Reverse-mode NCX Research Articles

Inhibition of the Sodium-Calcium Exchanger Reverse Mode Activity Reduces Alcohol Consumption in Rats.

Excessive and uncontrolled consumption of alcohol can cause alcohol use disorder (AUD), but its pharmacological mechanisms are not fully understood. Inhibiting the reverse mode activity of the sodium-calcium exchanger (NCX) can reduce the risk of alcohol withdrawal seizures, suggesting that NCX could play a role in controlling alcohol consumption. Here, we investigated how two potent inhibitors of NCX reverse mode activity, SN-6 (NCX1) and KB-R7943 (NCX3), affect voluntary alcohol consumption in adult male and female rats using the intermittent alcohol access two-bottle choice paradigm. Initially, animals were trained to drink 7.5% ethanol and water for four weeks before administering SN-6 and KB-R7934. Afterward, their alcohol intake, preference, and water intake were recorded 2 and 24 h after exposure to water and 7.5% ethanol. SN-6 significantly reduced alcohol consumption by 48% in male and 36% in female rats without affecting their water intake. Additionally, SN-6 significantly reduced alcohol preference in females by 27%. However, KB-R7943 reduced alcohol consumption by 42% in female rats and did not affect alcohol preference or water intake. These findings suggest that alcohol exposure increased NCX reverse activity, and targeting NCX1 could be an effective strategy for reducing alcohol consumption in subjects susceptible to withdrawal seizures.

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.

Alterations of the Ca2+ clearing mechanisms by type 2 diabetes in aortic smooth muscle cells of Zucker diabetic fatty rat.

Type 2 Diabetes Mellitus (T2DM) is a rapidly rising disease with cardiovascular complications constituting the most common cause of death among diabetic patients. Chronic hyperglycemia can induce vascular dysfunction through damage of the components of the vascular wall, such as vascular smooth muscle cells (VSMCs), which regulate vascular tone and contribute to vascular repair and remodeling. These functions are dependent on intracellular Ca2+ changes. The mechanisms by which T2DM affects Ca2+ handling in VSMCs still remain poorly understood. Therefore, the objective of this study was to determine whether and how T2DM affects Ca2+ homeostasis in VSMCs. We evaluated intracellular Ca2+ signaling in VSMCs from Zucker Diabetic Fatty rats using Ca2+ imaging with Fura-2/AM. Our results indicate that T2DM decreases Ca2+ release from the sarcoplasmic reticulum (SR) and increases the activity of store-operated channels (SOCs). Moreover, we were able to identify an enhancement of the activity of the main Ca2+ extrusion mechanisms (SERCA, PMCA and NCX) during the early stage of the decay of the ATP-induced Ca2+ transient. In addition, we found an increase in Ca2+ entry through the reverse mode of NCX and a decrease in SERCA and PMCA activity during the late stage of the signal decay. These effects were appreciated as a shortening of ATP-induced Ca2+ transient during the early stage of the decay, as well as an increase in the amplitude of the following plateau. Enhanced cytosolic Ca2+ activity in VSMCs could contribute to vascular dysfunction associated with T2DM.

Sodium regulates PLC and IP3 R-mediated calcium signaling in invasive breast cancer cells.

Intracellular Ca2+ signaling and Na+ homeostasis are inextricably linked via ion channels and co-transporters, with alterations in the concentration of one ion having profound effects on the other. Evidence indicates that intracellular Na+ concentration ([Na+ ]i ) is elevated in breast tumors, and that aberrant Ca2+ signaling regulates numerous key cancer hallmark processes. The present study therefore aimed to determine the effects of Na+ depletion on intracellular Ca2+ handling in metastatic breast cancer cell lines. The relationship between Na+ and Ca2+ was probed using fura-2 and SBFI fluorescence imaging and replacement of extracellular Na+ with equimolar N-methyl-D-glucamine (0Na+ /NMDG) or choline chloride (0Na+ /ChoCl). In triple-negative MDA-MB-231 and MDA-MB-468 cells and Her2+ SKBR3 cells, but not ER+ MCF-7 cells, 0Na+ /NMDG and 0Na+ /ChoCl resulted in a slow, sustained depletion in [Na+ ]i that was accompanied by a rapid and sustained increase in intracellular Ca2+ concentration ([Ca2+ ]i ). Application of La3+ in nominal Ca2+ -free conditions had no effect on this response, ruling out reverse-mode NCX activity and Ca2+ entry channels. Moreover, the Na+ -linked [Ca2+ ]i increase was independent of membrane potential hyperpolarization (NS-1619), but was inhibited by pharmacological blockade of IP3 receptors (2-APB), phospholipase C (PLC, U73122) or following depletion of endoplasmic reticulum Ca2+ stores (cyclopiazonic acid). Thus, Na+ is linked to PLC/IP3 -mediated activation of endoplasmic reticulum Ca2+ release in metastatic breast cancer cells and this may have an important role in breast tumors where [Na+ ]i is perturbed.

