Laurent Counillon

Protective effects of HOE642, a selective sodium-hydrogen exchange subtype 1 inhibitor, on cardiac ischaemia and reperfusion

OBJECTIVE The aim was to characterise the new compound HOE642 as a selective and cardioprotective Na+/H+ exchange inhibitor in various models. METHODS The effect of HOE642 was tested in the osmotically activated Na+/H+ exchange of rabbit erythrocytes and in propionate induced swelling of human thrombocytes. Recovery of pH after an NH4Cl prepulse and effects on other ion transport systems by patch clamp technique were investigated in rat cardiomyocytes. NHE subtype specifity of the compound was determined by 22Na+ uptake inhibition in a fibroblast cell line separately expressing subtype isoforms 1-3. Protective effects of HOE642 in cardiac ischaemia and reperfusion by ligation of coronary artery were investigated in isolated working rat hearts and in anaesthetised rats. RESULTS HOE642 concentration dependently inhibited the amiloride sensitive sodium influx in rabbit erythrocytes, reduced the swelling of human platelets induced by intracellular acidification, and delayed pH recovery in rat cardiomyocytes. In the isolated working rat heart subjected to ischaemia and reperfusion HOE642 dose dependently reduced the incidence and the duration of reperfusion arrhythmias. It also reduced the the release of lactate dehydrogenase and creatine kinase, and preserved the tissue content of glycogen, ATP, and creatine phosphate. In anaesthetised rats undergoing coronary artery ligation intravenous and oral pretreatment with HOE642 caused a dose dependent reduction or a complete prevention of ventricular premature beats, ventricular tachycardia, and ventricular fibrillation. The compound was well tolerated and neutral to circulatory variables. Other cardiovascular agents tested in this model were not, or were only partly, effective at doses showing marked cardiodepressive effects. CONCLUSIONS HOE642 is a very selective NHE subtype 1 inhibitor showing cardioprotective and antiarrhythmic effects in ischaemic and reperfused hearts. Further development of well tolerated compounds like HOE642 could lead to a new therapeutic approach in clinical indications related to cardiac ischaemia and reperfusion.

The Expanding Family of Eucaryotic Na+/H+Exchangers

Maintaining intracellular pH values close to neutrality is a crucial task for a wide variety of cells. Hence, various mechanisms for pH regulation have been selected early in evolution and are ubiquitously distributed. Among the actors in this scene, the members of the Na/H exchanger gene family (NHE isoforms) are widely expressed and constitute extremely efficient systems for protecting cells against internal acidification. To date, at least six genes have been identified in mammalian cells, and to various extents, the corresponding proteins have been molecularly and functionally characterized. In this short review, we will update our current knowledge on these NHE family members and highlight the most important aspects of the basic function of these transporters. Then, in a broader physiological context, we will present what we think are the most prominent specific features of the different NHE isoforms.

Structure/function studies of the epithelial isoforms of the mammalian Na+/H+ exchanger gene family

Na +/H + exchangers or antiporters are plasma membrane transport proteins, which in eukaryotes exchange extracellular Na + for intracellular H + with a stoichiometry of 1 : 1 [31,56]. In intact cells, Na + enters down the Na-K-ATPase generated electrochemical Na + gradient. All eukaryotic cells studied have plasma membrane Na +/H + exchangers, including yeast, Caenorhabditis elegans and crustaceans [1, 37, 49]. Prokaryotes have functionally similar Na +/H + exchanger proteins which regulate the intracellular Na + ion concentration and pH [38, 60]. In contrast to eukaryotic Na+/H + exchangers, prokaryotic Na+/H + exchangers are electrogenic, exchanging two intracellular Na + for 1 H + ; usually utilizing the intracellular H + ion electromotive force. In eukaryotic cells, the plasma membrane Na+/H + exchangers have multiple functions, including pH homeostasis, volume regulation, cell proliferation, and transcellular Na + absorption [reviewed in 31]. In no cell is it the only mechanism for any one of these functions. For instance, multiple mechanisms of pH homeostasis are present in most eukaryotic cells including a C1-/HCO3 exchanger, a NaHCO3 co-transporter, a Na+-dependent CI-/HCO3 exchanger and multiple mechanisms of H + extrusion [reviewed in 41], including the H-KATPase pump. In this review, we will focus on recent advances

