NCX1 Expression Research Articles

An In Vivo Model of Estrogen Supplementation Concerning the Expression of Ca2+-Dependent Exchangers and Mortality, Vitality and Survival After Myocardial Infarction in Ovariectomized Rats

Background: Ischemic-reperfusion damage of cardiomyocytes due to myocardial infarction (MI) often leads to the death of an individual. Premenopausal women have been observed to have a significantly lower risk of cardiovascular disease (CVD) than men of the same age. In menopausal women, this trend is significantly reversed, and the risk of CVD increases up to 10-fold. Estrogens affect the development and function of the heart muscle, and as they decrease, the risk and poor prognosis of CVD increase. This study is focused on the effects of estrogen supplementation on morbidity, vitality, and NCX1 expression after MI on a model system. Methods: In this study, female Sprague Dawley rats (n = 58), which were divided into three experimental groups (NN—control group, non-supplemented; OVX-N—ovariectomized, non-supplemented; OVX-S—ovariectomized, supplemented), received left thoracotomy in the fourth intercostal space. The left anterior descendent coronary artery was ligated 2 mm from its origin with an 8.0 suture. An immunohistological analysis as well as an RT-PCR analysis of NCX1 expression were performed. Results: A higher survival rate was recorded in the OVX-N group (86%) in comparison with the OVX-S group (53%) (p < 0.05). In addition, higher NCX1 expression 7 days/14 days after MI in the OVX-S group in comparison with the NN and OVX-N (p < 0.001 and p < 0.05) groups was recorded. Seven days after MI, a significantly higher expression (p < 0.005) of mRNA NCX1 in the OVX-N group was also recorded in comparison with the NN group. Conclusions: This study provides a comprehensive description of the effect of estrogen supplementation on NCX1 expression and overall vitality in ovariectomized rats that survived MI.

Electroacupuncture pretreatment enhances the calcium efflux activity of Na+/Ca2+ exchanger to attenuate cerebral injury by PI3K/Akt-mediated NCX1 upregulation after focal cerebral ischaemia

Electroacupuncture pretreatment is considered as an optimal strategy for inducing cerebral ischaemic tolerance. However, the underlying neuroprotective mechanism of this approach has never been explored from the perspective of calcium homeostasis. Intracellular calcium overload is a key inducer of cascade neuronal injury in the early stage after cerebral ischaemia attack and the Na+/Ca2+ exchanger (NCX) is the main plasma membrane calcium extrusion pathway maintaining post-ischaemic calcium homeostasis. This study aims to investigate whether the regulation of NCX-mediated calcium transport contributes to the cerebroprotective effect of electroacupuncture pretreatment against ischaemic injury and to elucidate the underlying mechanisms involved in this process. Following five days of repeated electroacupuncture stimulation on Baihui (GV20), Neiguan (PC6), and Sanyinjiao (SP6) acupoints in rats, in vivo and in vitro models of cerebral ischaemia were induced through middle cerebral artery occlusion and oxygen/glucose deprivation (OGD), respectively. Firstly, we verified the neuroprotective effect of electroacupuncture pretreatment from the perspective of neurological score, infarct volume and neuronal apoptosis. Our findings from brain slice patch-clamp indicated that electroacupuncture pretreatment enhanced the Ca2+ efflux capacity of NCX after OGD. NCX1 expression in the ischaemic penumbra exhibited a consistent decline from 1 to 24 h in MCAO rats. Electroacupuncture pretreatment upregulated the expression of NCX1, especially at 24 h, and silencing NCX1 by short hairpin RNA (shRNA) administration reversed the protective effect of electroacupuncture pretreatment against cerebral ischaemic injury. Furthermore, we administered LY294002, a phosphatidylinositol 3 kinase (PI3K) inhibitor, prior to inducing ischaemia to investigate the upstream regulatory mechanism of electroacupuncture pretreatment on NCX1 expression. Electroacupuncture pretreatment activates PI3K/Akt pathway, leading to an increase in the expression of NCX1, which facilitates calcium extrusion and exerts a neuroprotective effect against cerebral ischaemia. These findings provided a novel insight into the prevention of ischemic stroke and other similar conditions characterized by brain ischaemia or hypoperfusion.

