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Frontiers in Plant Science 2023Salt stress affects plant growth and development, resulting in the loss of crop yield across the world, and sodium-proton antiporters (NHXs) are one of the genes known...
Salt stress affects plant growth and development, resulting in the loss of crop yield across the world, and sodium-proton antiporters (NHXs) are one of the genes known to promote salt tolerance in transgenic plants. In this study, we conducted a comprehensive genome-wide analysis and expression profile of genes in wheat under salinity stress. We identified 30 genes in wheat based on the Na/H exchanger domain, with all genes containing an amiloride motif except one, a known for inhibiting Na ions in plants. Phylogenetic analysis classified these genes into three classes with subfamilies: 12 were localized in vacuoles, while 18 were in the endoplasmic reticulum and plasma membrane. Promoter analysis revealed stress-related -acting elements, indicating their potential role in abiotic stress tolerance. The non-synonymous (K)/synonymous (K) ratios highlighted that the majority of genes experienced robust purifying selection throughout their evolutionary history. Transcriptomis data analysis and qRT-PCR demonstrated distinct expression patterns for genes across various tissues when subjected to salt stress. Additionally, we predicted 20 different miRNA candidates targeting the identified genes. Protein-protein interaction prediction revealed NHX6's involvement in the SOS1 pathway, while gene exhibit proton antiporter activity. Molecular dynamics (MD) simulations were also conducted to examine the interactions of , , and . These results represent a significant advancement in our understanding of the molecular mechanisms governing Na transporters. This may also offer promising avenues for future studies aimed at unraveling the intricate details of their biological roles and applications.
PubMed: 38111881
DOI: 10.3389/fpls.2023.1266699 -
BioRxiv : the Preprint Server For... Dec 2023N-methyl-D-aspartate receptor (NMDAR) are amino acid receptors that are well studied in brain physiology; however, their role in kidney is poorly understood....
BACKGROUND
N-methyl-D-aspartate receptor (NMDAR) are amino acid receptors that are well studied in brain physiology; however, their role in kidney is poorly understood. Nonetheless, NMDAR inhibitors can increase serum K+ and reduce GFR, which suggests they have an important physiological role in the kidney. We hypothesized that NMDARs in the distal nephron induce afferent-arteriole vasodilation through the vasodilator mechanism connecting-tubule-glomerular feedback (CNTGF) that involves ENaC activation.
METHODS AND RESULTS
Using a tubule-specific transcriptome database combined with molecular biology and microscopy techniques, we showed kidney expression of NMDAR subunits along the nephron and specifically in ENaC-positive cells. This receptor is expressed in both male and female mice, with higher abundance in females (p=0.02). Microperfusing NMDAR agonists into the connecting tubule induced afferent-arteriole vasodilation (EC 10.7 vs. 24.5 mM; p<0.001) that was blunted or eliminated with the use of NMDAR blocker MK-801 or with the ENaC inhibitor Benzamil, indicating a dependence on CNTGF of the NMDAR-induced vasodilation. In vivo, we confirmed this CNTGF-associated vasodilation using kidney micropuncture (Stop-flow pressure 37.9±2.6 vs. 28.6±1.9 mmHg, NMDAR agonist vs vehicle; p<0.01). We explored NMDAR and ENaC channel interaction by using mpkCCD cells and split-open connecting tubules. We observed increased amiloride-sensitive current following NMDAR activation that was prevented by MK-801 (1.14 vs. 0.4 μAmp; p=0.03). In split-open tubules, NMDAR activation increased ENaC activity (Npo Vehicle vs. NMDA; p=0.04).
CONCLUSION
NMDARs are expressed along the nephron, including ENaC-positive cells, with higher expression in females. Epithelial NMDAR mediates renal vasodilation through the connecting-tubule-glomerular feedback, by increasing ENaC activity.
PubMed: 38106229
DOI: 10.1101/2023.12.04.569973 -
Signal Transduction and Targeted Therapy Dec 2023Metastasis is the leading cause of cancer-related deaths. Transforming growth factor beta (TGF-β) signaling drives metastasis and is strongly enhanced during cancer...
