NHE Inhibitor Research Articles

Inhibition of sodium/hydrogen exchanger 3 in the gastrointestinal tract by tenapanor reduces paracellular phosphate permeability.

Hyperphosphatemia is common in patients with chronic kidney disease and is increasingly associated with poor clinical outcomes. Current management of hyperphosphatemia with dietary restriction and oral phosphate binders often proves inadequate. Tenapanor, a minimally absorbed, small-molecule inhibitor of the sodium/hydrogen exchanger isoform 3 (NHE3), acts locally in the gastrointestinal tract to inhibit sodium absorption. Because tenapanor also reduces intestinal phosphate absorption, it may have potential as a therapy for hyperphosphatemia. We investigated the mechanism by which tenapanor reduces gastrointestinal phosphate uptake, using in vivo studies in rodents and translational experiments on human small intestinal stem cell-derived enteroid monolayers to model ion transport physiology. We found that tenapanor produces its effect by modulating tight junctions, which increases transepithelial electrical resistance (TEER) and reduces permeability to phosphate, reducing paracellular phosphate absorption. NHE3-deficient monolayers mimicked the phosphate phenotype of tenapanor treatment, and tenapanor did not affect TEER or phosphate flux in the absence of NHE3. Tenapanor also prevents active transcellular phosphate absorption compensation by decreasing the expression of NaPi2b, the major active intestinal phosphate transporter. In healthy human volunteers, tenapanor (15 mg, given twice daily for 4 days) increased stool phosphorus and decreased urinary phosphorus excretion. We determined that tenapanor reduces intestinal phosphate absorption predominantly through reduction of passive paracellular phosphate flux, an effect mediated exclusively via on-target NHE3 inhibition.

Linaclotide improves gastrointestinal transit in cystic fibrosis mice by inhibiting sodium/hydrogen exchanger 3.

Gastrointestinal dysfunction in cystic fibrosis (CF) is a prominent source of pain among patients with CF. Linaclotide, a guanylate cyclase C (GCC) receptor agonist, is a US Food and Drug Administration-approved drug prescribed for chronic constipation but has not been widely used in CF, as the cystic fibrosis transmembrane conductance regulator (CFTR) is the main mechanism of action. However, anecdotal clinical evidence suggests that linaclotide may be effective for treating some gastrointestinal symptoms in CF. The goal of this study was to determine the effectiveness and mechanism of linaclotide in treating CF gastrointestinal disorders using CF mouse models. Intestinal transit, chloride secretion, and intestinal lumen fluidity were assessed in wild-type and CF mouse models in response to linaclotide. CFTR and sodium/hydrogen exchanger 3 (NHE3) response to linaclotide was also evaluated. Linaclotide treatment improved intestinal transit in mice carrying either F508del or null Cftr mutations but did not induce detectable Cl- secretion. Linaclotide increased fluid retention and fluidity of CF intestinal contents, suggesting inhibition of fluid absorption. Targeted inhibition of sodium absorption by the NHE3 inhibitor tenapanor produced improvements in gastrointestinal transit similar to those produced by linaclotide treatment, suggesting that inhibition of fluid absorption by linaclotide contributes to improved gastrointestinal transit in CF. Our results demonstrate that linaclotide improves gastrointestinal transit in CF mouse models by increasing luminal fluidity through inhibiting NHE3-mediated sodium absorption. Further studies are necessary to assess whether linaclotide could improve CF intestinal pathologies in patients. GCC signaling and NHE3 inhibition may be therapeutic targets for CF intestinal manifestations. NEW & NOTEWORTHY Linaclotide's primary mechanism of action in alleviating chronic constipation is through cystic fibrosis transmembrane conductance regulator (CFTR), negating its use in patients with cystic fibrosis (CF). For the first time, our findings suggest that in the absence of CFTR, linaclotide can improve fluidity of the intestinal lumen through the inhibition of sodium/hydrogen exchanger 3. These findings suggest that linaclotide could improve CF intestinal pathologies in patients.

Intracellular acidosis via activation of Akt-Girdin signaling promotes post ischemic angiogenesis during hyperglycemia

