. Cell viability and apoptosis were studied after exposure to different increasing concentrations of the drug at 24, 48, and 72 hours by colorimetric MTT and annexin V/propidium iodide double-labeling assays, respectively. Amiloride significantly inhibited cell growth in the PTCL cell lines, depending on increasing drug concentration and time. Inhibition was statistically significant (P<0.05) from 0.15 mM for KARPAS-299, SR-786, and HH at 24, 48, and 72 hours and from 0.3 mM for HuT-78 at 48 and 72 hours. KARPAS-299 and SR-786 were the most sensitive cell lines to amiloride exposure, whereas HuT-78 cells were less sensitive, although with the highest concentration of amiloride, its cell viability decreased to levels below 50% and 40% at 48 and 72 hours, respectively. Induction of cell death by apoptosis was detected in all cell lines, with apoptosis levels up to 25% at 24 hours and more than 80% at 48 and 72 hours using intermediate doses of the drug. 1) Amiloride significantly decreased the cell viability of PTCL. 2) ALCL cell lines were the most sensitive to amiloride exposure. 3) The cytotoxic effect induced by amiloride in PTCL was mediated by apoptosis. Ongoing Objectives: To evaluate apoptosis as the possible mechanism of cell death induction through the analysis of caspases, synergism with drugs commonly used in the treatment of PTCL, and changes in gene and protein expression induced by amiloride. Regional Health Management of Castilla y León (GRS 2142/A/2020);); Carlos III Health Institute European Union (PI19/00674); AECC (PROYE20047GUTI).

Abstract P335: Contribution Of The Gut To The Salt-sensitive Phenotype Of Dahl S Rats Is Reflected In Increased Sodium Absorption In The Distal Colon

Salt-sensitive hypertension in Dahl sensitive (Dahl S) rats is primarily attributed to sodium mishandling associated with kidney dysfunction, and very few studies interrogated sodium absorption in the gut of Dahl S rats and its potential contribution to the salt-sensitive phenotype. To assess this, we compared sodium channel (ENaC)-driven sodium (Na + ) currents, as a measure of Na + transport across the distal colon, the primary sodium absorption site in the GI tract, between Dahl S and Dahl salt-resistant (Dahl R) rats ex vivo . In parallel, we evaluated fecal sodium levels to assess overall fecal sodium loss in Dahl S and R rats. Male adult (12-14wo) Dahl S and R were administered a low sodium diet (0.3% NaCl, Teklad) ad libitum from weaning. Prior to the experiment, food intake/rat was measured every day for three consecutive days. On the day of experiment, rats were euthanized by an overdose of isoflurane. Distal colons were dissected and transferred to the Ussing chamber with oxygenated warm Krebs solution containing d-glucose. Increasing concentrations of the ENaC blocker amiloride (0.01-1000uM) were applied to the Ussing chamber on the apical side of the distal colon in 10-minute intervals. Maximum decreases in total ionic current across the distal colon were measured in real time following addition of amiloride to quantify ENaC Na + current across the distal colon. Fecal samples were homogenized in distilled water, and sodium levels per gram of fecal mass were determined by flame photometry. Despite no differences in food intake, we observed a dose-dependent 2 to 4-fold higher responses to amiloride in the distal colon of Dahl S compared to Dahl R rats, suggesting elevated Na + current in the Dahl S. Conversely, a ~2-fold lower Na + concentration was detected in the feces of Dahl S compared to Dahl R rats. In conclusion, elevated ENaC-driven Na + current in the distal colon, coupled with reduced fecal Na + concentration, suggest higher absorption/transport of Na + across the distal colon of Dahl S compared to Dahl R rats, which was independent of salt intake. Considering that the circulating Na + levels are reportedly maintained in the Dahl S by the overworked kidney, elevated colonic sodium absorption may contribute to kidney disease and salt-sensitive phenotype in Dahl S.

Novel intranasal treatment for anxiety disorders using amiloride, an acid-sensing ion channel antagonist: Pharmacokinetic modeling and simulation

To develop a physiologically based pharmacokinetic (PBPK) model for amiloride, an acid-sensing ion channel (ASIC) antagonist, and to simulate its pharmacokinetics in plasma and the central nervous system following intranasal administration in a virtual human population. We first developed a PBPK model of amiloride after oral administration and optimized the model using data from five clinical studies. Next, we added a nasal compartment to the amiloride oral PBPK model and parameterized using data from previous clinical studies. We simulated amiloride's pharmacokinetics in plasma, brain, and cerebrospinal fluid (CSF) after intranasal administration of amiloride at various doses in a virtual human population. The target amiloride concentration in the central nervous system required for maximal ASIC inhibition was achieved with a 75-mg intranasal amiloride dose. However, this finding is based on simulations performed using a mathematical model and needs to be further validated with appropriate clinical data. The nasal PBPK model of amiloride could be used to design future clinical studies and allow for successful clinical translation of intranasal amiloride formulation.