Abstract P1094: Sexual Dimorphism In Histone Deacetylases Activity Regulates Ventricular Calcium Handling Properties

Introduction: There is a sexual dimorphism in cardiac excitation contraction coupling (ECC) with female ventricular myocytes (F VM ) exhibiting an attenuated basal calcium concentration ([Ca] i ), Ca transient (CaT) amplitude, and a prolonged CaT decay constant (τ). Increased expression levels of the sodium-calcium exchanger (NCX) have been described in F VM however, the role NCX expression plays in sexual dimorphism of physiological and pathophysiological Ca handling properties, and how NCX expression is regulated in a sex specific manner remains to be determined. Here we tested the hypothesis, that a sexual dimorphism in HDAC activity regulates NCX expression and NCXs differential contribution to the CaT adds to the sexual dimorphism in ventricular ECC. Method: [Ca] i was quantified in field stimulated, freshly isolated ventricular myocytes (VMs) from male (M VM ) and female (F VM ) FVB/N mice loaded with the Ca sensitive fluorescent dye Fluo-4/AM. Changes in NCX expression were quantified by qPCR and western blotting. Results: Under basal conditions F VM exhibited an attenuated CaT amplitude (ΔF/F 0 ; M VM :1.8±0.08, F VM :1.4±0.06, p=0.038) and prolonged CaT decay constant (τ: M VM :0.37±0.01ms, F VM :0.46±0.02ms, p<0.01). The caffeine induced Ca transient (CaT caf ) amplitude was comparable in F VM and M VM , but the CaT caf decay constant (τ caf: M VM : 3.34±0.22s ; F VM :4.81±0.36s) was longer in F VM although increased NCX expression was determined on the protein and mRNA level. Consistent with increased NCX expression, F VM showed an attenuated increase in basal [Ca] i (F/F 0 ; M= 60%, F=44%, p=0.0033) and τ (M=0.37±0.01s, F=0.46±0.02s, p<0.0001) when SERCA activity was attenuated (CPA: 10μM). Also during conditions favoring NCX reverse mode ([Na] o =93, 46 mM) F VM exhibited a larger increase in CaT amplitude (ΔF/F 0 ; M=81%, F=133%, p<0.01) and an increased propensity for spontaneous Ca release events. Inhibition of HDAC class II (LMK: 5mg/kg/day) did not change Ca handling properties in F VM but slowed τ (M LMK : 0.44±0.03ms) and τ caf in M VM (M LMK =5.1±0.33s) making them comparable to F VM . Conclusion: The sexual dimorphism in ventricular ECC is mediated by attenuated HDAC activity in F VM s and facilitates NCX Ca overload.

Identification and characterization of the promoter and transcription factors regulating the expression of cerebral sodium/calcium exchanger 2 (NCX2) gene

The isoform 2 of sodium-calcium exchanger family (NCX2) is selectively expressed in neuronal and glial cells where it participates in Ca2+-clearance following neuronal depolarization, synaptic plasticity, hippocampal-dependent learning and memory consolidation processes. On the other hand, NCX2 is also involved in a neuroprotective effect following stroke. Despite the relevance of this antiporter under physiological and pathophysiological conditions, no studies have been reported on its genetic/epigenetic regulation. Therefore, we identified, cloned, and characterized a transcriptional regulatory region (R3) of rat Slc8a2 gene encoding for NCX2. In particular, R3 sequence displayed a promoter activity in PC12, SH-SY5Y and U87MG cell lines consistent with their endogenous NCX2 expression levels. On the other hand, R3 failed to induce detectable luciferase activity in BHK cell line that does not express NCX2 under control conditions. These data support the hypothesis that R3 represents the promoter region of NCX2. Moreover, among several conserved binding sequences for transcription factors identified by in-silico analysis, we evaluated the transcriptional regulation and the binding sites of Sp1, Sp4, NFkB1, GATA2 and CREB1 on R3 sequence by using site-direct mutagenesis and ChIP assays. In particular, transfection of Sp1, Sp4, and CREB1 enhanced both R3 promoter activity and NCX2 transcription in PC12 cell line. More important, CREB1 transfection also enhanced NCX2 protein levels and NCX reverse mode activity in PC12 cells. Altogether, these data suggested that: (i) the identified region contained the regulatory promoter of the antiporter; (ii) NCX2 might represent a downstream effector of transcription factors involved in synaptic plasticity and neuronal survival.