AimsThe impaired angiogenesis is the major cause of diabetic delayed wound healing. The molecular insight remains unknown. Previous study has shown that high glucose (HG) activates Na+/H+ exchanger 1 (NHE1) and induces intracellular alkalinization, resulting in endothelial dysfunction. The aim of this study is to investigate whether activation of NHE1 in endothelial cells by HG damages the angiogenesis in vitro and in vivo. Methods and resultsWe used western blot to detect the phosphorylations of both Akt and Girdin, and pH-sensitive BCECF fluorescence to assay NHE1 activity and pHi value, respectively. The angiogenesis was evaluated by measuring the number of tube formation in vitro, and blood perfusion by laser doppler and neovascularization by staining CD31 in vivo. Our results indicated that induction of intracellular acidosis (IA) increased p-Akt and p-Girdin in human umbilical vein endothelial cells (HUVEC). HG activated NHE1 and increased pHi value in a time-dependent manner, associated with the decreased phosphorylations of both Akt and Gridin, while inhibition of NHE1 by amiloride abolished the HG-induced reductions of p-Akt and p-Girdin. However, silence of Akt by siRNA transfection or pharmacological inhibitors (wortmannin and LY294002) bypassed IA-induced Girdin phosphorylation. Overexpression of constitutively active Akt abolished HG-reduced Girdin phosphorylation. In addition, upregulation of Akt or inhibition of NHE1 remarkably attenuated HG-impaired tube formation in HUVEC. In vivo study revealed that amiloride dramatically rescued hyperglycemia-delayed blood perfusion and neovascularization by augmenting ischemia-induced angiogenesis. ConclusionIA promotes ischemia-induced angiogenesis via Akt-dependent Girdin phosphorylation in diabetic mice.

SLC26A3 inhibitor identified in small molecule screen blocks colonic fluid absorption and reduces constipation.

SLC26A3 (downregulated in adenoma; DRA) is a Cl-/anion exchanger expressed in the luminal membrane of intestinal epithelial cells, where it facilitates electroneutral NaCl absorption. SLC26A3 loss of function in humans or mice causes chloride-losing diarrhea. Here, we identified slc26a3 inhibitors in a screen of 50,000 synthetic small molecules done in Fischer rat thyroid (FRT) cells coexpressing slc26a3 and a genetically encoded halide sensor. Structure-activity relationship studies were done on the most potent inhibitor classes identified in the screen: 4,8-dimethylcoumarins and acetamide-thioimidazoles. The dimethylcoumarin DRAinh-A250 fully and reversibly inhibited slc26a3-mediated Cl- exchange with HCO3-, I-, and thiocyanate (SCN-), with an IC50 of ~0.2 μM. DRAinh-A250 did not inhibit the homologous anion exchangers slc26a4 (pendrin) or slc26a6 (PAT-1), nor did it alter activity of other related proteins or intestinal ion channels. In mice, intraluminal DRAinh-A250 blocked fluid absorption in closed colonic loops but not in jejunal loops, while the NHE3 (SLC9A3) inhibitor tenapanor blocked absorption only in the jejunum. Oral DRAinh-A250 and tenapanor comparably reduced signs of constipation in loperamide-treated mice, with additive effects found on coadministration. DRAinh-A250 was also effective in loperamide-treated cystic fibrosis mice. These studies support a major role of slc26a3 in colonic fluid absorption and suggest the therapeutic utility of SLC26A3 inhibition in constipation.

Role of sodium-hydrogen exchanger isoform 1 in regulating hepatocyte apoptosis induced by hyperammonaemia

BackgroundThe “secondary injury” theory of liver failure indicated that hyperammonaemia due to liver failure causes further deterioration of hepatocytes. Our previous studies have demonstrated that high blood ammonia levels may lead to hepatocyte apoptosis, as NH4Cl loading caused metabolic acidosis and an increase in sodium-hydrogen exchanger isoform 1 (NHE1). In this study, we established a hyperammonia hepatocyte model to determine the role of NHE1 in the regulation of hepatocyte apoptosis induced by NH4Cl. Materials and methodsIn current studies, intracellular pH (pHi) and NHE1 activity were analyzed using the pHi-sensitive dye BCECF-AM. The results showed that intracellular pH dropped and NHE1 activity increased in hepatocytes under NH4Cl treatment. As expected, decreased pHi induced by NH4Cl was associated with increased apoptosis, low cell proliferation and ATP depletion, which was exacerbated by exposure to the NHE1 inhibitor cariporide. We also found that NH4Cl treatment stimulated PI3K and Akt phosphorylation and this effect was considerably reduced by NHE1 inhibition. ConclusionThis study highlighted the significant role of NHE1 in the regulation of cell apoptosis induced by hyperammonaemia.

Empagliflozin effects on ischemic contracture and I/R injury in isolated mouse hearts perfused with or without insulin

Background: Activation of sodium hydrogen exchanger (NHE) contributes to cardiac ischemia reperfusion (I/R) injury. We recently reported NHE inhibition properties of the diabetic drug SGLT2 inhibitor empagliflozin (EMPA). Classic NHE inhibitors delay ischemic contracture and reduce I/R injury in the heart, dependent on the degree of ischemic glycogen depletion. We examined in normal-/low-glycogen hearts whether EMPA delays ischemic contracture and reduces I/R injury.