Enhanced degradation of amiloride over Bi2FeNbO7/bisulfite process: Key factors and mechanism

Construction of Bi2FeNbO7/bisulfite system for abatement of pharmaceutical residue was achieved. An attempt to synthesize Bi2FeNbO7 through hydrothermal technique was confirmed by X-ray diffraction. The magnetic field experiment revealed that Bi2FeNbO7 possessed a saturation magnetization of 6.99 emu/g, indicating magnetic attributes of Bi2FeNbO7. Scanning electron microscopy images showed that Bi2FeNbO7 exhibited regular octahedra in the size of 200–300 nm. In a self-made device, the activation of sodium bisulfite using Bi2FeNbO7 for the disposal of amiloride has been carefully explored. The effects of solution pH, sodium bisulfite concentration, Bi2FeNbO7 dosage, amiloride concentration, coexisting ions, and water matrix on the performance of Bi2FeNbO7/bisulfite system was investigated. The catalytic performance of Bi2FeNbO7/bisulfite to degrade amiloride was considerably higher than that of traditional iron oxides. The maximum removal efficiency of amiloride was 97.9% in Bi2FeNbO7/bisulfite process. The involvement of Fe might be crucial for activating bisulfite to create active species. The dominating radical in Bi2FeNbO7/bisulfite process was identified as SO3•‒. With the help of UHPLC/MS/MS, three new degradation products of amiloride were found. Dehalogenation and deamination of amiloride might account for the formation of these transformation products. This work provides a highly efficient Bi2FeNbO7/bisulfite process for the disposal of pharmaceutical pollutants in water treatment.

Rainbow Trout (Oncorhynchus mykiss) Na+/H+ Exchangers tNhe3a and tNhe3b Display Unique Inhibitory Profiles Dissimilar from Mammalian NHE Isoforms.

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 Oncorhynchus mykiss. 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.

TRPM7 channel activity in Jurkat T lymphocytes during magnesium depletion and loading: implications for divalent metal entry and cytotoxicity.

TRPM7 is a cation channel-protein kinase highly expressed in T lymphocytes and other immune cells. It has been proposed to constitute a cellular entry pathway for Mg2+ and divalent metal cations such as Ca2+, Zn2+, Cd2+, Mn2+, and Ni2+. TRPM7 channels are inhibited by cytosolic Mg2+, rendering them largely inactive in intact cells. The dependence of channel activity on extracellular Mg2+ is less well studied. Here, we measured native TRPM7 channel activity in Jurkat T cells maintained in external Mg2+ concentrations varying between 400 nM and 1.4 mM for 1-3 days, obtaining an IC50 value of 54 μM. Maintaining the cells in 400 nM or 8 μM [Mg2+]o resulted in almost complete activation of TRPM7 in intact cells, due to cytosolic Mg2+ depletion. A total of 1.4 mM [Mg2+]o was sufficient to fully eliminate the basal current. Submillimolar concentrations of amiloride prevented cellular Mg2+ depletion but not loading. We investigated whether the cytotoxicity of TRPM7 permeant metal ions Ni2+, Zn2+, Cd2+, Co2+, Mn2+, Sr2+, and Ba2+ requires TRPM7 channel activity. Mg2+ loading modestly reduced cytotoxicity of Zn2+, Co2+, Ni2+, and Mn2+ but not of Cd2+. Channel blocker NS8593 reduced Co2+ and Mn2+ but not Cd2+ or Zn2+ cytotoxicity and interfered with Mg2+ loading as evaluated by TRPM7 channel basal activity. Ba2+ and Sr2+ were neither detectably toxic nor permeant through the plasma membrane. These results indicate that in Jurkat T cells, entry of toxic divalent metal cations primarily occurs through pathways distinct from TRPM7. By contrast, we found evidence that Mg2+ entry requires TRPM7 channels.

Stability of extemporaneously compounded amiloride nasal spray.

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.

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.

