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IScience Jan 2024Tumors maintain an alkaline intracellular environment to enable rapid growth. The proton exporter NHE1 participates in maintenance of this pH gradient. However, whether...
Tumors maintain an alkaline intracellular environment to enable rapid growth. The proton exporter NHE1 participates in maintenance of this pH gradient. However, whether targeting NHE1 could inhibit the growth of tumor cells remains unknown. Here, we report that the NHE1 inhibitor Hexamethylene amiloride (HA) efficiently suppresses the growth of AML cell lines. Moreover, HA combined with venetoclax synergized to efficiently inhibit the growth of AML cells. Interestingly, lysosomes are the main contributors to the synergism of HA and venetoclax in inhibiting AML cells. Most importantly, the combination of HA and venetoclax also had prominent anti-leukemia effects in both xenograft models and bone marrow samples from AML patients. In summary, our results provide evidence that the NHE1 inhibitor HA or its combination with venetoclax efficiently inhibits the growth of AML and .
PubMed: 38205254
DOI: 10.1016/j.isci.2023.108691 -
The Journal of Physical Chemistry. B Jan 2024The human Na/H exchanger (NHE1) plays a crucial role in maintaining intracellular pH by regulating the electroneutral exchange of a single intracellular H for one...
The human Na/H exchanger (NHE1) plays a crucial role in maintaining intracellular pH by regulating the electroneutral exchange of a single intracellular H for one extracellular Na across the plasma membrane. Understanding the molecular mechanisms governing ion transport and the binding of inhibitors is of importance in the development of anticancer therapeutics targeting NHE1. In this context, we performed molecular dynamics (MD) simulations based on the recent cryo-electron microscopy (cryo-EM) structures of outward- and inward-facing conformations of NHE1. These simulations allowed us to explore the dynamics of the protein, examine the ion-translocation pore, and confirm that Asp267 is the ion-binding residue. Our free energy calculations did not show a significant difference between Na and K binding at the ion-binding site. Consequently, Na over K selectivity cannot be solely explained by differences in ion binding. Our MD simulations involving NHE1 inhibitors (cariporide and amiloride analogues) maintained stable interactions with Asp267 and Glu346. Our study highlights the importance of the salt bridge between the positively charged acylguanidine moiety and Asp267, which appears to play a role in the competitive inhibitory mechanism for this class of inhibitors. Our computational study provides a detailed mechanistic interpretation of experimental data and serves the basis of future structure-based inhibitor design.
Topics: Humans; Molecular Dynamics Simulation; Cryoelectron Microscopy; Sodium-Hydrogen Exchangers; Ion Transport; Cell Membrane; Hydrogen-Ion Concentration
PubMed: 38185879
DOI: 10.1021/acs.jpcb.3c05863 -
BMC Research Notes Jan 2024Silicosis is an irreversible occupational lung disease resulting from crystalline silica inhalation. Previously, we discovered that Western diet (HFWD)-consumption...
OBJECTIVES
Silicosis is an irreversible occupational lung disease resulting from crystalline silica inhalation. Previously, we discovered that Western diet (HFWD)-consumption increases susceptibility to silica-induced pulmonary inflammation and fibrosis. This study investigated the potential of HFWD to alter silica-induced effects on airway epithelial ion transport and smooth muscle reactivity.
METHODS
Six-week-old male F344 rats were fed a HFWD or standard rat chow (STD) and exposed to silica (Min-U-Sil 5, 15 mg/m, 6 h/day, 5 days/week, for 39 d) or filtered air. Experimental endpoints were measured at 0, 4, and 8 weeks post-exposure. Transepithelial potential difference (V), short-circuit current (I) and transepithelial resistance (R) were measured in tracheal segments and ion transport inhibitors [amiloride, Na channel blocker; NPPB; Cl- channel blocker; ouabain, Na, K-pump blocker] identified changes in ion transport pathways. Changes in airway smooth muscle reactivity to methacholine (MCh) were investigated in the isolated perfused trachea preparation.
RESULTS
Silica reduced basal I at 4 weeks and HFWD reduced the I response to amiloride at 0 week compared to air control. HFWD + silica exposure induced changes in ion transport 0 and 4 weeks after treatment compared to silica or HFWD treatments alone. No effects on airway smooth muscle reactivity to MCh were observed.
Topics: Male; Rats; Animals; Amiloride; Silicon Dioxide; Diet, Western; Rats, Inbred F344; Epithelium; Ion Transport; Methacholine Chloride; Muscle, Smooth
PubMed: 38172968
DOI: 10.1186/s13104-023-06672-w -
European Journal of Medicinal Chemistry Feb 2024Lung selective inhibition of the endothelial sodium channel (ENaC) is a potential mutation agnostic treatment of Cystic Fibrosis (CF). We describe the discovery and...
