Exchange Inhibitor Research Articles

Pyrazine ring-based Na+/H+ exchanger (NHE) inhibitors potently inhibit cancer cell growth in 3D culture, independent of NHE1

The Na+/H+ exchanger-1 (NHE1) supports tumour growth, making NHE1 inhibitors of interest in anticancer therapy, yet their molecular effects are incompletely characterized. Here, we demonstrate that widely used pyrazinoylguanidine-type NHE1 inhibitors potently inhibit growth and survival of cancer cell spheroids, in a manner unrelated to NHE1 inhibition. Cancer and non-cancer cells were grown as 3-dimensional (3D) spheroids and treated with pyrazinoylguanidine-type (amiloride, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), 5-(N,N-dimethyl)-amiloride (DMA), and 5-(N,N-hexamethylene)-amiloride (HMA)) or benzoylguanidine-type (eniporide, cariporide) NHE1 inhibitors for 2–7 days, followed by analyses of viability, compound accumulation, and stress- and death-associated signalling. EIPA, DMA and HMA dose-dependently reduced breast cancer spheroid viability while cariporide and eniporide had no effect. Although both compound types inhibited NHE1, the toxic effects were NHE1-independent, as inhibitor-induced viability loss was unaffected by NHE1 CRISPR/Cas9 knockout. EIPA and HMA accumulated extensively in spheroids, and this was associated with marked vacuolization, apparent autophagic arrest, ER stress, mitochondrial- and DNA damage and poly-ADP-ribose-polymerase (PARP) cleavage, indicative of severe stress and paraptosis-like cell death. Pyrazinoylguanidine-induced cell death was partially additive to that induced by conventional anticancer therapies and strongly additive to extracellular-signal-regulated-kinase (ERK) pathway inhibition. Thus, in addition to inhibiting NHE1, pyrazinoylguanidines exert potent, NHE1-independent cancer cell death, pointing to a novel relevance for these compounds in anticancer therapy.

Empagliflozin Improves Cardiac Functional Recovery after Prolonged Cold Storage of Donor Hearts in an Isolated Working Rat Heart Model

Many donor hearts are unsuitable for transplantation after prolonged cold storage or donation after circulatory death. Improved donor heart recovery is observed after supplementation of preservation solutions with sodium-hydrogen exchange inhibitors (NHEI) cariporide/zoniporide (Gao et al, 2010, BJP; Cropper et al, 2003, JHLT). NHEI are not clinically used due to neurotoxicity observed in cariporide clinical trials however the search for suitable NHEI alternatives continues. One promising alternative is the sodium-glucose cotransporter 2 inhibitor Empagliflozin (Empa) - the first anti-diabetic drug to yield positive cardiovascular outcomes. Studies have shown Empa mimics NHEI and slows pH recovery in cardiomyocytes following an acidic load. This study tests the cardioprotective efficacy of Empa-supplementation in cardiac preservation solution during prolonged cold storage of donor hearts. Wistar rat (320-420g; n=5-7) hearts were perfused ex-vivo and baseline hemodynamic measurements acquired. Hearts were arrested and stored in Celsior±0.3-10µM Empa, or zoniporide (1µM) (6h, 4°C) then reperfused (37°C, Langendorff 15min, working 30min). Post-reperfusion aortic flow (AF) and cardiac output (CO) were expressed as a percentage of baseline measurements. Coronary effluent (pre-storage, 15, 30, and 45 min reperfusion) was assessed for lactate dehydrogenase (LDH) release. Donor hearts supplemented with Empa during cold storage showed a dose-dependent improvement in cardiac functional recovery. Compared to unsupplemented control hearts, 10µM Empa-supplemented hearts showed significantly improved AF (54±11% vs 10±7%, p=0.006) and CO (55±12 vs 16±8, p=0.03) recovery. Hearts supplemented with 1µM zoniporide also showed significantly improved AF (63±7%, p=0.002 vs control) and CO (65±9%, p=0.01) recovery. A trend for reduced LDH efflux was observed in hearts supplemented with 1-10µM Empa or zoniporide compared to control hearts. Supplementation of cardiac preservation solution with 10µM Empa yields significantly improved functional recovery after prolonged cold storage, with similar cardioprotective efficacy to the NHEI zoniporide. Given the approved clinical use of Empa, this study highlights its potential as a suitable alternative for NHEI in donor heart preservation.

Homogeneous Dual-Parametric-Coupled Assay for Simultaneous Nucleotide Exchange and KRAS/RAF-RBD Interaction Monitoring.