Alternative splicing in human cancer cells is modulated by the amiloride derivative 3,5-diamino-6-chloro-N-(N-(2,6-dichlorobenzoyl)carbamimidoyl)pyrazine-2-carboxide.

Alternative splicing (AS) is a process that enables the generation of multiple protein isoforms with different biological properties from a single mRNA. Cancer cells often use the maneuverability conferred by AS to produce proteins that contribute to growth and survival. In our previous studies, we identified that amiloride modulates AS in cancer cells. However, the effective concentration of amiloride required to modulate AS is too high for use in cancer treatment. In this study, we used computational algorithms to screen potential amiloride derivatives for their ability to regulate AS in cancer cells. We found that 3,5‐diamino‐6‐chloro‐N‐(N‐(2,6‐dichlorobenzoyl)carbamimidoyl)pyrazine‐2‐carboxamide (BS008) can regulate AS of apoptotic gene transcripts, including HIPK3, SMAC, and BCL‐X, at a lower concentration than amiloride. This splicing regulation involved various splicing factors, and it was accompanied by a change in the phosphorylation state of serine/arginine‐rich proteins (SR proteins). RNA sequencing was performed to reveal that AS of many other apoptotic gene transcripts, such as AATF, ATM, AIFM1, NFKB1, and API5, was also modulated by BS008. In vivo experiments further indicated that treatment of tumor‐bearing mice with BS008 resulted in a marked decrease in tumor size. BS008 also had inhibitory effects in vitro, either alone or in a synergistic combination with the cytotoxic chemotherapeutic agents sorafenib and nilotinib. BS008 enabled sorafenib dose reduction without compromising antitumor activity. These findings suggest that BS008 may possess therapeutic potential for cancer treatment.

Effect of a synthetic inhibitor of urokinase plasminogen activator on the migration and invasion of human cervical cancer cells in vitro.

As a notable feature of malignant tumors, invasion and metastasis are important events in the process of tumor progression. Amiloride, a synthetic inhibitor of urokinase plasminogen activator (uPA), is involved in these events. To evaluate the therapeutic value of amiloride in cervical cancer, HeLa cells were used as in vitro cellular models. The migration and invasion abilities of HeLa cells, in addition to the mRNA expression of matriptase, uPA, uPA receptor and 72 kDa type IV collagenase (MMP-2), were detected using scratch assays, Transwell chamber assays and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The results of RT-qPCR demonstrated that the mRNA expression of uPA and MMP-2 in HeLa cells was downregulated significantly in a dose-dependent manner when incubated with various concentrations of amiloride for 24 h. The migration distance of HeLa cells was significantly shorter at 6, 12 and 24 h following incubation with amiloride (P<0.01), and there was a positive correlation between cell migratory ability and cellular uPA protein expression level (r=0.955, P<0.01). The number of HeLa cells that penetrated the Matrigel following incubation for 24 h with different concentrations of amiloride decreased significantly compared with the control group, indicating that cell invasiveness was positively correlated with the protein expression level of uPA in the cells (r=0.993, P<0.01). The present study demonstrated that amiloride was able to specifically inhibit the mRNA expression levels of uPA in HeLa cells, and sequentially downregulate the mRNA expression of downstream MMP-2 in the uPA system, thereby suppressing the migratory and invasive ability of HeLa cells. Therefore, amiloride may be a promising therapeutic target for the treatment of cervical cancer.

Significant natriuretic and antihypertensive action of the epithelial sodium channel blocker amiloride in diabetic patients with and without nephropathy.

Diabetic nephropathy is associated with aberrant glomerular filtration of serine proteases. The study was designed to test the hypothesis that the epithelial sodium channel is activated proteolytically by urine plasmin in diabetic nephropathy and mediates renal sodium retention. In an open-label intervention study on type 1 diabetes patients on standardized NaCl intake (200 mmol/day) with (n = 15) and without diabetic nephropathy (control, n = 12), urinary Na excretion in response to oral amiloride (20 or 40 mg/day for 2 days) was compared. A total of 27 patients completed the study and nine diabetic nephropathy and eight control study participants were compliant (24-h urine Na excretion of 200 mmol ± 30%). Amiloride increased significantly total and fractional Na excretion in both groups. Total natriuresis and weight loss were significantly larger in the control group compared with diabetic nephropathy at day 1 of amiloride, whereas fractional Na excretion did not differ. Amiloride intervention increased plasma renin concentration only in diabetic nephropathy group; it reduced SBP in both groups, whereas DBP was reduced in diabetic nephropathy group only. Albuminuria was reduced significantly by amiloride in diabetic nephropathy group. Urine total amiloride concentration was not different between groups (12 ± 1 and 16 ± 1 μmol/l, respectively). Urine total plasminogen and active plasmin were reduced after amiloride in diabetic nephropathy. Amiloride increased renal Na excretion, reduced blood pressure, albuminuria, and total and active plasmin in urine. It is concluded that epithelial sodium channel is an attractive target to attain blood pressure control in long-term type I diabetes with no enhanced activity associated with nephropathy.