NCX1/Ca2+ promotes autophagy and decreases bortezomib activity in multiple myeloma through non-canonical NFκB signaling pathway

Although bortezomib (BTZ) is the cornerstone of anti-multiple myeloma (MM) therapy, the inevitable primary and secondary drug resistance still seriously affects the prognosis of patients. New treatment strategies are in need. Sodium-calcium exchanger 1 (NCX1) is a calcium-permeable ion transporter on the membrane, and our previous studies showed that low NCX1 confers inferior viability in MM cells and suppressed osteoclast differentiation. However, the effect of NCX1 on BTZ sensitivity of MM and its possible mechanism remain unclear. In this study, we investigated the effect of NCX1 on BTZ sensitivity in MM, focusing on cellular processes of autophagy and cell viability. Our results provide evidence that NCX1 expression correlates with MM disease progression and low NCX1 expression increases BTZ sensitivity. NCX1/Ca2+ triggered autophagic flux through non-canonical NFκB pathway in MM cells, leading to attenuated the sensitivity of BTZ. Knockdown or inhibition of NCX1 could potentiate the anti-MM activity of BTZ in vitro and vivo, and inhibition of autophagy sensitized NCX1-overexpressing MM cells to BTZ. In general, this work implicates NCX1 as a potential therapeutic target in MM with BTZ resistance and provides novel mechanistic insights into its vital role in combating BTZ resistance.

Effects of Walnut-Rich Diet on Cation-Handling Proteins in the Heart of Healthy and Metabolically Compromised Male Rats.

The transport of cations in the cardiomyocytes, crucial for the functioning of the heart, can be affected by walnut diet due to the high content of polyunsaturated fatty acids. Healthy and metabolically compromised rats (drinking 10% fructose solution) were subjected to a diet supplemented with 2.4 g of walnuts for 6 weeks to investigate the effect on proteins involved in cation transport in the heart cells. Fructose increased the level of the α1 subunit of Na+/K+-ATPase and the phosphorylation of extracellular signal-regulated kinase 1/2 in the heart of control and walnut-eating rats, while elevated L-type calcium channel α (LTCCα), sodium-calcium exchanger 1 (NCX1), and Maxi Kα level were observed only in rats that did not consume walnuts. However, walnuts significantly increased the cardiac content of LTCC, NCX1, and Maxi Kα, as well as Kir6.1 and SUR2B subunits of KATP channel, but only in fructose-naive rats. In animals that drank fructose, a significant increasing effect of walnuts was observed only in Akt kinase phosphorylation, which may be a part of the antiarrhythmic mechanism of decreasing cation currents in cardiomyocytes. The walnut diet-induced increase in LTCC and NCX1 expression in healthy rats may indicate intense cardiac calcium turnover, whereas the effect on Kir6.1 and SUR2B subunits suggests stimulation of KATP channel transport in the cardiac vasculature. The effects of walnuts on the cation-handling proteins in the heart, mostly limited to healthy animals, suggest the possible use of a walnut-supplemented diet in the prevention rather than the treatment of cardiological channelopathies.

Exploring the Role of NCX1 and NCX3 in an In Vitro Model of Metabolism Impairment: Potential Neuroprotective Targets for Alzheimer's Disease.

Alzheimer's disease (AD) is a widespread neurodegenerative disorder, affecting a large number of elderly individuals worldwide. Mitochondrial dysfunction, metabolic alterations, and oxidative stress are regarded as cooperating drivers of the progression of AD. In particular, metabolic impairment amplifies the production of reactive oxygen species (ROS), resulting in detrimental alterations to intracellular Ca2+ regulatory processes. The Na+/Ca2+ exchanger (NCX) proteins are key pathophysiological determinants of Ca2+ and Na+ homeostasis, operating at both the plasma membrane and mitochondria levels. Our study aimed to explore the role of NCX1 and NCX3 in retinoic acid (RA) differentiated SH-SY5Y cells treated with glyceraldehyde (GA), to induce impairment of the default glucose metabolism that typically precedes Aβ deposition or Tau protein phosphorylation in AD. By using an RNA interference-mediated approach to silence either NCX1 or NCX3 expression, we found that, in GA-treated cells, the knocking-down of NCX3 ameliorated cell viability, increased the intracellular ATP production, and reduced the oxidative damage. Remarkably, NCX3 silencing also prevented the enhancement of Aβ and pTau levels and normalized the GA-induced decrease in NCX reverse-mode activity. By contrast, the knocking-down of NCX1 was totally ineffective in preventing GA-induced cytotoxicity except for the increase in ATP synthesis. These findings indicate that NCX3 and NCX1 may differently influence the evolution of AD pathology fostered by glucose metabolic dysfunction, thus providing a potential target for preventing AD.