PKCζ-NADPH Oxidase-PKCα Dependent Kv1.5 Phosphorylation by Endothelin-1 Modulates Nav1.5-NCX1-Cav1.2 Axis in Stimulating Ca2+ Level in Caveolae of Pulmonary Artery Smooth Muscle Cells.

Endothelin-1 (ET-1) is a potent endogenously derived vasoconstrictor, which increases pulmonary hypertension via stimulation of [Ca2+]i level in pulmonary artery smooth muscle cells (PASMCs). In this communication, we sought to investigate the mechanism by which ET-1 causes stimulation of Ca2+ concentration in caveolae vesicles of bovine PASMCs (BPASMCs). ET-1 activates PKC-α in the caveolae vesicles by O2.- derived from PKCζ-NADPH oxidase dependent pathway. PKC-α phosphorylates Kv1.5 channels leading to a marked stimulation of Na+ and Ca2+ concentration in the caveolae vesicles. The stimulation of Ca2+ concentration in the caveolae vesicles by ET-1 occurs predominantly via Cav1.2 channels. Additionally, an increase in Na+ concentration by ET-1 due to stimulation of Nav1.5 channels marginally increases Ca2+ level in the caveolae vesicles via reverse-mode Na+/Ca2+ exchanger (NCX-1) and also through "slip-mode conductance" Nav1.5 channels. 4-AP, a well-known inhibitor of Kv channels, also increases Ca2+ concentration in the caveolae vesicles via Cav1.2 channels, reverse-mode NCX-1 and Nav1.5 channels by phosphorylation independent modulation of Kv1.5 channels without the involvement of PKCζ-NADPH oxidase-PKCα signaling axis. Overall, PKCζ-NADPH oxidase-PKCα dependent phosphorylation of Kv1.5 by ET-1 modulates Nav1.5-NCX1-Cav1.2 axis for stimulation of Ca2+ concentration in caveolae vesicles of BPASMCs, which provides a crucial mechanism for better understanding of ET-1-mediated modulation of pulmonary vascular tone.

NCX1 and EAAC1 transporters are involved in the protective action of glutamate in an in vitro Alzheimer's disease-like model

Increasing evidence suggests that metabolic dysfunctions are at the roots of neurodegenerative disorders such as Alzheimer’s disease (AD). In particular, defects in cerebral glucose metabolism, which have been often noted even before the occurrence of clinical symptoms and histopathological lesions, are now regarded as critical contributors to the pathogenesis of AD. Hence, the stimulation of energy metabolism, by enhancing the availability of specific metabolites, might be an alternative way to improve ATP synthesis and to positively affect AD progression. For instance, glutamate may serve as an intermediary metabolite for ATP synthesis through the tricarboxylic acid (TCA) cycle and the oxidative phosphorylation. We have recently shown that two transporters are critical for the anaplerotic use of glutamate: the Na+-dependent Excitatory Amino Acids Carrier 1 (EAAC1) and the Na+-Ca2+ exchanger 1 (NCX1). Therefore, in the present study, we established an AD-like phenotype by perturbing glucose metabolism in both primary rat cortical neurons and retinoic acid (RA)-differentiated SH-SY5Y cells, and we explored the potential of glutamate to halt cell damage by monitoring neurotoxicity, AD markers, ATP synthesis, cytosolic Ca2+ levels and EAAC1/NCX1 functional activities.We found that glutamate significantly increased ATP production and cell survival, reduced the increase of AD biomarkers (amyloid β protein and the hyperphosphorylated form of tau protein), and recovered the increase of NCX reverse-mode activity. The RNA silencing of either EAAC1 or NCX1 caused the loss of the beneficial effects of glutamate, suggesting the requirement of a functional interplay between these transporters for glutamate-induced protection.Remarkably, our results indicate, as proof‐of‐principle, that facilitating the use of alternative fuels, like glutamate, may be an effective approach to overcome deficits in glucose utilization and significantly slow down neuronal degenerative process in AD.