Canavanine increases glucose uptake in C2 C12 cells through the activation of imidazoline I-2B receptors.

Canavanine is a guanidinium derivative that contains the basic structure of the ligand(s) of imidazoline receptor (I-R). Canavanine has been reported to activate the imidazoline I-3 receptor (I-3R) both in vivo and in vitro. Additionally, the activation of the imidazoline I-2B receptor (I-2BR) by guanidinium derivatives may increase glucose uptake. Therefore, the effect of canavanine on the I-2BR was investigated in the present study. Glucose uptake into cultured C2 C12 cells was determined using the radio-ligated tracer 2-[(14) C]-deoxy-glucose. The changes in 5' AMP-activated protein kinase (AMPK) expression were also identified using Western blotting analysis. The canavanine-induced glucose uptake was inhibited in a dose-dependent manner by BU224 (0.01-1 μmol/L), which is a specific I-2BR antagonist, in the C2 C12 cells. Additionally, the canavanine-stimulated AMPK phosphorylation and glucose transporter (GLUT4) expression were also sensitive to BU224 inhibition in the C2 C12 cells. Moreover, both canavanine-stimulated glucose uptake and AMPK phosphorylation were attenuated by high concentrations of amiloride (1-2 μmol/L), which is another established I-2BR inhibitor, in a dose-dependent manner in C2 C12 cells. Additionally, compound C abolished the canavanine-induced glucose uptake and AMPK phosphorylation at a concentration (0.1 μmol/L) sufficient to inhibit AMPK. In conclusion, these data demonstrated that canavanine has an ability to activate I-2BR through the AMPK pathway to increase glucose uptake, which indicates I-2BR as a new target for diabetic therapy.

Evans Blue is not a suitable inhibitor of the epithelial sodium channel δ-subunit

The Epithelial Sodium Channel (ENaC) is a heterotrimeric ion channel which can be either formed by assembly of its α-, β- and γ-subunits or, alternatively, its δ-, β- and γ-subunits. The physiological function of αβγ-ENaC is well established, but the function of δβγ-ENaC remains elusive. The azo-dye Evans Blue (EvB) has been routinely used to discriminate between the two channel isoforms by decreasing transmembrane currents and amiloride-sensitive current fractions of δβγ-ENaC expressing Xenopus oocytes. Even though these results could be reproduced, it was found by precipitation experiments and spectroscopic methods that the cationic amiloride and the anionic EvB directly interact in solution, forming a strong complex. Thereby a large amount of pharmacologically available amiloride is removed from physiological buffer solutions and the effective amiloride concentration is reduced. This interaction did not occur in the presence of albumin. In microelectrode recordings, EvB was able to abrogate the block of δβγ-ENaC by amiloride or its derivative benzamil.In sum, EvB reduces amiloride-sensitive ion current fractions in electrophysiological experiments. This is not a result of a specific inhibition of δβγ-ENaC but rather represents a pharmacological artefact. EvB should therefore not be used as an inhibitor of δ-ENaC.

Aberrant glomerular filtration of urokinase-type plasminogen activator in nephrotic syndrome leads to amiloride-sensitive plasminogen activation in urine.

In nephrotic syndrome, aberrant glomerular filtration of plasminogen and conversion to active plasmin in preurine are thought to activate proteolytically epithelial sodium channel (ENaC) and contribute to sodium retention and edema. The ENaC blocker amiloride is an off-target inhibitor of urokinase-type plasminogen activator (uPA) in vitro. It was hypothesized that uPA is abnormally filtered to preurine and is inhibited in urine by amiloride in nephrotic syndrome. This was tested by determination of Na(+) balance, uPA protein and activity, and amiloride concentration in urine from rats with puromycin aminonucleoside (PAN)-induced nephrotic syndrome. Urine samples from 6 adult and 18 pediatric patients with nephrotic syndrome were analyzed for uPA activity and protein. PAN treatment induced significant proteinuria in rats which coincided with increased urine uPA protein and activity, increased urine protease activity, and total plasminogen/plasmin concentration and Na(+) retention. Amiloride (2 mg·kg(-1)·24 h(-1)) concentration in urine was in the range 10-20 μmol/l and reduced significantly urine uPA activity, plasminogen activation, protease activity, and sodium retention in PAN rats, while proteinuria was not altered. In paired urine samples, uPA protein was significantly elevated in urine from children with active nephrotic syndrome compared with remission phase. In six adult nephrotic patients, urine uPA protein and activity correlated positively with 24 h urine protein excretion. In conclusion, nephrotic syndrome is associated with aberrant filtration of uPA across the injured glomerular barrier. Amiloride inhibits urine uPA activity which attenuates plasminogen activation and urine protease activity in vivo. Urine uPA is a relevant target for amiloride in vivo.