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Activation of the Vascular Smooth Muscle GLP-1/NCX1 Pathway Regulates Cytoplasmic Ca2+ Homeostasis and Improves Blood Pressure Variability in Hypertension

Background: Hypertension is the most common cardiovascular disease, and its main harmful effect is chronic damage to target organs. In some patients with well-controlled blood pressure, target organ damage still occurs. GLP-1 agonists have significant cardiovascular benefits, but their antihypertensive effect is limited. The cardiovascular protective effect of GLP-1 is worth studying. Methods: The ambulatory blood pressure of spontaneously hypertensive rats (SHRs) was detected by ambulatory blood pressure monitoring, and the characteristics of blood pressure and the effect of subcutaneous intervention with a GLP-1R agonist on blood pressure were observed. To explore the mechanism of the cardiovascular benefit of GLP-1R agonists in SHRs, we evaluated the effects of GLP-1R agonists on vasomotor function and calcium homeostasis in vascular smooth muscle cells (VSMCs) in vitro. Results: Although the blood pressure of SHRs was significantly higher than that of WKY rats, the blood pressure variability of SHRs was also significantly higher than that of the control group. The GLP-1R agonist significantly reduced blood pressure variability in SHRs, but the antihypertensive effect was not obvious. GLP-1R agonists can significantly improve the cytoplasmic calcium overload of VSMCs in SHRs by upregulating the expression of NCX1, improving the systolic and diastolic functions of arterioles, and reducing blood pressure variability. Conclusions: Taken together, these results provide evidence that GLP-1R agonists improved VSMC cytoplasmic Ca2+ homeostasis through upregulated NCX1 expression in SHRs, which plays a key role in blood pressure stability and broad cardiovascular benefits.

Higher Na+-Ca2+ Exchanger Function and Triggered Activity Contribute to Male Predisposition to Atrial Fibrillation.

Male sex is one of the most important risk factors of atrial fibrillation (AF), with the incidence in men being almost double that in women. However, the reasons for this sex difference are unknown. Accordingly, in this study, we sought to determine whether there are sex differences in intracellular Ca2+ homeostasis in mouse atrial myocytes that might help explain male predisposition to AF. AF susceptibility was assessed in male (M) and female (F) mice (4–5 months old) using programmed electrical stimulation (EPS) protocols. Males were 50% more likely to develop AF. The Ca2+ transient amplitude was 28% higher in male atrial myocytes. Spontaneous systolic and diastolic Ca2+ releases, which are known sources of triggered activity, were significantly more frequent in males than females. The time to 90% decay of Ca2+ transient was faster in males. Males had 54% higher Na+-Ca2+ exchanger (NCX1) current density, and its expression was also more abundant. L-type Ca2+ current (ICaL) was recorded with and without BAPTA, a Ca2+ chelator. ICaL density was lower in males only in the absence of BAPTA, suggesting stronger Ca2+-dependent inactivation in males. CaV1.2 expression was similar between sexes. This study reports major sex differences in Ca2+ homeostasis in mouse atria, with larger Ca2+ transients and enhanced NCX1 function and expression in males resulting in more spontaneous Ca2+ releases. These sex differences may contribute to male susceptibility to AF by promoting triggered activity.

Suppressing Effect of Na+/Ca2+ Exchanger (NCX) Inhibitors on the Growth of Melanoma Cells.