Contribution of the neuronal sodium channel NaV1.8 to sodium- and calcium-dependent cellular proarrhythmia

ObjectiveIn myocardial pathology such as heart failure a late sodium current (INaL) augmentation is known to be involved in conditions of arrhythmogenesis. However, the underlying mechanisms of the INaL generation are not entirely understood. By now evidence is growing that non-cardiac sodium channel isoforms could also be involved in the INaL generation. The present study investigates the contribution of the neuronal sodium channel isoform NaV1.8 to arrhythmogenesis in a clearly-defined setting of enhanced INaL by using anemone toxin II (ATX-II) in the absence of structural heart disease. MethodsElectrophysiological experiments were performed in order to measure INaL, action potential duration (APD), SR-Ca2+-leak and cellular proarrhythmic triggers in ATX-II exposed wild-type (WT) and SCN10A−/− mice cardiomyocytes. In addition, WT cardiomyocytes were stimulated with ATX-II in the presence or absence of NaV1.8 inhibitors. INCX was measured by using the whole cell patch clamp method. ResultsIn WT cardiomyocytes exposure to ATX-II augmented INaL, prolonged APD, increased SR-Ca2+-leak and induced proarrhythmic triggers such as early afterdepolarizations (EADs) and Ca2+-waves. All of them could be significantly reduced by applying NaV1.8 blockers PF-01247324 and A-803467. Both blockers had no relevant effects on cellular electrophysiology of SCN10A−/− cardiomyocytes. Moreover, in SCN10A−/−-cardiomyocytes, the ATX-II-dependent increase in INaL, SR-Ca2+-leak and APD prolongation was less than in WT and comparable to the results which were obtained with WT cardiomyocytes being exposed to ATX-II and NaV1.8 inhibitors in parallel. Moreover, we found a decrease in reverse mode NCX current and reduced CaMKII-dependent RyR2-phosphorylation after application of PF-01247324 as an underlying explanation for the Na+-mediated Ca2+-dependent proarrhythmic triggers. ConclusionThe current findings demonstrate that NaV1.8 is a significant contributor for INaL-induced arrhythmic triggers. Therefore, NaV1.8 inhibition under conditions of an enhanced INaL constitutes a promising antiarrhythmic strategy which merits further investigation.

Type 1 Sodium Calcium Exchanger Forms a Complex with Carbonic Anhydrase IX and Via Reverse Mode Activity Contributes to pH Control in Hypoxic Tumors.

Hypoxia and acidosis are among the key microenvironmental factors that contribute to cancer progression. We have explored a possibility that the type 1Na+/Ca2+ exchanger (NCX1) is involved in pH control in hypoxic tumors. We focused on changes in intracellular pH, co-localization of NCX1, carbonic anhydrase IX (CA IX), and sodium proton exchanger type 1 (NHE1) by proximity ligation assay, immunoprecipitation, spheroid formation assay and migration of cells due to treatment with KB-R7943, a selective inhibitor of the reverse-mode NCX1. In cancer cells exposed to hypoxia, reverse-mode NCX1 forms a membrane complex primarily with CA IX and also with NHE1. NCX1/CA IX/NHE1 assembly operates as a metabolon with a potent ability to extrude protons to the extracellular space and thereby facilitate acidosis. KB-R7943 prevents formation of this metabolon and reduces cell migration. Thus, we have shown that in hypoxic cancer cells, NCX1 operates in a reverse mode and participates in pH regulation in hypoxic tumors via cooperation with CAIX and NHE1.

Hyperglycemia Enhances Constriction of Retinal Venules via Activation of the Reverse-Mode Sodium-Calcium Exchanger.