Lung selective inhibition of the endothelial sodium channel (ENaC) is a potential mutation agnostic treatment of Cystic Fibrosis (CF). We describe the discovery and development of BI 1265162, the first ENaC inhibitor devoid of the amiloride structural motif that entered clinical trials. The design of BI 1265162 focused on its suitability for inhalation via the Respimat® Soft Mist™ Inhaler and a long duration of action. A convergent and scalable route for the synthesis of BI 1265162 as dihydrogen phosphate salt is presented, that was applied to support clinical trials. A phase 2 study with BI 1265162 did not provide a clear sign of clinical benefit. Whether ENaC inhibition will be able to hold its promise for CF patients remains an open question.
Topics: Humans; Cystic Fibrosis; Sodium Channel Blockers; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Sodium Channels; Amiloride; Sodium
PubMed: 38157597
DOI: 10.1016/j.ejmech.2023.116038 -
American Journal of Physiology. Renal... Feb 2024Dyslipidemia, with changes in plasma membrane (PM) composition, is associated with hypertension, while rising PM cholesterol induces Na channel activity. We hypothesize...
Dyslipidemia, with changes in plasma membrane (PM) composition, is associated with hypertension, while rising PM cholesterol induces Na channel activity. We hypothesize that ablation of renal tubular ABCA1, a cholesterol efflux protein, leads to cholesterol- and Na-dependent changes in blood pressure (BP). Transgenic mice () expressing a doxycycline (dox)-inducible CRE recombinase were bred with mice expressing floxed ABCA1 to generate renal tubules deficient in ABCA1 (ABCA1FF). Tail-cuff systolic BP (SBP) was measured in mice on specific diets. Immunoblotting was performed on whole and PM protein lysates of kidney from mice completing experimental diets. Cortical PM of ABCA1FF showed reduced ABCA1 (60 ± 28%; = 10, < 0.05) compared with wild-type littermates (WT; = 9). Tail-cuff SBP of ABCA1FF ( = 11) was not only greater post dox, but also during cholesterol or high Na feeding ( < 0.05) compared with WT mice ( = 15). A Na-deficient diet abolished the difference, while 6 wk of cholesterol diet raised SBP in ABCA1FF compared with mice before cholesterol feeding ( < 0.05). No difference in α-ENaC protein abundance was noted in kidney lysate; however, γ-ENaC increased in ABCA1FF mice versus WT mice. In kidney membranes, NKCC2 abundance was greater in ABCA1FF versus WT mice. Cortical lysates of ABCA1FF mouse kidneys expressed less renin and angiotensin I receptor than WT mouse kidneys. Furosemide injection induced a greater diuretic effect in ABCA1FF ( = 7; 45.2 ± 8.7 µL/g body wt) versus WT ( = 7; 33.1 ± 6.9 µL/g body wt; < 0.05) but amiloride did not. Tubular ABCA1 deficiency induces cholesterol-dependent rise in SBP and modest Na sensitivity of SBP, which we speculate is partly related to Na transporters and channels. Cholesterol has been linked to greater Na channel activity in kidney cells, which may predispose to systemic hypertension. We showed that when ABCA1, a protein that removes cholesterol from tissues, is ablated from mouse kidneys, systemic blood pressure is greater than normal mice. Dietary cholesterol further increases blood pressure in transgenic mice, whereas low dietary salt intake reduced blood pressure to that of normal mice. Thus, we speculate that diseases and pharmaceuticals that reduce renal ABCA1 expression, like diabetes and calcineurin inhibitors, respectively, contribute to the prominence of hypertension in their clinical presentation.
Topics: Animals; Male; Mice; Blood Pressure; Cholesterol; Epithelial Sodium Channels; Hypertension; Mice, Knockout; Mice, Transgenic; Sodium
PubMed: 38153852
DOI: 10.1152/ajprenal.00154.2023 -
Cancer Communications (London, England) Feb 2024Small extracellular vesicles (sEVs) mediate intercellular communication that contributes to hepatocellular carcinoma (HCC) progression via multifaceted pathways. The...
BACKGROUND
Small extracellular vesicles (sEVs) mediate intercellular communication that contributes to hepatocellular carcinoma (HCC) progression via multifaceted pathways. The success of cell entry determines the effect of sEV on recipient cells. Here, we aimed to delineate the mechanisms underlying the uptake of sEV in HCC.
METHODS
Macropinocytosis was examined by the ability of cells to internalize dextran and sEV. Macropinocytosis was analyzed in Na(+)/H(+) exchanger 7 (NHE7)-knockdown and -overexpressing cells. The properties of cells were studied using functional assays. pH biosensor was used to evaluate the intracellular and endosomal pH. The expression of NHE7 in patients' liver tissues was examined by immunofluorescent staining. Inducible silencing of NHE7 in established tumors was performed to reveal the therapeutic potential of targeting NHE7.