The role of calcium ion (Ca2+) signaling in tumorigenicity has received increasing attention in melanoma research. Previous Ca2+ signaling studies focused on Ca2+ entry routes, but rarely explored the role of Ca2+ extrusion. Functioning of the Na+/Ca2+ exchanger (NCX) on the plasma membrane is the major way of Ca2+ extrusion, but very few associations between NCX and melanoma have been reported. Here, we explored whether pharmacological modulation of the NCX could suppress melanoma and promise new therapeutic strategies. Methods included cell viability assay, Ca2+ imaging, immunoblotting, and cell death analysis. The NCX inhibitors SN-6 and YM-244769 were used to selectively block reverse operation of the NCX. Bepridil, KB-R7943, and CB-DMB blocked either reverse or forward NCX operation. We found that blocking the reverse NCX with SN-6 or YM-244769 (5–100 μM) did not affect melanoma cells or increase cytosolic Ca2+. Bepridil, KB-R7943, and CB-DMB all significantly suppressed melanoma cells with IC50 values of 3–20 μM. Bepridil and KB-R7943 elevated intracellular Ca2+ level of melanoma. Bepridil-induced melanoma cell death came from cell cycle arrest and enhanced apoptosis, which were all attenuated by the Ca2+ chelator BAPTA-AM. As compared with melanoma, normal melanocytes had lower NCX1 expression and were less sensitive to the cytotoxicity of bepridil. In conclusion, blockade of the forward but not the reverse NCX leads to Ca2+-related cell death in melanoma and the NCX is a potential drug target for cancer therapy.

Effect of C reactive protein on the sodium-calcium exchanger 1 in cardiomyocytes.

Numerous previous studies have found that C-reactive protein (CRP) is associated with cardiac arrhythmia and cardiac remodeling. However, the underlying mechanisms of this association remain unclear. Sodium-calcium exchanger 1 (NCX1) serves an important role in the regulation of intracellular calcium concentration, which is closely related with cardiac arrhythmia and cardiac remodeling. The present study aimed to evaluate the effects of CRP on NCX1 and intracellular calcium concentration in cardiomyocytes. Primary neonatal mouse ventricular cardiomyocytes were cultured and treated with varying concentrations of CRP (0, 5, 10, 20 and 40 µg/ml). The cardiomyocytes were also treated with NF-κB-specific inhibitor PTDC and a specific inhibitor of the reverse NCX1 KB-R7943 before their intracellular calcium concentrations were measured. mRNA and protein expression levels of NCX1 were detected by reverse transcription-quantitative PCR and western blotting, respectively and intracellular calcium concentration was evaluated by flow cytometry. CRP treatment significantly increased mRNA and protein expression levels of NCX1 in myocytes (P=0.024), as well as intracellular calcium concentration (P=0.01). These results were significantly attenuated by the NF-κB-specific inhibitor PDTC and a specific inhibitor of the reverse NCX1, KB-R7943. CRP significantly upregulated NCX1 expression and increased intracellular calcium concentration in cardiomyocytes via the NF-κB pathway, suggesting that CRP may serve a pro-arrhythmia role via direct influence on the calcium homeostasis of cardiomyocytes.

Gateways for Glutamate Neuroprotection in Parkinson's Disease (PD): Essential Role of EAAT3 and NCX1 Revealed in an In Vitro Model of PD.

Increasing evidence suggests that metabolic alterations may be etiologically linked to neurodegenerative disorders such as Parkinson’s disease (PD) and in particular empathizes the possibility of targeting mitochondrial dysfunctions to improve PD progression. Under different pathological conditions (i.e., cardiac and neuronal ischemia/reperfusion injury), we showed that supplementation of energetic substrates like glutamate exerts a protective role by preserving mitochondrial functions and enhancing ATP synthesis through a mechanism involving the Na+-dependent excitatory amino acid transporters (EAATs) and the Na+/Ca2+ exchanger (NCX). In this study, we investigated whether a similar approach aimed at promoting glutamate metabolism would be also beneficial against cell damage in an in vitro PD-like model. In retinoic acid (RA)-differentiated SH-SY5Y cells challenged with α-synuclein (α-syn) plus rotenone (Rot), glutamate significantly improved cell viability by increasing ATP levels, reducing oxidative damage and cytosolic and mitochondrial Ca2+ overload. Glutamate benefits were strikingly lost when either EAAT3 or NCX1 expression was knocked down by RNA silencing. Overall, our results open the possibility of targeting EAAT3/NCX1 functions to limit PD pathology by simultaneously favoring glutamate uptake and metabolic use in dopaminergic neurons.