Diabetes is associated with hyperglycemia and impairment of retinal microvascular function. However, the impact of hyperglycemia on retinal venular constriction remains unknown. We examined retinal venular responsiveness to endogenous vasoconstrictors and the contribution of the reverse-mode sodium-calcium exchanger (NCX) to these responses during hyperglycemia. Retinal venules were isolated from pigs with streptozocin-induced diabetes (2 weeks, in vivo hyperglycemia) and age-matched control pigs for vasoreactivity and molecular studies. For in vitro hyperglycemia, vessels from euglycemic pigs were exposed to high glucose (25 mmol/L) for 2 h, and 5 mmol/L glucose served as the control. Constrictions of venules from euglycemic pigs to endothelin-1 (ET-1), thromboxane analog U46619, and norepinephrine were mediated by ETA, thromboxane, and α2-adrenergic receptors, respectively, and were insensitive to reverse-mode NCX blockade (KB-R7943). In vivo hyperglycemia enhanced these vasoconstrictions without altering respective receptor mRNA expression. Similarly, in vitro hyperglycemia augmented venular constrictions. Enhanced vasoconstrictions during hyperglycemia were prevented by KB-R7943, while mRNA expression of venular NCX isoforms was unaltered. In vivo hyperglycemia increased vitreous levels of ET-1 but not thromboxane B2 In conclusion, both in vitro and in vivo hyperglycemia enhance retinal venular responses to endogenous vasoconstrictors by activating reverse-mode NCX. Therapies targeting this vascular molecule may alleviate retinal complications during diabetes.

Neurokinin-1 receptor antagonist decreases [Ca2+]i in airway smooth muscle cells by reducing the reverse-mode Na+/Ca2+ exchanger current

Airway smooth muscle (ASM) is involved in asthma airway inflammation. The aim of this study was to evaluate the effect of substance P and neurokinin-1 receptor (NK-1R) antagonist on intracellular calcium concentration ([Ca2+]i) in airway smooth muscle cells (ASMCs), ASMC contraction, and the effect on reverse-mode Na+–Ca2+ exchanger (NCX) currents in ASMCs. In our study, primary rat ASMCs were cultured. ASMCs were identified by immunofluorescence. [Ca2+]i variations were measured by fluorescence microscopy. Cell shortening (%) and relaxation (%) were analyzed with phase-contrast microscopy. Patch clamp techniques were used to assess NCX currents in ASMCs. We found that substance P increased, and NK-1R antagonist decreased [Ca2+]i in ASMCs. Substance P induced ASMCs contraction, and NK-1R antagonist can make ASMC relax. Patch clamp techniques were implemented to analyze NCX currents in ASMCs. Substance P increased reverse-mode NCX currents in ASMCs but the current density was lower than the one treated with acetylcholine (Ach). NK-1R antagonist reduced reverse-mode NCX current activity in ASMCs, and the current density was similar to the one treated with the reversed NCX inhibitor. So, we concluded that substance P increased [Ca2+]i in ASMCs by promoting the reverse-mode NCX current and stimulating ASMCs, whereas NK-1R antagonist decreased [Ca2+]i in ASMCs by decreasing the reverse-mode NCX current to make ASMCs relax.

Na+ microdomains and sparks: Role in cardiac excitation-contraction coupling and arrhythmias in ankyrin-B deficiency

Cardiac sodium (Na+) potassium ATPase (NaK) pumps, neuronal sodium channels (INa), and sodium calcium (Ca2+) exchangers (NCX1) may co-localize to form a Na+ microdomain. It remains controversial as to whether neuronal INa contributes to local Na+ accumulation, resulting in reversal of nearby NCX1 and influx of Ca2+ into the cell. Therefore, there has been great interest in the possible roles of a Na+ microdomain in cardiac Ca2+-induced Ca2+ release (CICR). In addition, the important role of co-localization of NaK and NCX1 in regulating localized Na+ and Ca2+ levels and CICR in ankyrin-B deficient (ankyrin-B+/−) cardiomyocytes has been examined in many recent studies. Altered Na+ dynamics may contribute to the appearance of arrhythmias, but the mechanisms underlying this relationship remain unclear. In order to investigate this, we present a mechanistic canine cardiomyocyte model which reproduces independent local dyadic junctional SR (JSR) Ca2+ release events underlying cell-wide excitation-contraction coupling, as well as a three-dimensional super-resolution model of the Ca2+ spark that describes local Na+ dynamics as governed by NaK pumps, neuronal INa, and NCX1. The model predicts the existence of Na+ sparks, which are generated by NCX1 and exhibit significantly slower dynamics as compared to Ca2+ sparks. Moreover, whole-cell simulations indicate that neuronal INa in the cardiac dyad plays a key role during the systolic phase. Rapid inward neuronal INa can elevate dyadic [Na+] to 35–40 mM, which drives reverse-mode NCX1 transport, and therefore promotes Ca2+ entry into the dyad, enhancing the trigger for JSR Ca2+ release. The specific role of decreased co-localization of NaK and NCX1 in ankyrin-B+/− cardiomyocytes was examined. Model results demonstrate that a reduction in the local NCX1- and NaK-mediated regulation of dyadic [Ca2+] and [Na+] results in an increase in Ca2+ spark activity during isoproterenol stimulation, which in turn stochastically activates NCX1 in the dyad. This alteration in NCX1/NaK co-localization interrupts the balance between NCX1 and NaK currents in a way that leads to enhanced depolarizing inward current during the action potential plateau, which ultimately leads to a higher probability of L-type Ca2+ channel reopening and arrhythmogenic early-afterdepolarizations.