RESULTS
The data revealed that macropinocytosis controlled the internalization of sEVs and their oncogenic effect on recipient cells. It was found that metastatic HCC cells exhibited the highest efficiency of sEV uptake relative to normal liver cells and non-metastatic HCC cells. Attenuation of macropinocytic activity by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) limited the entry of sEVs and compromised cell aggressiveness. Mechanistically, we delineated that high level of NHE7, a sodium-hydrogen exchanger, alkalized intracellular pH and acidized endosomal pH, leading to the maturation of macropinosomes. Inducible inhibition of NHE7 in established tumors developed in mice delayed tumor development and suppressed lung metastasis. Clinically, NHE7 expression was upregulated and linked to dismal prognosis of HCC.
CONCLUSIONS
This study advances the understanding that NHE7 enhances sEV uptake by macropinocytosis to promote the malignant properties of HCC cells. Inhibition of sEV uptake via macropinocytosis can be exploited as a treatment alone or in combination with conventional therapeutic approaches for HCC.
Topics: Animals; Humans; Mice; Carcinoma, Hepatocellular; Cell Line; Liver Neoplasms; Sodium-Hydrogen Exchangers; Up-Regulation
PubMed: 38152992
DOI: 10.1002/cac2.12515 -
Cell Chemical Biology Apr 2024Mycobacterial bioenergetics is a validated target space for antitubercular drug development. Here, we identify BB2-50F, a 6-substituted 5-(N,N-hexamethylene)amiloride...
Mycobacterial bioenergetics is a validated target space for antitubercular drug development. Here, we identify BB2-50F, a 6-substituted 5-(N,N-hexamethylene)amiloride derivative as a potent, multi-targeting bioenergetic inhibitor of Mycobacterium tuberculosis. We show that BB2-50F rapidly sterilizes both replicating and non-replicating cultures of M. tuberculosis and synergizes with several tuberculosis drugs. Target identification experiments, supported by docking studies, showed that BB2-50F targets the membrane-embedded c-ring of the FF-ATP synthase and the catalytic subunit (substrate-binding site) of succinate dehydrogenase. Biochemical assays and metabolomic profiling showed that BB2-50F inhibits succinate oxidation, decreases the activity of the tricarboxylic acid (TCA) cycle, and results in succinate secretion from M. tuberculosis. Moreover, we show that the lethality of BB2-50F under aerobic conditions involves the accumulation of reactive oxygen species. Overall, this study identifies BB2-50F as an effective inhibitor of M. tuberculosis and highlights that targeting multiple components of the mycobacterial respiratory chain can produce fast-acting antimicrobials.
Topics: Humans; Mycobacterium tuberculosis; Succinate Dehydrogenase; Antitubercular Agents; Tuberculosis; Adenosine Triphosphate; Enzyme Inhibitors; Succinates
PubMed: 38151019
DOI: 10.1016/j.chembiol.2023.12.002 -
American Journal of Physiology. Cell... Feb 2024Vitamin D deficiency is a risk factor for exacerbation of obstructive airway disease, a hallmark of which is mucus dehydration and plugging. Calcitriol (the active form...
Vitamin D deficiency is a risk factor for exacerbation of obstructive airway disease, a hallmark of which is mucus dehydration and plugging. Calcitriol (the active form of vitamin D) deficiency in cultured human airway epithelia resulted in increased and mRNAs encoding subunits of ENaC and the Na-K pump compared with supplemented epithelia. These drive the absorption of airway surface liquid. Consistently, calcitriol-deficient epithelia absorbed liquid faster than supplemented epithelia. Calcitriol deficiency also increased amiloride-sensitive and without altering Na-K pump activity, indicating the changes in amiloride-sensitivity arose from ENaC. ENaC activity can be regulated by trafficking, proteases, and channel abundance. We found the effect was likely not induced by changes to endocytosis of ENaC given that calcitriol did not affect the half-lives of amiloride-sensitive and . Furthermore, trypsin nominally increased produced by epithelia ± calcitriol, suggesting calcitriol did not affect proteolytic activation of ENaC. Consistent with mRNA and functional data, calcitriol deficiency resulted in increased γENaC protein. These data indicate that the vitamin D receptor response controls ENaC function and subsequent liquid absorption, providing insight into the relationship between vitamin D deficiency and respiratory disease. It is unknown why calcitriol (active vitamin D) deficiency worsens pulmonary disease outcomes. Results from mRNA, immunoblot, Ussing chamber, and absorption experiments indicate that calcitriol deficiency increases ENaC activity in human airway epithelia, decreasing apical hydration. Given that epithelial hydration is required for mucociliary transport and airway innate immune function, the increased ENaC activity observed in calcitriol-deficient epithelia may contribute to respiratory pathology observed in vitamin D deficiency.