Abstract 1786: High expression of NCX-1 in triple negative breast cancer cell lines identifies a potential biomarker for sensitivity to cardiac glycosides

Abstract Triple negative breast cancers (TNBCs) represent 15-20% of all breast cancers and are defined by lack of the estrogen and progesterone receptors and HER2 gene amplification. Therefore, TNBC patients do not benefit from available therapies targeting these receptors. The significant heterogeneity of TNBC precludes the expectation that a single molecular target will be identified for this disease. Our goal is to identify compounds with selective cytotoxic effects in subsets of TNBC cells to identify new therapeutic targets for TNBC subgroups. Our studies show that the TNBC cell lines BT-549 and Hs578T are distinct from other TNBC cells in that they are highly sensitive to 9 cardiac glycosides/cardenolides isolated from Calotropis gigantea as well as digoxin. BT-549 cells are 15-times more sensitive to the isolated cardiac glycoside calotropin and 9-times more sensitive to digoxin as compared to MDA-MB-231 TNBC cells. Cardiac glycosides bind to the Na+/K+ ATPase and inhibit its activity, leading to increased intracellular Na+, which results in higher intracellular Ca2+ through reversal of the Na+/Ca2+ exchanger (NCX). Notably, among the 10 cardiac glycosides/cardenolides, there was a significant correlation between potency for inhibition of purified Na+/K+ ATPase and cytotoxic potency and selectivity for BT-549 cells. These data suggest that inhibition of Na+/K+ ATPase is critical for the selective cytotoxic effects of these compounds. BT-549 and Hs578T cells express high levels of non-specific TRPC1/4 cation channels1 and low levels of SERCA2 Ca2+ pumps2 compared to other TNBC cells, suggesting that they have impaired ability to handle intracellular Ca2+. Consistent with this hypothesis, within 3 h, concentrations of calotropin and digoxin that are selectivity cytotoxic each initiated a significant increase in intracellular Ca2+ in BT-549 cells. To test whether sensitivity to cardiac glycosides was due to higher Na+/K+ ATPase expression, message and protein levels were evaluated in 10 TNBC cell lines. The results show that there is no correlation between levels of Na+/K+ ATPase and sensitivity to cardiac glycosides. In contrast, NCX-1 expression was found to be 120 and 60-fold higher in the sensitive BT-549 and Hs578T cells, respectively, as compared to the resistant TNBC cell lines. Ongoing studies are evaluating whether genetic manipulation of NCX-1 is sufficient to modulate sensitivity to cardiac glycosides. These findings demonstrate that NCX-1 expression is associated with the selective sensitivity of a subgroup of TNBC cell lines to the cytotoxic effects of cardiac glycosides. Our results suggest that NCX-1 could be a biomarker to identify TNBC patients that could ultimately benefit from use of a cardiac glycoside for anticancer indications. 1. Grant CV, et al. Breast Cancer Res Treat. 2019;177(2):345. 2. Varga K, et al. BMC Cancer. 2018;18(1):1029. Citation Format: Petra E. Pederson, Shengxin Cai, Chase M. Carver, Tanja Grkovic, Barry R. O'Keefe, April L. Risinger, Robert H. Cichewicz, Susan L. Mooberry. High expression of NCX-1 in triple negative breast cancer cell lines identifies a potential biomarker for sensitivity to cardiac glycosides [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1786.

Genetic knockout and pharmacologic inhibition of NCX1 attenuate hypoxia-induced pulmonary arterial hypertension

The Na+/Ca2+ exchanger type-1 (NCX1) is a bidirectional transporter that is controlled by membrane potential and transmembrane gradients of Na+ and Ca2+. Vascular smooth muscle NCX1 plays an important role in intracellular Ca2+ homeostasis and Ca2+ signaling. We found that NCX1 was upregulated in the pulmonary arteries of mice exposed to chronic hypoxia (10% O2 for 4 weeks). Hence, we investigated the pathophysiological role of NCX1 in hypoxia-induced pulmonary arterial hypertension (PAH), using NCX1-heterozygous (NCX1+/−) mice, in which NCX1 expression is reduced by half, and SEA0400, a specific NCX1 inhibitor. NCX1+/− mice exhibited attenuation of hypoxia-induced PAH and right ventricular (RV) hypertrophy compared with wild-type mice. Furthermore, continuous administration of SEA0400 (0.5 mg/kg/day for 4 weeks) to wild-type mice by osmotic pumps significantly suppressed hypoxia-induced PAH and pulmonary vessel muscularization, with a slight reduction in RV hypertrophy. These findings indicate that the upregulation of NCX1 contributes to the development of hypoxia-induced PAH, suggesting that NCX1 inhibition might be a novel approach for the treatment of PAH.