Metastasis is the leading cause of cancer-related deaths. Transforming growth factor beta (TGF-β) signaling drives metastasis and is strongly enhanced during cancer progression. Yet, the use of on-target TGF-β signaling inhibitors in the treatment of cancer patients remains unsuccessful, highlighting a gap in the understanding of TGF-β biology that limits the establishment of efficient anti-metastatic therapies. Here, we show that TGF-β signaling hyperactivation in breast cancer cells is required for metastasis and relies on increased small extracellular vesicle (sEV) secretion. Demonstrating sEV's unique role, TGF-β signaling levels induced by sEVs exceed the activity of matching concentrations of soluble ligand TGF-β. Further, genetic disruption of sEV secretion in highly-metastatic breast cancer cells impairs cancer cell aggressiveness by reducing TGF-β signaling to nearly-normal levels. Otherwise, TGF-β signaling activity in non-invasive breast cancer cells is inherently low, but can be amplified by sEVs, enabling invasion and metastasis of poorly-metastatic breast cancer cells. Underscoring the translational potential of inhibiting sEV trafficking in advanced breast cancers, treatment with dimethyl amiloride (DMA) decreases sEV secretion, TGF-β signaling activity, and breast cancer progression in vivo. Targeting both the sEV trafficking and TGF-β signaling by combining DMA and SB431542 at suboptimal doses potentiated this effect, normalizing the TGF-β signaling in primary tumors to potently reduce circulating tumor cells, metastasis, and tumor self-seeding. Collectively, this study establishes sEVs as critical elements in TGF-β biology, demonstrating the feasibility of inhibiting sEV trafficking as a new therapeutic approach to impair metastasis by normalizing TGF-β signaling levels in breast cancer cells.
Topics: Humans; Female; Cell Line, Tumor; Transforming Growth Factor beta; Breast Neoplasms; Receptors, Transforming Growth Factor beta; Extracellular Vesicles
PubMed: 38105247
DOI: 10.1038/s41392-023-01711-1 -
Molecular Medicine Reports Feb 2024Liddle syndrome is an autosomal dominant form of monogenic hypertension that is caused by mutations in , or , which respectively encode the α, β and γ subunits of...
Liddle syndrome is an autosomal dominant form of monogenic hypertension that is caused by mutations in , or , which respectively encode the α, β and γ subunits of the epithelial sodium channel. In the present study, DNA was extracted from leukocytes in peripheral blood obtained from all members of a family with Liddle syndrome. Whole‑exome sequencing and Sanger sequencing were performed to assess the candidate variant and a co‑segregation analysis was conducted. A frameshift mutation in (NM_ 000336: c.1806dupG, p.Pro603Alafs*5) in the family was identified, characterized by early‑onset hypertension and hypokalemia. The mutation led to the truncation of the β subunit of the epithelial sodium channel and a lack of the conservative PY motif. Furthermore, a systematic review of follow‑up data from patients with Liddle syndrome with mutations was performed. The follow‑up data of 108 patients with pathogenic mutations from 47 families were summarized. Phenotypic heterogeneity was evident in patients with Liddle syndrome and early‑onset hypertension was the most frequent symptom. Patients responded well to targeted amiloride therapy with significant improvements in blood pressure and serum potassium concentration. The present study demonstrates that confirmatory genetic testing and targeted therapy can prevent premature onset of clinical endpoint events in patients with Liddle syndrome.
Topics: Humans; Liddle Syndrome; Epithelial Sodium Channels; Frameshift Mutation; Mutation; Hypertension; Potassium
PubMed: 38099339
DOI: 10.3892/mmr.2023.13142 -
Molecules (Basel, Switzerland) Nov 2023This study was undertaken to investigate the interaction between the sodium channel blocker amiloride (AML) and human serum albumin (HSA). A combination of...
This study was undertaken to investigate the interaction between the sodium channel blocker amiloride (AML) and human serum albumin (HSA). A combination of multi-spectroscopic techniques and computational methods were employed to identify the AML binding site on HSA and the forces responsible for the formation of the HSA-AML complex. Our findings revealed that AML specifically binds to Sudlow's site II, located in subdomain IIIA of HSA, and that the complex formed is stabilized using van der Waals hydrogen-bonding and hydrophobic interactions. FRET analysis showed that the distance between AML and Trp214 was optimal for efficient quenching. UV-Vis spectroscopy and circular dichroism indicated minor changes in the structure of HSA after AML binding, and molecular dynamics simulations (MDS) conducted over 100 ns provided additional evidence of stable HSA-AML-complex formation. This study enhances understanding of the interaction between AML and HSA and the mechanism responsible.