Topics: Humans; Amiloride; Vitamin D; Calcitriol; Epithelial Sodium Channels; Lung; Vitamins; Vitamin D Deficiency; RNA, Messenger
PubMed: 38145296
DOI: 10.1152/ajpcell.00369.2023 -
Heart (British Cardiac Society) Dec 2023Resistant hypertension is a condition where blood pressure levels remain elevated above target despite changes in lifestyle and concurrent use of at least three...
Resistant hypertension is a condition where blood pressure levels remain elevated above target despite changes in lifestyle and concurrent use of at least three antihypertensive agents, including a long-acting calcium channel blocker (CCB), a blocker of the renin-angiotensin system (ACE inhibitor or angiotensin receptor blocker) and a diuretic. To be diagnosed as resistant hypertension, maintaining adherence to therapy is required along with confirmation of blood pressure levels above target by out-of-office blood pressure measurements and exclusion of secondary causes of hypertension. The key management points of this condition include lifestyle changes such as reduced sodium and alcohol intake, regular physical activity, weight loss and discontinuation of substances that can interfere with blood pressure control. It is also recommended that current treatment be rationalised, including single pill combination treatment where antihypertensive drugs should be provided at the maximum tolerated dose. It is further recommended that current drugs be replaced with a more appropriate and less difficult treatment regimen based on the patient's age, ethnicity, comorbidities and risk of drug-drug interactions. The fourth line of treatment for patients with resistant hypertension should include mineralocorticoid receptor antagonists such as spironolactone, as demonstrated in the PATHWAY-2 trial and meta-analyses. Alternatives to spironolactone include amiloride, doxazosin, eplerenone, clonidine and beta-blockers, as well as any other antihypertensive drugs not already in use. New approaches under research are selective non-steroidal mineralocorticoid receptor antagonists such as finerenone, esaxerenone and ocedurenone, selective aldosterone synthase inhibitors such as baxdrostat, and dual endothelin antagonist aprocitentan.
PubMed: 38135468
DOI: 10.1136/heartjnl-2022-321730 -
Journal of Neurophysiology Jan 2024Previous work has shown that activation of tiger salamander retinal radial glial cells by extracellular ATP induces a pronounced extracellular acidification, which has...
Previous work has shown that activation of tiger salamander retinal radial glial cells by extracellular ATP induces a pronounced extracellular acidification, which has been proposed to be a potent modulator of neurotransmitter release. This study demonstrates that low micromolar concentrations of extracellular ATP similarly induce significant H effluxes from Müller cells isolated from the axolotl retina. Müller cells were enzymatically isolated from axolotl retina and H fluxes were measured from individual cells using self-referencing H-selective microelectrodes. The increased H efflux from axolotl Müller cells induced by extracellular ATP required activation of metabotropic purinergic receptors and was dependent upon calcium released from internal stores. We further found that the ATP-evoked increase in H efflux from Müller cells of both tiger salamander and axolotl were sensitive to pharmacological agents known to interrupt calmodulin and protein kinase C (PKC) activity: chlorpromazine (CLP), trifluoperazine (TFP), and W-7 (all calmodulin inhibitors) and chelerythrine, a PKC inhibitor, all attenuated ATP-elicited increases in H efflux. ATP-initiated H fluxes of axolotl Müller cells were also significantly reduced by amiloride, suggesting a significant contribution by sodium-hydrogen exchangers (NHEs). In addition, α-cyano-4-hydroxycinnamate (4-cin), a monocarboxylate transport (MCT) inhibitor, also reduced the ATP-induced increase in H efflux in both axolotl and tiger salamander Müller cells, and when combined with amiloride, abolished ATP-evoked increase in H efflux. These data suggest that axolotl Müller cells are likely to be an excellent model system to understand the cell-signaling pathways regulating H release from glia and the role this may play in modulating neuronal signaling. Glial cells are a key structural part of the tripartite synapse and have been suggested to regulate synaptic transmission, but the regulatory mechanisms remain unclear. We show that extracellular ATP, a potent glial cell activator, induces H efflux from axolotl retinal Müller (glial) cells through a calcium-dependent pathway that is likely to involve calmodulin, PKC, Na/H exchange, and monocarboxylate transport, and suggest that such H release may play a key role in modulating neuronal transmission.
Topics: Animals; Ependymoglial Cells; Ambystoma mexicanum; Calmodulin; Calcium; Amiloride; Adenosine Triphosphate; Neuroglia; Retina
PubMed: 38116604
DOI: 10.1152/jn.00321.2023