Blockade of ENaCs by amiloride induces c-Fos activation of the area postrema

Epithelial sodium channels (ENaCs) are strongly expressed in the circumventricular organs (CVOs), and these structures may play an important role in sensing plasma sodium levels. Here, the potent ENaC blocker amiloride was injected intraperitoneally in rats and 2h later, the c-Fos activation pattern in the CVOs was studied. Amiloride elicited dose-related activation in the area postrema (AP) but only ~10% of the rats showed c-Fos activity in the organum vasculosum of the lamina terminalis (OVLT) and subfornical organ (SFO). Tyrosine hydroxylase-immunoreactive (catecholamine) AP neurons were activated, but tryptophan hydroxylase-immunoreactive (serotonin) neurons were unaffected. The AP projects to FoxP2-expressing neurons in the dorsolateral pons which include the pre-locus coeruleus nucleus and external lateral part of the parabrachial nucleus; both cell groups were c-Fos activated following systemic injections of amiloride. In contrast, another AP projection target – the aldosterone-sensitive neurons of the nucleus tractus solitarius which express the enzyme 11-β-hydroxysteriod dehydrogenase type 2 (HSD2) were not activated. As shown here, plasma concentrations of amiloride used in these experiments were near or below the IC50 level for ENaCs. Amiloride did not induce changes in blood pressure, heart rate, or regional vascular resistance, so sensory feedback from the cardiovascular system was probably not a causal factor for the c-Fos activity seen in the CVOs. In summary, amiloride may have a dual effect on sodium homeostasis causing a loss of sodium via the kidney and inhibiting sodium appetite by activating the central satiety pathway arising from the AP.

Reply to the Editor— Amiloride Concentrations in Clinical Practice

Reply to the Editor— Amiloride Concentrations in Clinical Practice

To the Editor— Amiloride concentrations in clinical practice

To the Editor— Amiloride concentrations in clinical practice

Effect of Amiloride to Retinal Toxicity Induced by Tissue Plasminogen Activator

PurposeThe effects of amiloride on cellular toxicity caused by tissue plasminogen activator (tPA) in mouse primary retinal cells were investigated.MethodsPrimary retinal cell cultures were maintained using glial conditioned medium. Commercial tPA and L-arginine were added, and the level of cyclic guanosine monophosphate (cyclic-GMP) in the culture supernatant was assessed using an ELISA assay. We measured the cell viability of cultured retinal cells pretreated with three different concentrations of amiloride (1, 10, and 100 µm) in addition to commercial tPA or L-arginine treatment.ResultsAfter exposing the cultured mouse retinal cells to tPA plus L-arginine or L-arginine alone, cyclic-GMP concentrations were 61.9 ± 5.1 pmole/mL and 63.1 ± 6.1 pmole/mL, respectively. However, the control group had a significantly lower concentration of cyclic-GMP (37.2 ± 3.4 pmole/mL, p < 0.01). The cyclic GMP-dissolved solution did not cause retinal cell death. In the control group and the group treated with 1 µm amiloride and tPA containing L-arginine, the cell viability was 43.7% and 44.5%, respectively. However, cell viability increased to 70.6% with 10 µm amiloride and 78.4% with 100 µm amiloride (p = 0.015).ConclusionsL-arginine increases intracellular cyclic-GMP and may give rise to retinal cells through this mechanism. In addition, amiloride in concentrations greater than 10 µm protects against L-arginine-induced retinal cell death.