We have developed a rapid and sensitive single-well dual-parametric method introduced in linked RAS nucleotide exchange and RAS/RAF-RBD interaction assays. RAS mutations are frequent drivers of multiple different human cancers, but the development of therapeutic strategies has been challenging. Traditionally, efforts to disrupt the RAS function have focused on nucleotide exchange inhibitors, GTP-RAS interaction inhibitors, and activators increasing GTPase activity of mutant RAS proteins. As the amount of biological knowledge grows, targeted biochemical assays enabling high-throughput screening have become increasingly interesting. We have previously introduced a homogeneous quenching resonance energy transfer (QRET) assay for nucleotide binding studies with RAS and heterotrimeric G proteins. Here, we introduce a novel homogeneous signaling technique called QTR-FRET, which combine QRET technology and time-resolved Förster resonance energy transfer (TR-FRET). The dual-parametric QTR-FRET technique enables the linking of guanine nucleotide exchange factor-induced Eu3+-GTP association to RAS, monitored at 615 nm, and subsequent Eu3+-GTP-loaded RAS interaction with RAF-RBD-Alexa680 monitored at 730 nm. Both reactions were monitored in a single-well assay applicable for inhibitor screening and real-time reaction monitoring. This homogeneous assay enables separable detection of both nucleotide exchange and RAS/RAF interaction inhibitors using low nanomolar protein concentrations. To demonstrate a wider applicability as a screening and real-time reaction monitoring method, the QTR-FRET technique was also applied for G(i)α GTP-loading and pertussis toxin-catalyzed ADP-ribosylation of G(i)α, for which we synthesized a novel γ-GTP-Eu3+ molecule. The study indicates that the QTR-FRET detection technique presented here can be readily applied to dual-parametric assays for various targets.

Novel Na+/Ca2+ Exchanger Inhibitor ORM-10962 Supports Coupled Function of Funny-Current and Na+/Ca2+ Exchanger in Pacemaking of Rabbit Sinus Node Tissue.

Background and PurposeThe exact mechanism of spontaneous pacemaking is not fully understood. Recent results suggest tight cooperation between intracellular Ca2+ handling and sarcolemmal ion channels. An important player of this crosstalk is the Na+/Ca2+ exchanger (NCX), however, direct pharmacological evidence was unavailable so far because of the lack of a selective inhibitor. We investigated the role of the NCX current in pacemaking and analyzed the functional consequences of the If-NCX coupling by applying the novel selective NCX inhibitor ORM-10962 on the sinus node (SAN).Experimental ApproachCurrents were measured by patch-clamp, Ca2+-transients were monitored by fluorescent optical method in rabbit SAN cells. Action potentials (AP) were recorded from rabbit SAN tissue preparations. Mechanistic computational data were obtained using the Yaniv et al. SAN model.Key ResultsORM-10962 (ORM) marginally reduced the SAN pacemaking cycle length with a marked increase in the diastolic Ca2+ level as well as the transient amplitude. The bradycardic effect of NCX inhibition was augmented when the funny-current (If) was previously inhibited and vice versa, the effect of If was augmented when the Ca2+ handling was suppressed.Conclusion and ImplicationsWe confirmed the contribution of the NCX current to cardiac pacemaking using a novel NCX inhibitor. Our experimental and modeling data support a close cooperation between If and NCX providing an important functional consequence: these currents together establish a strong depolarization capacity providing important safety factor for stable pacemaking. Thus, after individual inhibition of If or NCX, excessive bradycardia or instability cannot be expected because each of these currents may compensate for the reduction of the other providing safe and rhythmic SAN pacemaking.

Thiol/disulfide exchange occurs in rotavirus structural proteins during contact with intestinal villus cell surface.

Protein disulfide isomerase (PDI) is an enzyme that catalyzes disulfide bond reduction or formation and rearrangements of disulfide bridges, and also functions as a chaperone. During entry of some of the viruses PDI participates in thiol-disulfide exchange. Previous reports show that rotavirus entry is interfered by impermeant thiol/disulfide exchange inhibitors and antibodies against PDI. Our objective was to assess the interaction between PDI and triple-layered particles (TLPs) from rotavirus strains ECwt and RRV and from a human rotavirus isolate (HI) during the early steps of virus entry in a system of isolated small intestinal villi. Purified soluble PDI was incubated with either isolated intestinal villi or cell membrane-enriched fractions in the presence or absence of thiol/disulfide inhibitors such as bacitracin, DTNB or N- ethylmaleimide followed by the assessment of the PDI interactions with TLPs and rotavirus structural proteins in terms of their redox state changes. Soluble and membrane-bound PDI was found to interact with TLPs from all the rotaviruses assayed and also with the isolated structural proteins represented by the recombinant rVP5* (a tryptic cleavage product of VP4), rVP6 and the native VP7. PDI interaction with TLPs and rotavirus structural proteins was decreased by the presence of thiol/disulfide exchange inhibitors. Interactions of cell membrane-enriched fractions with TLPs produced rearrangements in the disulfide bridges of rotavirus structural proteins. We conclude that PDI interacts with rotavirus virions through redox reactions that could facilitate the rotavirus entry into the host cell. Keywords: cell surface PDI; thiol-disulfide exchange; rotavirus TLPs; virus entry; bacitracin; DTNB.