Glutamate as a potential \u201csurvival factor\u201d in an in vitro model of neuronal hypoxia/reoxygenation injury: leading role of the Na+/Ca2+ exchanger

In brain ischemia, reduction in oxygen and substrates affects mitochondrial respiratory chain and aerobic metabolism, culminating in ATP production impairment, ionic imbalance, and cell death. The restoration of blood flow and reoxygenation are frequently associated with exacerbation of tissue injury, giving rise to ischemia/reperfusion (I/R) injury. In this setting, the imbalance of brain bioenergetics induces important metabolic adaptations, including utilization of alternative energy sources, such as glutamate. Although glutamate has long been considered as a neurotoxin, it can also be used as intermediary metabolite for ATP synthesis, and both the Na+/Ca2+ exchanger (NCX) and the Na+-dependent excitatory amino-acid transporters (EAATs) are essential in this pathway. Here we analyzed the role of NCX in the potential of glutamate to improve metabolism and survival of neuronal cells subjected to hypoxia/reoxygenation (H/R). In SH-SY5Y neuroblastoma cells differentiated into a neuron-like state, H/R produced a significant cell damage, a decrease in ATP cellular content, and intracellular Ca2+ alterations. Exposure to glutamate at the onset of the reoxygenation phase attenuated H/R-induced cell damage and evoked a significant raise in intracellular ATP levels. Furthermore, we found that in H/R cells NCX reverse-mode activity was reduced, and that glutamate limited such reduction. All the effects induced by glutamate supplementation were lost when cells were transfected with small interfering RNA against NCX1 and EAAT3, suggesting the need of a specific functional interplay between these proteins for glutamate-induced protection. Collectively, our results revealed the potential beneficial effect of glutamate in an in vitro model of H/R injury and focused on the essential role exerted by NCX1. Although preliminary, these findings could be a starting point to further investigate in in vivo systems such protective effect in ischemic settings, shedding a new light on the classical view of glutamate as detrimental factor.

Ca2+ Entry Through Reverse Mode Na+/Ca2+ Exchanger Contributes to Store Operated Channel-Mediated Neointima Formation After Arterial Injury

BackgroundNa+/Ca2+ exchange (NCX) reversal-mediated Ca2+ entry is a critical pathway for stimulating proliferation in many cell lines. However, the role of reverse-mode NCX1 in neointima formation and atherosclerosis remains unclear. The aims of the present study were to investigate the functional role of NCX1 in the pathogenesis of atherosclerosis and vascular smooth muscle cell (VSMC) proliferation, and to determine the interaction between NCX1 and store depletion in VSMCs. MethodsA rat balloon injury model was established to examine the effect of the knockdown of NCX1 on neointima formation after injury. VSMCs were cultured to verify that NCX1 knockdown suppressed serum-induced VSMC proliferation. ResultsThe results showed that balloon injury induced neointima formation and upregulated NCX1 expression at 7 and 14 days after injury in rat carotid arteries (1.18- and 1.45-fold, respectively). A lentivirus vector expressing short hairpin (sh)RNA against rat NCX1 dramatically downregulated NCX1, proliferating cell nuclear antigen (PCNA) and Ki-67 expression, and suppressed neointima formation in vivo (62% at 7 days and 70% at 14 days). KB-R7943 (an inhibitor of reverse-mode NCX1) and NCX1 knockdown significantly inhibited serum-induced VSMC proliferation (65% at 72 hours and 41% at 72 hours, respectively), determined according to PCNA and Ki-67 expression and cell counting in vitro, and markedly suppressed store depletion-mediated Ca2+ entry and peripheral cytosolic Na+ transients in VSMCs. ConclusionsReverse-mode NCX1 is activated by store depletion and is required for proliferative VSMC proliferation and neointima formation after arterial injury.

Critical contribution of Na+-Ca2+ exchanger to the Ca2+-mediated vasodilation activated in endothelial cells of resistance arteries.