Alleviative effect of fucoxanthin-containing extract from brown seaweed Laminaria japonica on renal tubular cell apoptosis through upregulating Na+/H+ exchanger NHE1 in chronic kidney disease mice

Ethnopharmacological relevanceBrown seaweed is a common food for Asians, and the bioactive ingredient fucoxanthin exerts anti-apoptotic activities in several cell types. Renal tubular cell apoptosis is one of the common cellular events leading to renal fibrosis and chronic kidney disease (CKD). However, the influence of fucoxanthin-containing brown seaweed extract on CKD is still unknown. We intended to evaluate the inhibitory effect of fucoxanthin-containing extract from brown seaweed on renal apoptosis under CKD condition and its molecular mechanism. Materials and methodsThe fucoxanthin-containing brown seaweed extract (LJE) was prepared from Laminaria japonica. We investigated how LJE influences on both doxorubicin-treated rat renal tubular cells (NRK-52E) and the renal symptoms of nephrectomy-induced CKD mice. ResultsLJE inhibited doxorubicin-induced apoptosis and upregulated Na+/H+ exchanger isoform 1 (NHE1) expression in NRK-52E cells, which were blocked by the NHE1 inhibitor cariporide. LJE also upregulated peroxisome proliferator-activated receptor alpha (PPARα). PPARα siRNA transfection inhibited LJE-induced NHE1 expression and anti-apoptotic effect. In CKD mice, LJE increased NHE1 expression in renal tubules and reduced apoptotic renal tubular cells, but not in PPARα knockout mice. The inhibitory effect of LJE on apoptosis also reduced renal tubulointerstitial fibrosis and improved renal function in CKD mice. ConclusionWe demonstrated that LJE inhibits renal apoptosis via NHE1 upregulation. The anti-apoptotic effect of LJE also improves renal function in CKD mice. Therefore, fucoxanthin-containing brown seaweed may have a therapeutic potential for CKD patients.

Sodium Transporters Are Involved in Lithium Influx in Brain Endothelial Cells.

Variability in drug response to lithium (Li+) is poorly understood and significant, as only 40% of patients with bipolar disorder highly respond to Li+. Li+ can be transported by sodium (Na+) transporters in kidney tubules or red blood cells, but its transport has not been investigated at the blood-brain barrier (BBB). Inhibition and/or transcriptomic strategies for Na+ transporters such as NHE (SLC9), NBC (SLC4), and NKCC (SLC12) were used to assess their role on Li+ transport in human brain endothelial cells. Na+-free buffer was also used to examine Na+/Li+ countertransport (NLCT) activity. The BBB permeability of Li+ evaluated in the rat was 2% that of diazepam, a high passive diffusion lipophilic compound. Gene expression of several Na+ transporters was determined in hCMEC/D3 cells, human hematopoietic stem-cell-derived BBB models (HBLEC), and human primary brain microvascular endothelial cells (hPBMECs) and showed the following rank order with close expression profile: NHE1 > NKCC1 > NHE5 > NBCn1, while NHE2-4, NBCn2, and NBCe1-2 were barely detected. Li+ influx in hCMEC/D3 cells was increased in Na+-free buffer by 3.3-fold, while depletion of chloride or bicarbonate had no effect. DMA (NHE inhibitor), DIDS (anionic carriers inhibitor), and bumetanide (NKCC inhibitor) decreased Li+ uptake significantly in hCMEC/D3 by 52, 51, and 47%, respectively, while S0859 (NBC inhibitor) increased Li+ influx 2.3-fold. Zoniporide (NHE1 inhibitor) and siRNA against NHE1 had no effect on Li+ influx in hCMEC/D3 cells. Our study shows that NHE1 and/or NHE5, NBCn1, and NKCC1 may play a significant role in the transport of Li+ through the plasma membrane of brain endothelial cells.

Can Less Be More: Evaluating the Synergistic Relationship between Chemotherapy Agents and NHE1 Inhibitors in Ovarian Cancer Cells