Placebo and Nocebo Responses in Randomized, Controlled Trials of Medications for ADHD: A Systematic Review and Meta-Analysis

Background: The nature and magnitude of placebo and nocebo responses to ADHD medications is unclear. The extent to which response to active medications and placebo are inter-correlated has not been explored. Methods: We searched scientific literature until June 26, 2019 for published/unpublished double-blind, randomised placebo-controlled trials (RCTs) of ADHD medications in children, adolescents and adults. Authors and drug manufacturers were contacted for additional information. We assessed placebo effects on efficacy and nocebo effects on tolerability outcomes using aggregate data from trials and random-effects meta-analysis. We assessed the association of study design and patient features with placebo/nocebo response. Findings: We analysed 128 RCTs and found significant and heterogenous placebo effects for all efficacy outcomes, with no publication bias. We found nocebo effects on some tolerability outcomes. Apart from self-ratings, efficacy outcomes from other raters showed significant positive correlations between the baseline to endpoint placebo effects and the baseline to endpoint drug effects, suggesting that response to placebo and ADHD medications are influenced by similar non-specific factors. Baseline severity and type of rating scale influenced the magnitude of placebo response. Interpretation: Placebo effects are highly significant and response to placebo and ADHD medications are correlated, suggesting that shared non-specific factors influence response to both placebo and active medication. Although ADHD medications are superior to placebo, and placebo treatment in clinical practice is not feasible, clinicians should attempt to incorporate factors associated with placebo effects into clinical care, and future studies should explore how such effects influence response to medication treatment. Funding Statement: This was not a funded study. Declaration of Interests: In the past year, Dr. Faraone received income, potential income, travel expenses continuing education support and/or research support from Takeda, OnDosis, Tris, Otsuka, Arbor, Ironshore, Rhodes, Akili Interactive Labs, Enzymotec, Sunovion, Supernus and Genomind. With his institution, he has US patent US20130217707 A1 for the use of sodium-hydrogen exchange inhibitors in the treatment of ADHD. He also receives royalties from books published by Guilford Press: Straight Talk about Your Child’s Mental Health, Oxford University Press: Schizophrenia: The Facts and Elsevier: ADHD: Non-Pharmacologic Interventions. He is Program Director of www.adhdinadults.com. In the past year, Dr. Newcorn is/has been an advisor and/or consultant for Adlon Therapeutics, Arbor, Eisai, NLS, OnDosis, Rhodes, Shire/Takeda, and Supernus. He was a DSMB member for Pfizer and Sunovion, and received research funds from Otsuka, Shire and Supernus. He also has received speaker fees from Shire/Takeda for disease-state presentations and served as a consultant for the US National Football League. Dr. Cipriani has received research and consultancy fees from INCiPiT (Italian Network for Paediatric Trials), CARIPLO Foundation and Angelini Pharma. Dr. Brandeis serves as an unpaid scientific consultant for an EUfunded neurofeedback trial. Anna Kaiser reports has no financial disclosures. Sarah Hohmann has no financial disclosures. Dr. Hage received conference support, speaker´s fee and/or served in an advisory role for Shire/Takeda and Lily. He was involved as investigator in clinical trials by Shire, Janssen-Cilag, Otsuka, Sunovion, Servier, Lundbeck, Takeda,Nuvelution, Gedeon Richter, and Emalex. The present work is unrelated to the above relationships. Dr. Cortese declares reimbursement for travel and accommodation expenses from the Association for Child and Adolescent Central Health (ACAMH) in relation to lectures delivered for ACAMH, Canadian ADHD Resource Alliance (CADDRA), British Association of Psychopharmacology (BAP), and from Healthcare Convention for educational activity on ADHD.

Palmitic Acid Methyl Ester Induces G2/M Arrest in Human Bone Marrow-Derived Mesenchymal Stem Cells via the p53/p21 Pathway.