The Degree of t-System Remodeling Predicts Negative Force-Frequency Relationship and Prolonged Relaxation Time in Failing Human Myocardium.

The normally positive cardiac force-frequency relationship (FFR) becomes flat or negative in chronic heart failure (HF). Here we explored if remodeling of the cardiomyocyte transverse tubular system (t-system) is associated with alterations in FFR and contractile kinetics in failing human myocardium. Left-ventricular myocardial slices from 13 failing human hearts were mounted into a biomimetic culture setup. Maximum twitch force (F), 90% contraction duration (CD90), time to peak force (TTP) and time to relaxation (TTR) were determined at 37°C and 0.2–2 Hz pacing frequency. F1Hz/F0.5Hz and F2Hz/F0.5Hz served as measures of FFR, intracellular cardiomyocyte t-tubule distance (ΔTT) as measure of t-system remodeling. Protein levels of SERCA2, NCX1, and PLB were quantified by immunoblotting. F1Hz/F0.5Hz (R2 = 0.82) and F2Hz/F0.5Hz (R2 = 0.5) correlated negatively with ΔTT, i.e., samples with severe t-system loss exhibited a negative FFR and reduced myocardial wall tension at high pacing rates. PLB levels also predicted F1Hz/F0.5Hz, but to a lesser degree (R2 = 0.49), whereas NCX1 was not correlated (R2 = 0.02). CD90 correlated positively with ΔTT (R2 = 0.39) and negatively with SERCA2/PLB (R2 = 0.42), indicating that both the t-system and SERCA activity are important for contraction kinetics. Surprisingly, ΔTT was not associated with TTP (R2 = 0) but rather with TTR (R2 = 0.5). This became even more pronounced when interaction with NCX1 expression was added to the model (R2 = 0.79), suggesting that t-system loss impairs myocardial relaxation especially when NCX1 expression is low. The degree of t-system remodeling predicts FFR inversion and contraction slowing in failing human myocardium. Moreover, together with NCX, the t-system may be important for myocardial relaxation.

Na/Ca exchange in the atrium: Role in sinoatrial node pacemaking and excitation-contraction coupling

Na/Ca exchange is the dominant calcium (Ca) efflux mechanism in cardiac myocytes. Although our knowledge of exchanger function (NCX1 in the heart) was originally established using biochemical and electrophysiological tools such as cardiac sarcolemmal vesicles and the giant patch technique [1–4], many advances in our understanding of the physiological/pathophysiological roles of NCX1 in the heart have been obtained using a suite of genetically modified mice. Early mouse studies focused on modification of expression levels of NCX1 in the ventricles, with transgenic overexpressors, global NCX1 knockout (KO) mice (which were embryonic lethal if homozygous), and finally ventricular-specific NCX1 KO [5–12]. We found, to our surprise, that ventricular cardiomyocytes lacking NCX1 can survive and function by engaging a clever set of adaptations to minimize Ca entry, while maintaining contractile function through an increase in excitation-contraction (EC) coupling gain [5,6,13]. Having studied ventricular NCX1 ablation in detail, we more recently focused on elucidating the role of NCX1 in the atria through altering NCX1 expression. Using a novel atrial-specific NCX1 KO mouse, we found unexpected changes in atrial cell morphology and calcium handling, together with dramatic alterations in the function of sinoatrial node (SAN) pacemaker activity. In this review, we will discuss these findings and their implications for cardiac disease.

Nuclear Factor-κB Increases Intracellular Calcium by Upregulation of Na+-Ca2+ Exchanger 1 in Cerulein-Induced Acute Pancreatitis.