Ovarian cancer is the seventh most prevalent cancer found in women, resulting in over 200,000 new cases and close to 140,000 deaths annually. In order to combat the risk of mortality associated with this disease, extensive chemotherapy regimes are employed. These measures often include the use of therapeutic agents such as paclitaxel, rucaparib, olaparib, and cisplatin. The mechanisms of action for each of these oncogenic therapeutics differs substantially. Paclitaxel stabilizes microtubule polymerization, effectively preventing mitotic completion. Rucaparib and olaparib both inhibit PARP, an enzyme associated with repairing damaged DNA. Cisplatin also hinders DNA repair. However, it does so by crosslinking the purine bases on DNA to promote cellular apoptosis. Although the use of such chemotherapy agents has been instrumental in reducing cancer‐related deaths, the dosage intensity of these cytostatics can procure adverse side effects in the form of hair loss, anemia, nausea, and vomiting. Because of this reality, it has become of interest to investigate methodologies for reducing the required chemotherapy drug dosage, while still maintaining the same rate of cancer cell death. The Na+‐H+ Exchanger Isoform 1 (NHE1) is a plasma membrane protein that exchanges 1 extracellular Na+ for 1 intracellular H+, thus playing a primary role in pH regulation. It is hypothesized that such a synergistic effect can be produced through the pairing of chemotherapy agents with NHE1 inhibitors. In cancer cells, NHE1 is often hyperactivated leading to the development of a pH inversion. This dysregulation contributes to increased cancer cell proliferation, migration and invasion. Inhibition of this transport protein typically results in intracellular acidification, a mechanism that supports the progression of apoptosis. Applying this inhibition, with an accompanying cytostatic, has thus interested researchers as a possible therapeutic approach towards mitigating neoplastic development. The potential of these drug‐inhibitor pairs will be measured primarily via flow cytometry. Through the addition of a fluorescent dye, this instrument is able to differentiate viable cells from compromised cells within a single population. Such analysis allows for a quantitative assessment of each inhibitor‐drug combination.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Empagliflozin Inhibits NHE3 Activity in the Proximal Tubule of Normotensive and Hypertensive Rats

BackgroundA growing body of evidence suggests an interplay between extracellular volume and glucose homeostasis. NHE3 is the main pathway for Na reabsorption in the proximal tubule (PT). The sodium glucose cotransporter SGLT2 reabsorbs most of the glucose filtered by the kidneys and colocalizes with NHE3 in the apical membrane of PT. Thus, we tested the hypothesis that SGLT2 and NHE3 functionally interact. Given that SGLT2 inhibitors confer blood‐pressure lowering effects in T2DM patients, we also tested the hypothesis that the antihypertensive effects of SGLT2 inhibitors may be associated with PT NHE3 inhibition.MethodsThe acute and long‐term effects of the SGLT2 inhibitor empagliflozin on PT NHE3 activity were determined by in vivo stationary microperfusion in fourteen‐week‐old male Wistar rats and SHR‐treated rats. Additionally, twelve‐week‐old SHRs were treated with empagliflozin (10 mg/kg/day) or vehicle (control) for two weeks. Tail‐cuff blood pressure and renal function were measured before (baseline) and after treatment (post‐treatment). NHE3 expression and phosphorylation levels were evaluated by immunoblotting.ResultsEmpagliflozin acutely inhibited PT NHE3 activity in both Wistar (1.73 ± 0.07 vs. 2.55 ± 0.14 nmol/cm2.s, P < 0.001) and SHR (0.82 ± 0.07 vs. 1.21 ± 0.10 nmol/cm2.s, P < 0.01). Blood pressure (BP) of empagliflozin‐treated SHR was lower than baseline levels (176 ± 4 vs. 194 ± 4 mm Hg, P < 0.01) whereas no change in BP was observed in vehicle‐treated SHR (194 ± 6 vs. 192 ± 5 mm Hg). Compared to baseline, empagliflozin‐treatment enhanced cumulative urinary flow, calcium, sodium and glucose excretion whereas in vehicle‐treated SHR these parameters remained unchanged. Lower BP in empagliflozin‐treated SHR was associated with lower PT NHE3 activity (0.67 ± 0.10 vs. 1.18 ± 0.10 nmol/cm2.s, P < 0.001). No differences were observed on renal cortical NHE3 protein expression or NHE3 phosphorylation levels between the two groups of rats.ConclusionThese results suggest that NHE3 and SGLT2 functionally interact in the PT. Inhibition of NHE3 by SGLT2 inhibitors may underlie, at least in part, the antihypertensive effect of empagliflozin.Support or Funding InformationFAPESPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Empagliflozin effects on ischemic contracture and I/R injury in isolated mouse hearts perfused with or without insulin