Bone marrow-derived mesenchymal cells (BM-MSCs) are able to differentiate into adipocytes, which can secrete adipokines to affect BM-MSC proliferation and differentiation. Recent evidences indicated that adipocytes can secrete fatty acid metabolites, such as palmitic acid methyl ester (PAME), which is able to cause vasorelaxation and exerts anti-inflammatory effects. However, effects of PAME on BM-MSC proliferation remain unclear. The aim of this study was to investigate the effect of PAME on human BM-MSC (hBM-MSC) proliferation and its underlying molecular mechanisms. hBM-MSCs were treated with PAME for 48 h and then subjected to various analyses. The results from the present study show that PAME significantly reduced the levels of G2/M phase regulatory proteins, cyclin-dependent kinase 1 (Cdk1), and cyclin B1 and inhibited proliferation in hBM-MSCs. Moreover, the level of Mdm2 protein decreased, while the levels of p21 and p53 protein increased in the PAME-treated hBM-MSCs. However, PAME treatment did not significantly affect apoptosis/necrosis, ROS generation, and the level of Cdc25C protein. PAME also induced intracellular acidosis and increased intracellular Ca2+ levels. Cotreatment with PAME and Na+/H+ exchanger inhibitors together further reduced the intracellular pH but did not affect the PAME-induced decreases of cell proliferation and increases of the cell population at the G2/M phase. Cotreatment with PAME and a calcium chelator together inhibited the PAME-increased intracellular Ca2+ levels but did not affect the PAME-induced cell proliferation inhibition and G2/M cell cycle arrest. Moreover, the half-life of p53 protein was prolonged in the PAME-treated hBM-MSCs. Taken together, these results suggest that PAME induced p53 stabilization, which in turn increased the levels of p53/p21 proteins and decreased the levels of Cdk1/cyclin B1 proteins, thereby preventing the activation of Cdk1, and eventually caused cell cycle arrest at the G2/M phase. The findings from the present study might help get insight into the physiological roles of PAME in regulating hBM-MSC proliferation.

Long-term effects of Na+ /Ca2+ exchanger inhibition with ORM-11035 improves cardiac function and remodelling without lowering blood pressure in a model of heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is increasingly common but there is currently no established pharmacological therapy. We hypothesized that ORM-11035, a novel specific Na+ /Ca2+ exchanger (NCX) inhibitor, improves cardiac function and remodelling independent of effects on arterial blood pressure in a model of cardiorenal HFpEF. Rats were subjected to subtotal nephrectomy (NXT) or sham operation. Eight weeks after intervention, treatment for 16 weeks with ORM-11035 (1mg/kg body weight) or vehicle was initiated. At 24 weeks, blood pressure measurements, echocardiography and pressure-volume loops were performed. Contractile function, Ca2+ transients and NCX-mediated Ca2+ extrusion were measured in isolated ventricular cardiomyocytes. NXT rats (untreated) showed a HFpEF phenotype with left ventricular (LV) hypertrophy, LV end-diastolic pressure (LVEDP) elevation, increased brain natriuretic peptide (BNP) levels, preserved ejection fraction and pulmonary congestion. In cardiomyocytes from untreated NXT rats, early relaxation was prolonged and NCX-mediated Ca2+ extrusion was decreased. Chronic treatment with ORM-11035 significantly reduced LV hypertrophy and cardiac remodelling without lowering systolic blood pressure. LVEDP [14 ± 3 vs. 9± 2 mmHg; NXT (n= 12) vs. NXT + ORM (n= 12); P= 0.0002] and BNP levels [71 ± 12 vs. 49 ± 11 pg/mL; NXT (n= 12) vs. NXT + ORM (n= 12); P< 0.0001] were reduced after ORM treatment. LV cardiomyocytes from ORM-treated rats showed improved active relaxation and diastolic cytosolic Ca2+ decay as well as restored NCX-mediated Ca2+ removal, indicating NCX modulation with ORM-11035 as a promising target in the treatment of HFpEF. Chronic inhibition of NCX with ORM-11035 significantly attenuated cardiac remodelling and diastolic dysfunction without lowering systemic blood pressure in this model of HFpEF. Therefore, long-term treatment with selective NCX inhibitors such as ORM-11035 should be evaluated further in the treatment of heart failure.

Mechanisms of orexin 2 receptor-mediated depolarization in the rat paraventricular nucleus of the hypothalamus