Na+-Ca2+ exchanger (NCX) contributes to control the intracellular free Ca2+ concentration ([Ca2+]i), but the functional activation of NCX reverse mode (NCXrm) in endothelial cells is controversial. We evaluated the participation of NCXrm-mediated Ca2+ uptake in the endothelium-dependent vasodilation of rat isolated mesenteric arterial beds. In phenylephrine-contracted mesenteries, the acetylcholine (ACh)-induced vasodilation was abolished by treatment with the NCXrm blockers SEA0400, KB-R7943, or SN-6. Consistent with that, the ACh-induced hyperpolarization observed in primary cultures of mesenteric endothelial cells and in smooth muscle of isolated mesenteric resistance arteries was attenuated by KB-R7943 and SEA0400, respectively. In addition, both blockers abolished the NO production activated by ACh in intact mesenteric arteries. In contrast, the inhibition of NCXrm did not affect the vasodilator responses induced by the Ca2+ ionophore, ionomycin, and the NO donor, S-nitroso- N-acetylpenicillamine. Furthermore, SEA0400, KB-R7943, and a small interference RNA directed against NCX1 blunted the increase in [Ca2+]i induced by ACh or ATP in cultured endothelial cells. The analysis by proximity ligation assay showed that the NO-synthesizing enzyme, eNOS, and NCX1 were associated in endothelial cell caveolae of intact mesenteric resistance arteries. These results indicate that the activation of NCXrm has a central role in Ca2+-mediated vasodilation initiated by ACh in endothelial cells of resistance arteries.-Lillo, M. A., Gaete, P. S., Puebla, M., Ardiles, N. M., Poblete, I., Becerra, A., Simon, F., Figueroa, X. F. Critical contribution of Na+-Ca2+ exchanger to the Ca2+-mediated vasodilation activated in endothelial cells of resistance arteries.

Coupling between the TRPC3 ion channel and the NCX1 transporter contributed to VEGF-induced ERK1/2 activation and angiogenesis in human primary endothelial cells

It has been previously demonstrated that the bi-directional transporter Na+/Ca2+ exchanger (NCX) working in the reverse (Ca2+-influx) - mode promotes the activation of ERK1/2 in response to the key pro-angiogenic cytokine VEGF in human endothelial cells (ECs). However, the molecular event(s) that elicit NCX reversal in VEGF-stimulated ECs remain unclear. Here we investigated whether Na+ influx via the diacylglycerol (DAG) – activated non-selective cation channel TRPC3 was functionally associated with NCX and whether its activity was required for VEGF-induced ERK1/2 activation and angiogenesis. We provide evidence that TRPC3 inhibitors and siRNA attenuated ERK1/2 phosphorylation, reduced PKCα activity and partially suppressed Ca2+ transients in response to VEGF. Additionally, TRPC3 inhibitors and siRNA significantly suppressed endothelial tubular differentiation, an in vitro indicator of angiogenesis. We also report that simulating PLCγ activation downstream of VEGF receptor 2 by application of the cell-permeable DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) was sufficient to activate ERK1/2 and enhance tubular differentiation. OAG-induced ERK1/2 activation and tubulogenesis were significantly suppressed by TRPC3 and reverse-mode NCX inhibitors and siRNA. Moreover, whilst both reverse-mode NCX and TRPC3 inhibitors attenuated OAG-induced Ca2+ transients, only TRPC3 antagonists blunted Na+ influx in response to OAG. Importantly, when Na+ was increased in ECs by inhibiting the Na+-K+-ATPase, TRPC3 activity was dispensable for OAG-induced ERK1/2 phosphorylation. Collectively, our research suggests that DAG generation downstream of VEGF receptors activatesTRPC3 causing Na+ influx with subsequent reversal of NCX, ERK1/2 activation and ultimately contributes to enhanced angiogenesis. Targeting reverse-mode NCX and its upstream initiator TRPC3 could be clinically relevant in conditions characterised by abnormal VEGF signalling.

Effect of Docosahexaenoic Acid on Voltage-Independent Ca2+ Entry Pathways in Cultured Vascular Smooth Muscle Cells Stimulated with 5-Hydroxytryptamine.