IntroductionThe kidney‐targeted Empagliflozin (EMPA) has shown large unexpected cardiovascular benefits, which cannot be explained by general cardiovascular risk factors. We recently reported that EMPA has sodium hydrogen exchanger (NHE) inhibition properties in isolated cardiomyocytes. Classic NHE inhibitors such as Cariporide (CARI) delay ischemic contracture onset and reduce ischemia reperfusion (I/R) injury in the heart. Here we examined whether EMPA delays contracture onset and reduces I/R injury. Both normal‐ and low‐glycogen hearts were used, because the rate of glycogen depletion during ischemia affects contracture onset and IR injury.Methods/ResultsIsolated mouse hearts were perfused with and without 50 mU/L insulin, to create normal‐ and low‐glycogen hearts respectively, and subjected to 25 min I and 120 min R. 1μM EMPA, 10μM cariporide (CARI; positive control) or vehicle was administered prior to ischemia until 10 minutes post‐reperfusion. In normal‐glycogen hearts, EMPA and CARI did not change ischemic contracture (in seconds, vehicle 1056±56, EMPA 900±44, CARI 880±84) and IR injury (infarct size (%): vehicle 59±6, Empa 62±10, Cari 68±4), however in low‐glycogen hearts, both EMPA and CARI delayed ischemic contracture onset (vehicle 459±25, EMPA 559±22, CARI 588±31). Higher EMPA concentration further delayed contracture onset. Only CARI reduced I/R injury (from 51±6 to 34±5). The presence of insulin (same concentration used) did not alter NHE activity, nor the ability of CARI or EMPA to inhibit NHE, in isolated cardiomyocytes. The increased IR injury in normal‐glycogen hearts was associated with increased end‐ischemic lactate and glucose‐6‐phosphate accumulation, and consequently decreased mitochondrial hexokinase ll.ConclusionEMPA does not protect against I/R injury in isolated hearts, yet EMPA delayed ischemic contracture onset in low‐glycogen hearts, suggesting a direct cardiac, ATP preserving, effect of EMPA during ischemia. Cardiac glycogen content interferes with the effects of NHE inhibitors on I/R injury in isolated hearts. These data indicate that empagliflozin and cariporide may inhibit NHE in different manners.Support or Funding InformationNone.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rho Kinase and Na+/H+ Exchanger Mediate Endothelin‐1 Induced Pulmonary Arterial Smooth Muscle Cell Proliferation and Migration

ObjectiveRemodeling of the pulmonary vasculature, involving increased proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), is a key component of the pathology underlying pulmonary hypertension (PH). Endothelin‐1 (ET‐1) is known to be an important contributor to both pulmonary vasoconstriction and remodeling in PH. We aimed to determine whether ET‐1 modulates PASMC function via a pathway involving Rho kinase (ROCK) and the Na+/H+ exchanger (NHE).MethodsPASMCs were isolated from distal pulmonary arteries of male C57/B6 mice as well as male Wistar rats and expanded in culture. To measure NHE activity, cells were incubated with pH‐sensitive fluorescent dye and the Na+‐dependent recovery of intracellular pH (pHi) following acidification by ammonium pulse challenge was measured via fluorescence microscopy. NHE1 mRNA expression was measured via qRT‐PCR and protein expression was measured via immunoblot. Proliferation was assessed via ELISA for BrdU incorporation over 24 hr. Migration was measured over 24 hr using Transwell filters with 8 μm pores. Cells were treated with 10−8 M ET‐1 versus vehicle for 24–48 hr. NHE was pharmacologically inhibited with 10 μM ethyl‐isopropyl amiloride. ROCK was inhibited with 10 μM Y‐27632.ResultsET‐1 treatment, compared to vehicle, resulted in increased resting pHi and NHE activity in PASMCs. ET‐1 did not result in increased NHE1 mRNA or protein expression. ROCK inhibition prevented ET‐1‐induced NHE activation. PASMC proliferation and migration were both increased by ET‐1 treatment, compared to vehicle. ROCK inhibition attenuated PASMC proliferation and migration in the presence of ET‐1. Similarly, NHE inhibition also attenuated ET‐1‐induced PASMC proliferation and migration.ConclusionsET‐1 results in increased PASMC proliferation and migration via a pathway involving ROCK and NHE activation. This suggests a novel pathway which may contribute to vascular remodeling in PH.Support or Funding InformationNIH K08HL133475 & R01HL073859This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evaluating the Inhibition of Palmitoylation and NHE1 on Cell Proliferation and Migration

The Na+‐ H+ Exchanger Isoform I (NHE1) is a key regulator of cell proliferation and migration in a range of cell types from both healthy and diseased tissues. Activation of NHE1 is an initial step in the development of the transformed phenotypes of cancer cells and therefore a key driver for tumor development and cancer progression. NHE1 also contributes to two of the hallmarks of cancer, the activation of invasion and metastasis and sustained proliferative growth. We have identified for the first time, that NHE1 undergoes reversible palmitoylation, the addition of a palmitic acid residue to the cytoplasmic regulatory domain of NHE1. We hypothesize that this process regulates NHE1 activation and will play a role in the control of cell proliferation and migration. This suggests that targeting the potential palmitoylation sites on NHE1 may provide a therapeutic target for cancer treatment. Our work characterizes the role of the palmitoylation of NHE1 in cell growth and migration. Using the palmitoylation inhibitor, 2‐bromopalmitate (2BP), we evaluated cell growth and migration in the presence and absence of palmitoylation. In proliferation assays, the rate of cell growth increased approximately 5‐fold when the serum present in the growth media was increased from 0.5% to 10% and approximately 2‐fold when 50 μM lysophosphatidic acid was added to the growth media. Under both conditions, the addition of 15 μM 2BP or 10 μM cariporide dramatically reduced or eliminated the increased proliferation. Cell migration was evaluated using Electric Cell‐substrate Impedance Sensing (ECIS). Once again, the rate of cell migration was reduced by 50% or more in the presence of 15 μM 2BP or 10 μM cariporide. These data demonstrated that inhibiting the palmitoylation of NHE1 decreases both cell proliferation and the rate of cell migration.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Investigating the Potential for Na+‐H+ Exchanger Isoform 1 (NHE1) Inhibitors as Adjuvant Therapies in the Treatment of Ovarian Cancer