ObjectivesThe mechanisms underlying pathogenesis of acute pancreatitis (AP) are still not completely understood. An early, critical feature of AP is aberrant calcium (Ca2+) signaling, termed Ca2+ overload, within pancreatic acinar cells. This study aimed to develop a model system in rats for AP induction to study the contribution of the Na+-Ca2+ exchanger 1 (NCX1) ion channel in AP pathogenesis.MethodsTo establish a rat model of AP induction, cerulein or l-arginine were intraperitoneally injected and tissue was histologically analyzed by hematoxylin and eosin staining. A cell culture-based model for AP induction was similarly created through cerulein treatment of AR42J cells. Induction of AP was also examined following exposure to the NXC1-targeted inhibitor KB-R7943. The expression of each gene was detected by Western blotting, immunofluorescence, immunohistochemistry, or quantitative reverse transcription polymerase chain reaction. Transcriptional regulation by nuclear factor (NF)-κB was detected using an NCX1 promoter-fusion dual luciferase reporter system. Cytosolic Ca2+ was measured using a fluorescent calcium indicator.ResultsWe found that cerulein induced NCX1 expression via activation of nuclear factor NF-κB, which potentially binds to the NCX1 promoter to induce its transcription.ConclusionsOur findings reveal a regulatory pathway through NF-κB/NCX1 governing Ca2+ overload in AP development, thus providing potential targets for AP treatment.

High Salt Up‐Regulates Ca2+‐Sensing Receptor and Na+/Ca2+ Exchanger Expression in Kidneys of Dahl Salt‐Sensitive Rats

The G protein‐coupled Ca2+‐sensing receptor (CaSR) provides the primary mechanism for Ca2+e‐mediated regulation of the release of parathyroid hormone (PTH) as a result of parathyroid cells sensing small changes in the [Ca2+]e. However, the CaSR is also involved in the fine regulation of serum Ca2+ levels and renal Ca2+ excretion, independent of PTH secretion. The mechanisms underlying these latter effects are not clear. We studied the effect of high salt on urinary ion excretion, blood pressure and expression of the CaSR and NCX1in kidneys of Dahl SR and SS rats. Our results show that four weeks of feeding a high salt diet increased BP in SS, increased urinary Na+/Ca2+ excretion in SR and SS but reduced K+ excretion. The high salt diet caused minimal structural changes in the kidneys, as determined by haematoxylin/eosin (H&E) and periodic acid‐Schiff (PAS) staining. Immunohistochemical analysis of tissue sections showed localization of the CaSR and NCX1 in the thick ascending limb of the loop of Henle, collecting ducts, cortical distal tubules, which was up‐regulated by the high salt in SS animals. NCX1 expression and localization in this study tracks closely with the CaSR. Thus, expression levels and localization patterns of these protein suggest that CaSR modulation of ion transporter and channel proteins could account for the difference in ion excretion observed between SS and SR rats as well as their responses to the high salt diet.Support or Funding InformationNIH Grant SC1 HL136278This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NCX1 and NCX3 as potential factors contributing to neurodegeneration and neuroinflammation in the A53T transgenic mouse model of Parkinson\u2019s Disease

Na+-Ca2+ exchanger (NCX) isoforms constitute the major cellular Ca2+ extruding system in neurons and microglia. We herein investigated the role of NCX isoforms in the pathophysiology of Parkinson’s disease (PD). Their expression and activity were evaluated in neurons and glia of mice expressing the human A53T variant of α-synuclein (A53T mice), an animal model mimicking a familial form of PD. Western blotting revealed that NCX3 expression in the midbrain of 12-month old A53T mice was lower than that of wild type (WT). Conversely, NCX1 expression increased in the striatum. Immunohistochemical studies showed that glial fibrillary acidic protein (GFAP)-positive astroglial cells significantly increased in the substantia nigra pars compacta (SNc) and in the striatum. However, the number and the density of tyrosine hydroxylase (TH)-positive neurons decreased in both brain regions. Interestingly, ionized calcium binding adaptor molecule 1 (IBA-1)-positive microglial cells increased only in the striatum of A53T mice compared to WT. Double immunostaining studies showed that in A53T mice, NCX1 was exclusively co-expressed in IBA-1-positive microglial cells in the striatum, whereas NCX3 was solely co-expressed in TH-positive neurons in SNc. Beam walking and pole tests revealed a reduction in motor performance for A53T mice compared to WT. In vitro experiments in midbrain neurons from A53T and WT mice demonstrated a reduction in NCX3 expression, which was accompanied by mitochondrial overload of Ca2+ ions, monitored with confocal microscopy by X-Rhod-1 fluorescent dye. Collectively, in vivo and in vitro findings suggest that the reduction in NCX3 expression and activity in A53T neurons from midbrain may cause mitochondrial dysfunction and neuronal death in this brain area, whereas NCX1 overexpression in microglial cells may promote their proliferation in the striatum.