The paraventricular nucleus of the hypothalamus (PVN) contains dense orexin 2 (OX2) receptor. We examined the mechanisms of OX2 receptor-mediated excitation on electrophysiologically identified type I (putative magnocellular), low-threshold spikes (LTS)-expressing type II (putative preautonomic), and non-LTS type II (putative parvocellular neuroendocrine) neurons. In the presence of tetrodotoxin, an OX2 receptor agonist, ALOXB (30–1000 nM) depolarized 56% of type I, and 73–75% of type II neurons. In type I neurons, ALOXB-induced inward current displayed increased-conductance current-voltage (I–V) relationship and reversed polarity at −27.5 ± 4.8 mV. A Na+-Ca2+ exchanger (NCX) inhibitor, KBR-7943, attenuated ALOXB responses in the majority of type I neurons, while no attenuation was observed in nearly all type II neurons. Type II neurons exhibited three types of I–V relationships in response to ALOXB, characterized by decreased, increased, and unchanged conductance, respectively. The reversal potential of the decreased-conductance responses was near the equilibrium potential of K+ (Ek+) and became more positive in a high-K+ solution, suggesting that K+ conductance blockade is involved. In a low-Na+ solution, non-reversed I–V curves of increased-conductance responses became decreased-conductance responses and reversed polarity near Ek+, suggesting the involvement of both K+ conductance and non-selective cation conductance (NSCC). Approximately 35% of LTS-expressing type II neurons were vasopressin-immunoreactive and 71% of them responded to ALOXB. In conclusion, orexins may activate OX2 receptor on PVN neurons and cause depolarization by promoting NCX and/or NSCC in magnocellular neurons, and by decreasing K+ conductance and/or increasing NSCC in parvocellular neurons. Furthermore, the majority of vasopressinergic preautonomic neurons are under OX2 receptor regulation.

Novel insights into the potential role of ion transport in sensory perception in Acanthamoeba

BackgroundAcanthamoeba is well known to produce a blinding keratitis and serious brain infection known as encephalitis. Effective treatment is problematic, and can continue up to a year, and even then, recurrence can ensue. Partly, this is due to the capability of vegetative amoebae to convert into resistant cysts. Cysts can persist in an inactive form for decades while retaining their pathogenicity. It is not clear how Acanthamoeba cysts monitor environmental changes, and determine favourable conditions leading to their emergence as viable trophozoites.MethodsThe role of ion transporters in the encystation and excystation of Acanthamoeba remains unclear. Here, we investigated the role of sodium, potassium and calcium ion transporters as well as proton pump inhibitors on A. castellanii encystation and excystation and their effects on trophozoites.ResultsRemarkably 3′,4′-dichlorobenzamil hydrochloride a sodium–calcium exchange inhibitor, completely abolished excystation of Acanthamoeba. Furthermore, lanthanum oxide and stevioside hydrate, both potassium transport inhibitors, resulted in the partial inhibition of Acanthamoeba excystation. Conversely, none of the ion transport inhibitors affected encystation or had any effects on Acanthamoeba trophozoites viability.ConclusionsThe present study indicates that ion transporters are involved in sensory perception of A. castellanii suggesting their value as potential therapeutic targets to block cellular differentiation that presents a significant challenge in the successful prognosis of Acanthamoeba infections.

Cardiac Na+/Ca2+ exchange stimulators among cardioprotective drugs.

We previously reviewed our study of the pharmacological properties of cardiac Na+/Ca2+ exchange (NCX1) inhibitors among cardioprotective drugs, such as amiodarone, bepridil, dronedarone, cibenzoline, azimilide, aprindine, and benzyl-oxyphenyl derivatives (Watanabe et al. in J Pharmacol Sci 102:7-16, 2006). Since then we have continued our studies further and found that some cardioprotective drugs are NCX1 stimulators. Cardiac Na+/Ca2+ exchange current (INCX1) was stimulated by nicorandil (a hybrid ATP-sensitive K+ channel opener), pinacidil (a non-selective ATP-sensitive K+ channel opener), flecainide (an antiarrhythmic drug), and sodium nitroprusside (SNP) (an NO donor). Sildenafil (a phosphodiesterase-5 inhibitor) further increased the pinacidil-induced augmentation of INCX1. In paper, here I review the NCX stimulants that enhance NCX function among the cardioprotective agents we examined such as nicorandil, pinacidil, SNP, sildenafil and flecainide, in addition to atrial natriuretic (ANP) and dofetilide, which were reported by other investigators.

Orexin-A Excites Airway Vagal Preganglionic Neurons via Activation of Orexin Receptor Type 1 and Type 2 in Rats.