Each year in the United States over 200,000 women are diagnosed with ovarian cancer leading to more than 130,000 deaths. A major challenge in ovarian cancer treatment is the development of drug resistance to the therapeutic agents. Increases in the expression and activity Na+‐H+ Exchanger Isoform 1 (NHE1) was recently demonstrated to regulate cell proliferation in ovarian cancer patients. NHE1 is an 815 amino acid transmembrane transport protein that regulates intracellular pH. We hypothesize that combining common ovarian cancer therapeutic agents along with compounds that directly or indirectly inhibit NHE1 will enhance the ability of the therapeutic agents to slow ovarian cancer cell proliferation and migration. Three ovarian adenocarcinoma cell lines; CAOV‐3, SKOV‐3, and OVCAR‐3 are used in this research. Using a proliferation assay, we assessed the impact of different ovarian cancer therapeutics (Rucaparib, Olaparib, Paclitaxel and Cisplatin) in these three cell lines in the presence and absence of inhibitors that decrease NHE1 activity directly or indirectly. Our previous work demonstrated that the NHE1 inhibitor cariporide and a number of protein kinase inhibitors decreased cell proliferation rates in all three cell types. Treatment with Cisplatin reduced cell proliferation most significantly in the CAOV‐3 line. Paclitaxel and Cisplatin each had significant and comparable impacts on the SKOV‐3 and OVCAR‐3 cell lines. Cell migration was evaluated using electric cell‐substrate impedance sensing (ECIS). In the SKOV‐3 and CAOV‐3 cells, inhibition with cariporide and the Rsk inhibitor BID1870 each dramatically reduced the rate of cell migration. The research presented here supports the role of NHE1 in cell proliferation, and for the first time implicates NHE1 in the regulation of cell migration in ovarian cancer cells. Data will be presented that evaluated the potential for the inhibition of NHE1 as an adjuvant therapy in ovarian cancer.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evaluating the Role of the Na+‐H+ Exchanger Isoform 1 (NHE1) in Non‐small Cell Lung Cancer

Lung cancer is a debilitating disease with 222,000 new cases diagnosed each year and causing 156,000 deaths each year in the United States. Approximately 80% of lung cancers are categorized as non‐small cell lung cancer (NSCLC). Of these NSCLCs, 50% are comprised of adenocarcinomas, or tumors that originated within glandular epithelium, while 30% are due to squamous cell carcinomas, or a cancer that began at in a squamous epithelial lining. The Na+‐H+ Exchanger Isoform 1 (NHE1) acts as a key regulator in cellular proliferation, migration, and metastasis in a variety of solid tumors. NHE1 exchanges 1 extracellular Na+ ion for 1 intracellular H+ ion driving changes in both intracellular and extracellular pH in these developing tumors. To evaluate the role of NHE1 in NSCLC, we employed three cells types: 1) NCI‐H1299 a carcinoma cell line, 2) NCI‐H520 a squamous cell carcinoma cell line, and 3) NCI‐H23 and adenocarcinoma cell line. For each cell type, changing the media conditions from 0.5% serum to 10% serum increased the rate of cell proliferation 2 to 3 fold. This increased proliferation was blocked when the NHE1 inhibitors EIPA (ethylisopropylamiloride) or cariporide were present. Similarly, the change in serum concentration increase cell migrations rates in an NHE1‐dependent manner. To further evaluate the role of NHE1 in these cells, western blot analysis was completed to evaluate NHE1 expression in both low and normal serum conditions for each cell type. We also employed CRISPR‐Cas9 technology to develop an NHE1‐knockout (NHE1‐KO) cell line for each cell type. The rates of cell proliferation and migration where then evaluated in the NHE1‐KO cells as compared to NHE1 expressing cells.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Anion and fluid secretory response of the murine jejunum to the heat‐stable Eschericia coli enterotoxin (STa) analogue linaclotide: Involvement of NHE3, Slc26a6, CFTR, proteinkinase GII (cGKII) and NHERF1‐3.