Pharmacologic Doses of Amiloride Preferentially Induce Apoptosis and Growth Inhibition of Flt3-ITD Mutation Positive Acute Myeloid Leukemia Cell Lines

Abstract 5004 Introduction:Amiloride is an FDA approved potassium-sparing diuretic which targets Na+/H+ exchanger isoform 1 (NHE1). NHE1 is responsible for the regulation of the intracellular pH, as well as cell-cycle and apoptosis. In supra-pharmacologic concentrations, amiloride non-specifically inhibits protein kinases. Recent study demonstrated that proapoptotic effect of amiloride in CML cell lines is linked to the modulation of the alternative splicing of Bcl-x, HIPK3, and BCR/ABL genes and is independent of pHi.Here, we demonstrate that pharmacologic doses of amiloride preferentially induce growth inhibition, cell cycle arrest and apoptosis in Flt3-ITD positive acute myeloid leukemia cell lines as compared to Bcr-Abl positive leukemia cell line. Our data suggests that amiloride may have an effect on Flt3 signaling and that its treatment potential for Flt3-ITD positive acute myeloid leukemia needs to be explored. Methods:MV4-11, MOLM13 and K562 cells lines in log-phase growth were used for the experiments. Analysis of the baseline Flt3 expression and phosphorylation status was assessed via Flt3 immunoprecipitation and Western blotting for Flt3 and phosphotyrosine. Cells were incubated with various amiloride concentrations; equal volume dilutions of DMSO were used for control. Cell counting and trypan blue exclusion viability was performed on TC10 Bio-Rad automated cell counter. The cell cycle analysis was performed applying propidium iodide staining. To assess for apoptosis and cell death, we used annexin V/PI staining kit and flow cytometry. Results:MOLM13 and MV4-11 cell lines carry activating Flt3-ITD mutation. We confirmed the expression and constituative activation of Flt3 in MOLM13 and MV4-11 cells with Western blotting. Flt3 protein was not detectable in K562 cell line. Amiloride at 0.025 mM and 0.05 mM completely inhibited the growth of MV4-11 cells after 24 hrs of treatment with no significant increase in total or live cell numbers at 72 hrs, but only mildly affected K562 cell proliferation. While the above amiloride concentrations caused cell death in MV4-11 and MOLM13 cell lines, there was no increased cell death in K562 cells.Incubation of MOLM13 and MV4-11 cell lines with 0.05 mM amiloride for 20 hrs induced cell cycle arrest. In MV4-11 cell line, the proportion of S phase cells after amiloride treatment was 15.4% (SD=5.4%) as compared to 31.3% (SD=1.4%) in control. MOLM13 cell line demonstrated 15.3% (SD=4.7%) of cells in S after amiloride treatment as compared to 35.3% (SD=2.4%) cells in S phase in control treatment. In K562 cell line, there was less effect with 52% (SD=4.2%) of cells in S phase in control as compared to 37% (SD=8.9%) in amiloride treatment.MV4-11 and MOLM 13 cell lines were more sensitive than K562 cells to amiloride induced apoptosis with 28.8% (control 12.7%) of MV4-11 cells, 11.4% (control 7.4%) of MOLM13 cells, and 11.4% (control 8.6%) of K562 cells being apoptotic after 20 hr treatment with 0.05mM amiloride. At 72 hrs of amiloride treatment 34% (control 1.5%) of MV4-11 cells, 17% (control 5%) of MOLM13 cells and 11% of K562 cells (control 8.9%) were apoptotic. Amiloride had similar effect on the number of dead cells with no increase in total cell death in K562 cell line. Upon treatment with increasing amiloride concentrations, there was dose-dependent increase in cell death and apoptosis in all three cell lines with K562 line showing relative resistance to amiloride. Discussion:Our results demonstrate that amiloride induces cell cycle arrest and inhibits proliferation of Flt3-ITD positive cell lines MV4-11 and MOLM13 as well as K562 cell line at a pharmacologic concentration of 0.05 mM. Both, cell cycle arrest and antiproliferative effect are more pronounced in Flt3-ITD positive cells lines while it is mild in Bcr-Abl positive K562 cell line. Pharmacologic doses of amiloride induce cell death and apoptosis in Flt3-ITD positive cell lines but not in K562 cell line. Both, Bcr-Abl and Flt3 signaling stimulates proliferation and inhibits apoptosis in myeloid leukemia cells. Our study suggests that amiloride may induce cell cycle arrest and apoptosis via modulating Flt3 signaling cascade. We are currently investigating the effects of amiloride on Flt3 phosphorylation. In conclusion, our data suggests that amiloride presents a potential treatment option for Flt3-ITD positive acute myeloid leukemia. Disclosures:No relevant conflicts of interest to declare.