Airway vagal nerves play a predominant role in the neural control of the airway, and augmented airway vagal activity is known to play important roles in the pathogenesis of some chronic inflammatory airway diseases. Several lines of evidence indicate that dysfunctional central orexinergic system is closely related to the severity of airway diseases, however, whether orexins affect airway vagal activity is unknown. This study investigates whether and how orexin-A regulates the activity of medullary airway vagal preganglionic neurons (AVPNs). The expression of orexin receptor type 1 (OX1R) and type 2 (OX2R) was examined using immunofluorescent staining. The effects of orexin-A on functionally identified inspiratory-activated AVPNs (IA-AVPNs), which are critical in the control of airway smooth muscle, were examined using patch-clamp in medullary slices of neonatal rats. Airway vagal response to injection of orexin-A into the magna cisterna was examined using plethysmography in juvenile rats. The results show that retrogradely labeled AVPNs were immunoreactive to anti-OX1R antibody and anti-OX2R antibody. Orexin-A dose-dependently depolarized IA-AVPNs and increased their firing rate. In synaptically isolated IA-AVPNs, the depolarization induced by orexin-A was blocked partially by OX1R antagonist SB-334867 or OX2R antagonist TCS OX2 29 alone, and completely by co-application of both antagonists. The orexin-A-induced depolarization was also mostly blocked by Na+/Ca2+ exchanger inhibitor KB-R7943. Orexin-A facilitated the glutamatergic, glycinergic and GABAergic inputs to IA-AVPNs, and the facilitation of each type of input was blocked partially by SB-334867 or TCS OX2 29 alone, and completely by co-application of both antagonists. Injection of orexin-A into the magna cisterna of juvenile rats significantly increased the inspiratory and expiratory resistance of the airway and consequently decreased the dynamic compliance of the lungs, all of which were prevented by atropine sulfate or bilateral vagotomy. These results demonstrate that orexin-A excites IA-AVPNs via activation of both OX1R and OX2R, and suggest that increased central synthesis/release of orexins might participate in the pathogenesis of airway diseases via over-activation of AVPNs.

P4466Empagliflozin improves heart function after myocardial infarction in the rat

Abstract Background Selective Sodium-glucose cotransporter 2 (SGLT2) inhibition with Empagliflozin reduced progression of left ventricular dysfunction and improved tolerance of physical exercise in heart failure by normoglycemic rats. Here, we hypothesized that Empagliflozin prevents cardiac dysfunction after myocardial infarction (MI). Purpose This study aimed to investigate whether Empagliflozin protects the heart in the early phase after experimental MI in normoglycemic rats. Methods MI was induced in Wistar rats via permanent ligation of the left coronary artery. Treatment with Empagliflozin (1 mg/kg/daily per os) was started after MI and continued for 7 days. Sham operated and vehicle treated animals served as controls (n=8). Hemodynamic parameters were measured via transthoracic echocardiography and intracardiac Samba catheter. Glucose concentration was determined in serum and urine. Protein expression of Na+/H+ exchanger isoform-1 (NHE-1), sodium bicarbonate co-transporter (NBC), Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), transforming growth factor beta 1 (TGF-beta1), Smad2 in the left ventricle were also studied. Additionally, NHE-1 regulation was investigated in cardiomyocyte cell line H9c2. Results Systolic heart function was improved in Empagliflozin treated MI animals compared to vehicle as demonstrated by Global Longitudinal Strain (GLS) (20,9% vs. 16,6%; p&lt;0.05). E/A ratio was decreased by tendency and blood pressure was not affected. Ejection fraction (p&lt;0.05), fractional shortening (p&lt;0.05), stroke volume (p&lt;0.01) were increased in Empagliflozin treated control rats as compared with the sham group. Moreover, application of Empagliflozin (1mg/kg, i.v. bolus) to healthy rats in 30 min increased maximal pressure in the left ventricle as compared with vehicle (110,5±15,3 mmHg vs 79,1±11,9 mmHg; p&lt;0.05). Parallel, dP/dtmax was increased while dP/dtmin was decreased by tendency. Empagliflozin treatment did not affect glucose concentration in serum and urine. Treatment of cardiac H9c2 cells with Empagliflozin (1μM) down-regulated NHE-1 by 27%. Conclusion SGLT2 inhibitor Empagliflozin improved systolic function in the early phase post MI independently from glucose regulation. The cardioprotective mechanisms of SGLT2 inhibitors may involve cardiac NHE-1 exchanger inhibition.

Benzamil inhibits neuronal and heterologously expressed small conductance Ca2+-activated K+ channels

Small conductance Ca2+-activated K+ (SK) channels are expressed throughout the soma and dendrites of pyramidal neurons in the neocortex and hippocampal formation, where they participate in the local regulation of membrane excitability and synaptic signals. Through their inter-play with Ca2+ channels, SK channels regulate Ca2+ influx triggered by back-propagating action potentials in dendrites. Inhibition of SK channels affects both the amplitude and duration of Ca2+ transients, but the role of Ca2+ clearance mechanisms and their link to SK channel activity has not been established. Here we report the effect of the Na+/Ca2+ exchanger (NCX) inhibitor benzamil on Ca2+ extrusion and SK channels in the regulation of dendritic Ca2+ signals. Benzamil increased the duration and amplitude of dendritic Ca2+ transients elicited by back-propagating action potentials in hippocampal pyramidal neurons. This data is consistent with previous studies with SK channel blockers and suggests that benzamil inhibits SK channels in addition to the Na+/Ca2+ exchanger. Here we show that indeed both the neuronal SK-mediated IAHP current and the currents mediated by heterologously expressed SK channels were inhibited by benzamil. The inhibition of recombinant SK channels was seen with different K+ concentration gradients, and was stronger at negative voltages. The suppression of SK channels by benzamil is consistent with previous findings on the modulation of Ca2+ signals by SK channels in neurons. We additionally show that benzamil inhibits neuronal voltage-gated calcium currents. The results prompt a careful reassessment of the effects of benzamil on Ca2+ transients in native systems, given the spectrum of ion channels and exchangers this compound targets within a similar range of concentrations.