The different PDZ‐adaptors of the NHERF family differentially associate with intestinal electrolyte transporters and modulate their activity in an agonist‐specific fashion. STa induces secretory diarrhea via binding to the guanylate cyclase C, eliciting cGMP production, resulting both in a stimulation of CFTR‐mediated anion secretion and an inhibition of NHE3‐mediated fluid absorption, and these effects are modulated by different NHERF adaptor proteins in cell lines.AimWe studied the STa‐mediated short circuit current response in isolated jejunal mucosa as well as the STa action on in vivo fluid transport in anethstized mice, deficiet for NHE3, Slc26a6, CFTR and NHERF1‐3, to study how well in vitro and in vivo modulation of fluid transport by the above named transporters and NHERF proteins may correlate.Results10−7M of the STa analogue linacolide was applied to the luminal bath of chambered isolated jejunal mucosa of CFTR‐, NHE3‐, Slc26a6‐, cGKII, and NHERF1–3 deficient and WT mice, and short circuit current (Isc) response was measured. The same concentration of linaclotide was added to the jejunal perfusate during single pass perfusion of a jejunal segment in isoflurane‐anesthetized, acid/base‐ and blood pressure controlled KO and WT mice, and net fluid balance before and after linaclotide application assessed gravimetrically. The peak Isc response to linaclotide in chambered jejunal mucosa was absent in CFTR KO, reduced by 88% in cGKII KO, by 40% in NHERF1 KO, and not significantly in NHERF2, NHERF (PDZK1) and SLC26a6 KO isolated jejunal mucosa. In contrast, the magnitude of change from jejunal fluid absorption to secretion in vivo was reduced by 51% in cGKII KO, 49% in CFTR KO, 45% in NHERF1 KO, by 34% in NHERF2 KO, and not significantly in NHERF3 KO and Slc26a6 KO anesthetized mice. NHE3 deletion (48%), pharmacological NHE3 inhibition (50%) or Slc26a6 deletion (44%) also reduced the STa‐elicited change in fluid movement.ConclusionsThe results demonstrate that Isc measurements in isolated small intestinal mucosa are excellent models to selectively study agonist‐mediated CFTR activation, but do not accurately predict in vivo “secretory responses”, i.e. a relative decrease in the basal fluid absorptive flux. Activating the GCC receptor elicited a decrease in fluid absorption in CFTR KO mice, making this substance an interesting drug to improve gut fluidity in CF patients. Apart from direct interference with guanylate cyclase C activation, potential pharmacological strategies to prevent STa‐mediated fluid loss may be targeting both NHERF1 and NHERF2.Support or Funding InformationFunded by SFB621/C1, DFG grants SE460/13‐4, 19‐1 and 21‐1.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of Na+/H+ Exchanger With EMD 87580 does not Confer Greater Cardioprotection Beyond Preconditioning on Ischemia-Reperfusion Injury in Normal Dogs.

Postischemic accumulation of intracellular Na+ promotes calcium overload and contributes to cellular necrosis. Cardioprotection afforded by pharmacologic blockade of the sodium-hydrogen exchanger subtype 1 (NHE1) is thought to be more remarkable than that obtained by ischemic conditioning (IC). The window of protection provided by IC pretreatment is maintained even when performed up to 48 hours before ischemia. In addition, the perception exists that combined NHE1 inhibition plus IC produces greater than additive protection against ischemic injury. The current study compared the efficacy of NHE1 blockade by N-[2-methyl-4,5-bis(methylsulfonyl)-benzoyl]-guanidine (EMD 87580 5 mg/kg) combined with first- or second-window IC on ischemic tolerance in dogs subject to 90-minute acute ischemia and 180-minute reperfusion. Infarct size (tetrazolium staining), vascular responses, and myocardial perfusion (microspheres) were assessed. EMD 87580 given before ischemia or before reperfusion did not reduce infarct size (compared to vehicle-treated group). Significant protection against tissue necrosis was obtained by both first- and second-window IC, but additive cardioprotection (ie, greater than that afforded by IC) was not observed by treatment with EMD 87580. Vascular reactivity in the infarct-related artery was not preserved after ischemia-reperfusion in any of the experimental groups. Likewise, either the pharmacologic or the nonpharmacologic interventions did not modify myocardial perfusion. These data demonstrate that EMD 87580 did not protect against ischemia-reperfusion injury regardless of the time of drug administration. Combined EMD 87580 and IC did not antagonize protection that was achieved by either first- or second-window IC alone; no additive protection beyond preconditioning was obtained. Further study is necessary to assess the value of NHE1 blockers as protective agents against myocardial injury.

Effect of cariporide on ram sperm pH regulation and motility: possible role of NHE1.