Topics: Humans; Serum Albumin, Human; Molecular Docking Simulation; Amiloride; Protein Binding; Binding Sites; Circular Dichroism; Thermodynamics; Leukemia, Myeloid, Acute; Spectrometry, Fluorescence
PubMed: 38067419
DOI: 10.3390/molecules28237688 -
Frontiers in Oncology 2023Multiple myeloma (MM) is an incurable cancer of malignant plasma cells that engraft in the bone marrow (BM). It is more than likely that the poorly investigated physical...
INTRODUCTION
Multiple myeloma (MM) is an incurable cancer of malignant plasma cells that engraft in the bone marrow (BM). It is more than likely that the poorly investigated physical parameters of hypoxia and pH in the tumor microenvironment (TME) is critical for MM survival. Here, we explore the effects of a hypoxic environment on pH regulation and its role in MM survival.
METHODS
We used in vitro models of MM, in which the culturing medium was modified to specific pH and pO2 levels and then measured the effects on cell survival that was correlated with changes in intracellular (pHi) and extracellular pH (pHe). In a MM xenograft model, we used PET/CT to study hypoxia-mediated effects on tumor growth.
RESULTS
Hypoxia-mediated apoptosis of MM cells is correlated with acidic intracellular pHi (less than < 6.6) that is dependent on HIF activity. Using a polyamide HIF responsive element binding compound, a carbonic anhydrase inhibitor (acetazolamide), and an NHE-1 inhibitor (amiloride) acidified the pHi and lead to cell death. In contrast, treatment of cells with an alkalization agent, Na-lactate, rescued these cells by increasing the pHi (pH > 6.6). Finally, treatment of mice with acetazolamide decreased cell growth in the tumor nodules.
DISCUSSION
Targeting hypoxia and HIF have been proposed as an anti-tumor therapy but the clinical efficacy of such strategies are modest. We propose that targeting the pHi may be more effective at treating cancers within a hypoxic TME.
PubMed: 38023253
DOI: 10.3389/fonc.2023.1268421 -
BioRxiv : the Preprint Server For... Nov 2023Mitochondrial ion channels are essential for energy production and cell survival. To avoid depleting the electrochemical gradient used for ATP synthesis, channels so far...
Mitochondrial ion channels are essential for energy production and cell survival. To avoid depleting the electrochemical gradient used for ATP synthesis, channels so far described in the mitochondrial inner membrane open only briefly, are highly ion-selective, have restricted tissue distributions, or have small currents. Here, we identify a mitochondrial inner membrane conductance that has strikingly different behavior from previously described channels. It is expressed ubiquitously, and transports cations non-selectively, producing a large, up to nanoampere-level, current. The channel does not lead to inner membrane uncoupling during normal physiology because it only becomes active at depolarized voltages. It is inhibited by external Ca, corresponding to the intermembrane space, as well as amiloride. This large, ubiquitous, non-selective, amiloride-sensitive (LUNA) current appears most active when expression of the mitochondrial calcium uniporter is minimal, such as in the heart. In this organ, we find that LUNA current magnitude increases two- to threefold in multiple mouse models of injury, an effect also seen in cardiac mitochondria from human patients with heart failure with reduced ejection fraction. Taken together, these features lead us to speculate that LUNA current may arise from an essential protein that acts as a transporter under physiological conditions, but becomes a channel under conditions of mitochondrial stress and depolarization.
PubMed: 38014208
DOI: 10.1101/2023.11.15.567241 -
Metabolites Nov 2023Proton transporters play a key role in maintaining the acidic tumor microenvironment; hence, their inhibition has been proposed as a new therapeutic treatment, although...
Assessing the Therapeutic Efficacy of Proton Transport Inhibitors in a Triple-Negative Breast Cancer Murine Model with Magnetic Resonance Imaging-Chemical Exchange Saturation Transfer Tumor pH Imaging.
Proton transporters play a key role in maintaining the acidic tumor microenvironment; hence, their inhibition has been proposed as a new therapeutic treatment, although few methods can accurately assess their effect in vivo. In this study, we investigated whether MRI-CEST (Magnetic Resonance Imaging-Chemical Exchange Saturation Transfer) tumor pH imaging can be a useful tool to evaluate in vivo the therapeutic efficacy of several Proton Pump Inhibitors (PPIs) in breast cancer. Cell viability and extracellular pH assays were carried out in breast cancer cells cultured at physiological pH (7.4) or acid-adapted (pH of 6.5 and 6.8) following the exposure to inhibitors of V-ATPase (Lansoprazole, Esomeprazole) or NHE1 (Amiloride, Cariporide) at several concentrations. Next, triple-negative breast cancer 4T1 tumor-bearing mice were treated with Lansoprazole or Amiloride and MRI-CEST tumor pH imaging was utilized to assess the in vivo efficacy. Only Lansoprazole induced, in addition to breast cancer cell toxicity, a significant inhibition of proton extrusion. A significant reduction in tumor volume, prolonged survival, and increase in extracellular tumor pH after 1 and 2 weeks were observed after Lansoprazole treatment, whereas no significant changes were detected upon Amiloride treatment. Our results suggested that MRI-CEST tumor pH imaging can monitor the therapeutic efficacy of PPIs in breast cancer murine models.