Development of a Novel Sodium-Hydrogen Exchanger Inhibitor for Heart Failure

This study was designed to determine the potential therapeutic effects of a new sodium-hydrogen exchanger (NHE-1) inhibitor in the rat coronary artery ligation model of chronic heart failure. After the induction of acute myocardial infarction, rats were entered into a randomly designed pilot dose study from 0.3 mg/kg, 1.0 mg/kg, and 3.0 mg/kg. Solid state micrometer hemodynamics, echocardiographic, and pressure-volume relationships were measured after 6 weeks of treatment. Treatment with this NHE-¬1 inhibitor at 3 mg/kg increased (P<0.05) ejection fraction from 23±3% (N=6) to 33±2% (N=13), while the 1 mg/kg dose decreased (P<0.05) the infarct size in CHF rats from 21.7±1.4% (N=7) to 15.9±0.7% (N=3) and prevented (P<0.05) dilata¬tion of the left ventricle in CHF rats in diastole (1.0±0.1 cm, N=6) to 0.9±0.1 cm, N=10) and in systole (0.9±0.1 cm, N=6) to 0.8±0.1, N=10). These study results suggest that this new NHE-1 inhibitor may be potentially useful in treating CHF with an improvement in maladaptive left ventricle remodeling. Because the mechanism of action of this agent is entirely different than the currently applied approach in treating CHF that focuses on aggressive neuro-hormonal blockade and because this agent does not adversely affect important hemodynamic variables, further investigations with this agent may be warranted.

Front Cover: Catalytic Asymmetric Hydrogenation of Dehydroamino Acid Esters with Biscarbamate Protection and Its Application to the Synthesis of xCT Inhibitors (Chem. Eur. J. 20/2019)

A new cystine-glutamate exchanger inhibitor was created through the development of a new catalytic asymmetric synthesis method. Rh-catalyzed catalytic asymmetric hydrogenation of dehydro amino acid esters was expanded to the synthesis of chiral amino acid esters with bis-carbamate protection. The new chemistry was successfully applied to the synthesis of N-benzoyl diamino suberic acid, which showed potent inhibitory effects for cystine-glutamate exchangers, a promising molecular target for cancer therapy. More information can be found in the Communication by T. Shinada et al. on page 5145.

Functional Role of Basolateral ClC-2 Channels in the Regulation of Duodenal Anion Secretion in Mice.

Although ClC-2 channels are important in colonic Cl- secretion, it is unclear about their roles in small intestinal anion secretion. Therefore, we sought to examine whether ClC-2 channels play important roles in anion secretion, particularly duodenal bicarbonate secretion (DBS). Duodenal mucosae from mice were stripped of seromuscular layers and mounted in Ussing chambers. Both duodenal short-circuit current (Isc) and HCO3- secretion in vitro were simultaneously recorded. DBS in vivo was measured by a CO2-sensitive electrode. Lubiprostone, a selective ClC-2 activator, concentration-dependently increased both duodenal Isc and DBS only when applied basolaterally, but not when applied apically. Removal of extracellular Cl- abolished lubiprostone-induced duodenal Isc, but did not alter HCO3- secretion even in the presence of DIDS, a Cl-/HCO3- exchanger inhibitor. However, further addition of glibenclamide, a CFTR channel blocker, abolished lubiprostone-evoked HCO3- secretion. Moreover, lubiprostone-induced HCO3- secretion was impaired in CFTR-/- mice compared to wild-type littermates. Luminal perfusion of duodenal lumen with lubiprostone did not alter basal DBS in vivo, but lubiprostone (i.p.) was able to induce DBS, which was also significantly inhibited by Cd2+, a ClC-2 channel blocker. [Ca2+]cyt level, Ca2+-activated K+ channel- and cAMP-mediated duodenal Isc, and HCO3- secretion were unchanged by lubiprostone. We have provided the first evidence for the novel functional role of basolateral ClC-2 channels in the regulation of duodenal anion secretion.

Catalytic Asymmetric Hydrogenation of Dehydroamino Acid Esters with Biscarbamate Protection and Its Application to the Synthesis of xCT Inhibitors.

Catalytic asymmetric hydrogenation of dehydroamino acid esters with biscarbamate protection was examined for the first time to prepare optically active amino acids. The new method was successfully applied to the synthesis of new cystine-glutamate exchanger inhibitors.