Sperm motility, a feature essential for in vivo fertilization, is influenced by intracellular pH (pHi) homeostasis. Several mechanisms are involved in pHi regulation, among which sodium-hydrogen exchangers (NHEs), a family of integral transmembrane proteins that catalyze the exchange of Na+ for H+ across lipid bilayers. A preliminary characterization of NHE activity and kinetic parameters, followed by analysis of the expression and localization of the protein in ram spermatozoa was performed. NHE activity showed an apparent Km for external Na+ of 17.61 mM. Immunoblotting revealed a molecular mass of 85 kDa. Immunolocalization pattern showed some species-specific aspects, such as positive labeling at the equatorial region of the sperm head. Cariporide, a selective NHE1 inhibitor, significantly reduced pHi recovery (85%). Similarly, exposure to cariporide significantly inhibited different motility parameters, including those related to sperm capacitation. In vitro fertilization (IVF) was not affected by cariporide, possibly due to the non-dramatic, although significant, drop in motility and velocity parameters or due to prolonged exposure during IVF, which may have caused progressive loss of its inhibitory effect. In conclusion, this is the first study documenting, in a large animal model (sheep) of well-known translational relevance, a direct functional role of NHE on sperm pHi and motility. The postulated specificity of cariporide toward isoform 1 of the Na+/H+ exchanger seems to suggest that NHE1 may contribute to the observed effects on sperm cell functionality.

Glucose-dependent growth arrest of leukemia cells by MCT1 inhibition: Feeding Warburg’s sweet tooth and blocking acid export as an anticancer strategy

This study aims to investigate the utilization of The Warburg Effect, cancer’s “sweet tooth” and natural greed for glucose to enhance the effect of monocarboxylate transporter inhibition on cellular acidification. By simulating hyperglycemia with high glucose we may increase the effectiveness of inhibition of lactate and proton export on the dysregulation of cell pH homeostasis causing cell death or disruption of growth in cancer cells. MCT1 and MCT4 expression was determined in MCF7 and K562 cell lines using RT-PCR. Cell viability, growth, intracellular pH and cell cycle analysis was measured in the cell lines grown in 5 mM and 25 mM glucose containing media in the presence and absence of the MCT1 inhibitor AR-C155858 (1 μM) and the NHE1 inhibitor cariporide (10 μM). The MCT1 inhibitor, AR-C155858 had minimal effect on the viability, growth and intracellular pH of MCT4 expressing MCF7 cells. AR-C155858 had no effect on the viability of the MCT1 expressing K562 cells, but decreased intracellular pH and cell proliferation, by a glucose-dependent mechanism. Inhibition of NHE1 on its own had a no effect on cell growth, but together with AR-C155858 showed an additive effect on inhibition of cell growth. In cancer cells that only express MCT1, increased glucose concentrations in the presence of an MCT1 inhibitor decreased intracellular pH and reduced cell growth by G1 phase cell-cycle arrest. Thus we propose a transient hyperglycemic-clamp in combination with proton export inhibitors be evaluated as an adjunct to cancer treatment in clinical studies.

Activation and Inhibition of Sodium-Hydrogen Exchanger Is a Mechanism That Links the Pathophysiology and Treatment of Diabetes Mellitus With That of Heart Failure.

The mechanisms underlying the progression of diabetes mellitus and heart failure are closely intertwined, such that worsening of one condition is frequently accompanied by worsening of the other; the degree of clinical acceleration is marked when the 2 coexist. Activation of the sodium-hydrogen exchanger in the heart and vasculature (NHE1 isoform) and the kidneys (NHE3 isoform) may serve as a common mechanism that links both disorders and may underlie their interplay. Insulin insensitivity and adipokine abnormalities (the hallmarks of type 2 diabetes mellitus) are characteristic features of heart failure; conversely, neurohormonal systems activated in heart failure (norepinephrine, angiotensin II, aldosterone, and neprilysin) impair insulin sensitivity and contribute to microvascular disease in diabetes mellitus. Each of these neurohormonal derangements may act through increased activity of both NHE1 and NHE3. Drugs used to treat diabetes mellitus may favorably affect the pathophysiological mechanisms of heart failure by inhibiting either or both NHE isoforms, and drugs used to treat heart failure may have beneficial effects on glucose tolerance and the complications of diabetes mellitus by interfering with the actions of NHE1 and NHE3. The efficacy of NHE inhibitors on the risk of cardiovascular events may be enhanced when heart failure and glucose intolerance coexist and may be attenuated when drugs with NHE inhibitory actions are given concomitantly. Therefore, the sodium-hydrogen exchanger may play a central role in the interplay of diabetes mellitus and heart failure, contribute to the physiological and clinical progression of both diseases, and explain certain drug-drug and drug-disease interactions that have been reported in large-scale randomized clinical trials.