We previously reported that docosahexaenoic acid (DHA) inhibits an increase in intracellular Ca2+ concentration ([Ca2+]i) in cultured rat vascular smooth muscle cells (VSMCs) through a mechanism involving mainly voltage-dependent Ca2+ channels; however, the effect of DHA on voltage-independent pathways, such as store-operated and receptor-operated Ca2+ entry, and Ca2+ entry through Na+/Ca2+ exchanger (NCX), has not been clarified. In the present study, we investigated the effect of DHA treatment on the expression of transient receptor potential canonical (TRPC) channels, capacitative Ca2+ entry, and Ca2+ entry through NCX in rat cultured VSMCs stimulated with 5-hydroxytryptamine (5-HT). RT-PCR analysis detected TRPC1, TRPC4, and TRPC6 mRNA in cultured VSMCs. DHA treatment for 2 d slightly but significantly decreased TRPC1, but not TRPC4 and TRPC6, mRNA expression. Sarpogrelate, a selective serotonin 5-HT2A receptor inhibitor, completely inhibited the 5-HT-induced increase in [Ca2+]i in cultured VSMCs. Ca2+ influx by adding extracellular Ca2+ (1.3 mM) to the Ca2+-free condition in the presence of 5-HT was partially but significantly inhibited by sarpogrelate. DHA treatment for 2 d had no effect on Ca2+ influx when extracellular Ca2+ was added to the Ca2+-free condition in the presence of either 5-HT alone or 5-HT with sarpogrelate. KB-R7943, a selective inhibitor of reverse mode NCX, significantly suppressed the 5-HT-induced increase of [Ca2+]i. Furthermore, DHA treatment for 2 d significantly decreased NCX1 mRNA expression. These results suggest that DHA seems to have little effect on capacitative Ca2+ entry. Through decreasing NCX1 expression, DHA may suppress the 5-HT-induced increase in [Ca2+]i.

The effect of neropeptide substance P receptor antagonist can relieve the current of reverse-mode NCX in airway smooth muscle cells

Objective Investigate the effect of substance P and receptor antagonist on the current of NCX in ASMC. Methods Primary rat ASMC cultures were established.Immunofluorescence was used to identify ASMCs.Patch clamp exam was used to assess NCX currents in ASMC before and after intervented by Ach, substance P, NK-1R receptor antagonist and nimodiping, Voltage-current curves were drawn according to variation of voltage and current.PA indicates current amplitude, PF indicates cell area, and PA/PF indicates current density. Results As the voltage increased, the current density increased.When voltage up to-40mV the reverse current appeard.When the voltage is 60mV the current density in the Ach intervention group was highest, but lowest in the nimodipine intervention group; the current density in the substance P intervention group is more than in control group but lower than in Ach interevention group.The current density in the substance P receptor antagonist intervention group higher than nimodipine interevention goup, but lower than the other group(P<0.05). Conclusion In asthma airways, Ca2+ overload is related to the appearance of reverse-mode NCX.NK-1R antagonist plays a role in decreasing the Ca2+ concentration, which can relieve the current of reverse-mode NCX, and may benefit alleviating airway inflammation and responsiveness.As a result, NK-1R antagonist may be an attractive target for therapeutic approaches to asthma. Key words: Substance P; Receptor antagonist; Asthma; Airway smooth muscle cell; Sodium-calcium exchang

The reverse-mode NCX1 activity inhibitor KB-R7943 promotes prostate cancer cell death by activating the JNK pathway and blocking autophagic flux.

We explored the effects of KB-R7943, an inhibitor of reverse-mode NCX1 activity, in prostate cancer (PCa). NCX1 was overexpressed in PCa tissues and cell lines, and higher NCX1 levels were associated higher PCa grades. At concentrations greater than 10 μM, KB-R7943 dose-dependently decreased PC3 and LNCaP cell viability. KB-R7943 also increased cell cycle G1/S phase arrest and induced apoptosis in PC3 cells. KB-R7943 increased autophagosome accumulation in PCa cells as indicated by increases in LC3-II levels and eGFP-LC3 puncta. Combined treatment with chloroquine (CQ) and KB-R7943 decreased P62 and increased LC3-II protein levels in PC3 cells, indicating that KB-R7943 blocked autophagic flux. KB-R7943 induced autophagosome accumulation mainly by downregulating the PI3K/AKT/m-TOR pathway and upregulating the JNK pathway. In xenograft experiments, KB-R7943 inhibited tumor growth. Combined treatment with KB-R7943 and an autophagy inhibitor inhibited growth and increased apoptosis. These results indicate that KB-R7943 promotes cell death in PCa by activating the JNK signaling pathway and blocking autophagic flux.