The NCX1/TRPC6 Complex Mediates TGFβ-Driven Migration and Invasion of Human Hepatocellular Carcinoma Cells

TGFβ plays an important role in the progression and metastasis of hepatocellular carcinoma (HCC), yet the cellular and molecular mechanisms underlying this role are not completely understood. In this study, we investigated the roles of Na+/Ca2+ exchanger 1 (NCX1) and canonical transient receptor potential channel 6 (TRPC6) in regulating TGFβ in human HCC. In HepG2 and Huh7 cells, TGFβ-stimulated intracellular Ca2+ increases through NCX1 and TRPC6 and induced the formation of a TRPC6/NCX1 molecular complex. This complex-mediated Ca2+ signaling regulated the effect of TGFβ on the migration, invasion, and intrahepatic metastasis of human HCC cells in nude mice. TGFβ upregulated TRPC6 and NCX1 expression, and there was a positive feedback between TRPC6/NCX1 signaling and Smad signaling. Expression of both TRPC6 and NCX1 were markedly increased in native human HCC tissues, and their expression levels positively correlated with advancement of HCC in patients. These data reveal the role of the TRPC6/NCX1 molecular complex in HCC and in regulating TGFβ signaling, and they implicate TRPC6 and NCX1 as potential targets for therapy in HCC.Significance: TGFβ induces the formation and activation of a TRPC6/NCX1 molecular complex, which mediates the effects of TGFβ on the migration, invasion, and intrahepatic metastasis of HCC. Cancer Res; 78(10); 2564-76. ©2018 AACR.

Modulation of renal calcium and phosphate transporting proteins by dietary nitrogen and/or calcium in young goats.

In young goats, a reduction in dietary nitrogen (N) had an impact on mineral homeostasis although ruminants are able to recycle N effectively due to rumino-hepatic circulation. A solitary calcium (Ca) reduction stimulated calcitriol synthesis and Ca concentrations remained unchanged, whereas a dietary N reduction led to a decrease in calcitriol, which could not be prevented by a simultaneous reduction of N and Ca. In a previous study, it was shown that a reduced dietary N intake caused a decrease in intestinal Ca absorption due to a reduction of intestinal Ca transporting proteins. As no data on the potential role of the kidneys are available, it was the aim of the present study to evaluate whether an N- and/or Ca-reduced diet had an impact on renal Ca and phosphate (Pi) transporting protein expression in young goats. The animals were divided into 4 feeding groups, each receiving an adequate N and Ca supply, a reduced N supply, a reduced Ca supply, or a combined N and Ca reduction for 6 to 9 wk. The protein expression of the renal Ca channel transient receptor potential cation channel subfamily V member 5 (TRPV5) was diminished in N-reduced fed goats (P = 0.03), whereas in Ca restricted animals, the expression remained unaltered. The mRNA and protein expression of the Ca-binding protein calbindin-D28K (CaBPD28K) and the sodium-Ca exchanger 1 (NCX1) were significantly decreased due to the N-reduced feeding (mRNA, P = 0.003; P < 0.0001; protein, P = 0.002; P = 0.02), whereas dietary Ca reduction increased the CaBPD28K and NCX1 mRNA expression (P = 0.05; P = 0.01). The mRNA and protein expression of the parathyroid hormone receptor (PTHR) decreased due to the N-reduced feeding (P = 0.02; P = 0.03). These results confirm that a reduced dietary N intake led to decreased TRPV5, CaBPD28K, PTHR, and NCX1 expression levels, contributing to low levels of calcitriol and plasma Ca. In contrast to this, sodium-phosphate cotransporter type IIa expression and plasma Pi concentration were increased during dietary N reduction, thus indicating that Pi homeostasis is modulated in a calcitriol-independent manner. In conclusion, the modulation of Ca transporting proteins expression in the kidney is not able to prevent changes in mineral homeostasis in young goats receiving an N-reduced diet.