TRPA1 channel contributes to myocardial ischemia-reperfusion injury.

Myocardial ischemia-reperfusion (I/R) results in the generation of free radicals, accumulation of lipid peroxidation-derived unsaturated aldehydes, variable angina (pain), and infarction. The transient receptor potential ankyrin 1 (TRPA1) mediates pain signaling and is activated by unsaturated aldehydes, including acrolein and 4-hydroxynonenal. The contribution of TRPA1 (a Ca2+-permeable channel) to I/R-induced myocardial injury is unknown. We tested the hypothesis that cardiac TRPA1 confers myocyte sensitivity to aldehyde accumulation and promotes I/R injury. Although basal cardiovascular function in TRPA1-null mice was similar to that in wild-type (WT) mice, infarct size was significantly smaller in TRPA1-null mice than in WT mice (34.1 ± 9.3 vs. 14.3 ± 9.9% of the risk region, n = 8 and 7, respectively, P < 0.05), despite a similar I/R-induced area at risk (40.3 ±8.4% and 42.2 ± 11.3% for WT and TRPA1-null mice, respectively) after myocardial I/R (30 min of ischemia followed by 24 h of reperfusion) in situ. Positive TRPA1 immunofluorescence was present in murine and human hearts and was colocalized with connexin43 at intercalated disks in isolated murine cardiomyocytes. Cardiomyocyte TRPA1 was confirmed by quantitative RT-PCR, DNA sequencing, Western blot analysis, and electrophysiology. A role of TRPA1 in cardiomyocyte toxicity was demonstrated in isolated cardiomyocytes exposed to acrolein, an I/R-associated toxin that induces Ca2+ accumulation and hypercontraction, effects significantly blunted by HC-030031, a TRPA1 antagonist. Protection induced by HC-030031 was quantitatively equivalent to that induced by SN-6, a Na+/Ca2+ exchange inhibitor, further supporting a role of Ca2+ overload in acrolein-induced cardiomyocyte toxicity. These data indicate that cardiac TRPA1 activation likely contributes to I/R injury and, thus, that TRPA1 may be a novel therapeutic target for decreasing myocardial I/R injury. NEW & NOTEWORTHY Transient receptor potential ankyrin 1 (TRPA1) activation mediates increased blood flow, edema, and pain reception, yet its role in myocardial ischemia-reperfusion (I/R) injury is unknown. Genetic ablation of TRPA1 significantly decreased myocardial infarction after I/R in mice. Functional TRPA1 in cardiomyocytes was enriched in intercalated disks and contributed to acrolein-induced Ca2+ overload and hypercontraction. These data indicate that I/R activation of TRPA1 worsens myocardial infarction; TRPA1 may be a potential target to mitigate I/R injury.

P38 mitogen activated protein kinase mediates cardiac Na+/H+ exchanger inhibition induced by Sildenafil

Since the original description as potent antianginal compounds, phosphodiesterase 5A inhibitors have continuously increased their possible therapeutic applications. In the heart, Sildenafil was shown to protect against an ischemic insult by decreasing cardiac Na+/H+ exchanger (NHE1) activity, action that was mediated by protein kinase G. p38 mitogen activated protein kinase (p38MAPK) activation was described in cardiac ischemia, but its precise role remains elusive. It has been shown that p38MAPK is activated by protein kinase G (PKG) in certain non-cardiac tissues, while in others modulates NHE1 activity. Current study was aimed to seek the role of p38MAPK in the Sildenafil-triggered pathway leading to NHE1 inhibition in myocardium. Rat isolated papillary muscles were used to evaluate NHE1 activity during intracellular pH recovery from an acidic load. Protein kinases phosphorylation (activation) was determined by western blot. Sustained acidosis promoted NHE1 hyperactivity by enhancing Ser703 phosphorylation, effect that was blunted by Sildenafil. p38MAPK inhibition reversed the effect of Sildenafil on NHE1. Activation of p38MAPK, by Sodium Arsenite or Anisomycin, mimicked the inhibitory effect of Sildenafil on the exchanger. Consistently, Sildenafil induced p38MAPK phosphorylation/activation during acidosis. Neither Sildenafil nor p38MAPK inhibition affected extracellular signal–regulated kinases 1/2 phosphorylation, kinases upstream NHE1. Furthermore, inhibition of NHE1 after p38MAPK activation was precluded by preventing the activation of protein phosphatase 2A with Okadaic Acid. Taken together, these results suggest that activation of p38MAPK is a necessary step to trigger the inhibitory effect of Sildenafil on cardiac NHE1 activity, thorough a mechanism that involves protein phosphatase 2A-mediated exchanger dephosphorylation.