PubMed: 37999256
DOI: 10.3390/metabo13111161 -
American Journal of Cancer Research 2023The alkaline intracellular environment of cancer cells is critical for cell proliferation and controlled by various plasma membrane transporters including Na+/H+...
Inhibition of Na+/H+ exchanger (NHE) 7 by 5-(N-ethyl-N-isopropyl)-Amiloride displays anti-cancer activity in non-small cell lung cancer by disrupting cancer stem cell activity and downregulating PD-L1 expression.
The alkaline intracellular environment of cancer cells is critical for cell proliferation and controlled by various plasma membrane transporters including Na+/H+ exchangers (NHEs). NHEs can also mediate cell behavior by regulating signaling transduction. In this study, we investigated the role of NHE7 in cancer stem cell (CSC) activity in non-small cell lung cancer (NSCLC) cells and the potential therapeutic implications of targeting NHE7 and the associated immune checkpoint molecule PD-L1. By analyzing the database from The Cancer Genome Atlas, we found a positive correlation between mRNA levels (the gene encoding NHE7) and poor overall survival in lung adenocarcinoma patients. Using 5-(N-ethyl-N-isopropyl)-Amiloride (EIPA) to inhibit NHE7 activity, we observed disrupted cell cycle progression and suppressed NSCLC cell proliferation without inducing apoptosis. Furthermore, EIPA demonstrated a suppressive effect on CSC activity, evidenced by decreased tumorsphere numbers and inhibition of CSC markers such as ALDH1A2, ABCG2, CD44, and CD133. Flow cytometric analysis revealed that EIPA treatment or NHE7 knockdown in NSCLC cells led to downregulated PD-L1 expression, associated with inhibited STAT3 activity. Interestingly, EIPA's CSC-targeting activity was preferentially observed in NSCLC cells overexpressing BMI1, while increased PD-L1 expression was detected in BMI1-overexpressing NSCLC cells. Our findings suggest that targeting NHE7 with inhibitors like EIPA may have therapeutic potential in NSCLC treatment by disrupting cell cycle progression and suppressing CSC activity. The observed increase in PD-L1 expression in BMI1-overexpressing NSCLC cells upon EIPA treatment highlights the potential benefit of combining NHE7 inhibitors with anti-PD-L1 agents as a promising new therapeutic strategy for NSCLC.
PubMed: 37970357
DOI: No ID Found -
Plant Direct Nov 2023Plant / () genes enhance salt tolerance by preventing excessive Na accumulation in the cytosol through partitioning of Na ions into vacuoles or extracellular...
Plant / () genes enhance salt tolerance by preventing excessive Na accumulation in the cytosol through partitioning of Na ions into vacuoles or extracellular transport across the plasma membrane. However, there is limited detailed information regarding the salt stress responsive s in the most recent tomato genome. We investigated the role of this gene family's expression patterns in the open flower tissues under salt shock in using a genome-wide approach. A total of seven putative genes located on chromosomes 1, 4, 6, and 10 were identified, but no ortholog of the gene was identified in the tomato genome. Phylogenetic analysis revealed that these genes are divided into three different groups. SlNHX proteins with 10-12 transmembrane domains were hypothetically localized in vacuoles or cell membranes. Promoter analysis revealed that and are involved with the stress-related MeJA hormone in response to salt stress signaling. The structural motif analysis of SlNHX1, -2, -3, -4, and -6 proteins showed that they have highly conserved amiloride binding sites. The protein-protein network revealed that SlNHX7 and SlNHX8 interact physically with Salt Overly Sensitive (SOS) pathway proteins. Transcriptome analysis demonstrated that the and genes were substantially expressed in the open flower tissues. Moreover, quantitative PCR analysis indicated that all genes, particularly and , are significantly upregulated by salt shock in the open flower tissues. Our results provide an updated framework for future genetic research and development of breeding strategies against salt stress in the tomato.
PubMed: 37965196
DOI: 10.1002/pld3.543