Cisplatin-Induced Apoptosis Involves Membrane Fluidification via Inhibition of NHE1 in Human Colon Cancer Cells

We have previously shown that cisplatin triggers an early acid sphingomyelinase (aSMase)-dependent ceramide generation concomitantly with an increase in membrane fluidity and induces apoptosis in HT29 cells. The present study further explores the role and origin of membrane fluidification in cisplatin-induced apoptosis. The rapid increase in membrane fluidity following cisplatin treatment was inhibited by membrane-stabilizing agents such as cholesterol or monosialoganglioside-1. In HT29 cells, these compounds prevented the early aggregation of Fas death receptor and of membrane lipid rafts on cell surface and significantly inhibited cisplatin-induced apoptosis without altering drug intracellular uptake or cisplatin DNA adducts formation. Early after cisplatin treatment, Na+/H+ membrane exchanger-1 (NHE1) was inhibited leading to intracellular acidification, aSMase was activated, and ceramide was detected at the cell membrane. Treatment of HT29 cells with Staphylococcus aureus sphingomyelinase increased membrane fluidity. Moreover, pretreatment with cariporide, a specific inhibitor of NHE1, inhibited cisplatin-induced intracellular acidification, aSMase activation, ceramide membrane generation, membrane fluidification, and apoptosis. Finally, NHE1-expressing PS120 cells were more sensitive to cisplatin than NHE1-deficient PS120 cells. Altogether, these findings suggest that the apoptotic pathway triggered by cisplatin involves a very early NHE1-dependent intracellular acidification leading to aSMase activation and increase in membrane fluidity. These events are independent of cisplatin-induced DNA adducts formation. The membrane exchanger NHE1 may be another potential target of cisplatin, increasing cell sensitivity to this compound.

A mechanism for the activation of the Na/H exchanger NHE‐1 by cytoplasmic acidification and mitogens

Eukaryotic cells constantly have to fight against internal acidification. In mammals, this task is mainly performed by the ubiquitously expressed electroneutral Na+/H+ exchanger NHE‐1, which activates in a cooperative manner when cells become acidic. Despite its biological importance, the mechanism of this activation is still poorly understood, the most commonly accepted hypothesis being the existence of a proton‐sensor site on the internal face of the transporter. This work uncovers mutations that lead to a nonallosteric form of the exchanger and demonstrates that NHE‐1 activation is best described by a Monod–Wyman–Changeux concerted mechanism for a dimeric transporter. During intracellular acidification, a low‐affinity form of NHE‐1 is converted into a form possessing a higher affinity for intracellular protons, with no requirement for an additional proton‐sensor site on the protein. This new mechanism also explains the activation of the exchanger by growth signals, which shift the equilibrium towards the high‐affinity form.

NaV1.5 Na+ channels allosterically regulate the NHE-1 exchanger and promote the activity of breast cancer cell invadopodia

Summary The degradation of the extracellular matrix by cancer cells represents an essential step in metastatic progression and this is performed by cancer cell structures called invadopodia. NaV1.5 (also known as SCN5A) Na+ channels are overexpressed in breast cancer tumours and are associated with metastatic occurrence. It has been previously shown that NaV1.5 activity enhances breast cancer cell invasiveness through perimembrane acidification and subsequent degradation of the extracellular matrix by cysteine cathepsins. Here, we show that NaV1.5 colocalises with Na+/H+ exchanger type 1 (NHE-1) and caveolin-1 at the sites of matrix remodelling in invadopodia of MDA-MB-231 breast cancer cells. NHE-1, NaV1.5 and caveolin-1 co-immunoprecipitated, which indicates a close association between these proteins. We found that the expression of NaV1.5 was responsible for the allosteric modulation of NHE-1, rendering it more active at the intracellular pH range of 6.4–7; thus, it potentially extrudes more protons into the extracellular space. Furthermore, NaV1.5 expression increased Src kinase activity and the phosphorylation (Y421) of the actin-nucleation-promoting factor cortactin, modified F-actin polymerisation and promoted the acquisition of an invasive morphology in these cells. Taken together, our study suggests that NaV1.5 is a central regulator of invadopodia formation and activity in breast cancer cells.

Nongenomic effects of cisplatin: acute inhibition of mechanosensitive transporters and channels without actin remodeling

Cisplatin is an antineoplastic drug, mostly documented to cause cell death through the formation of DNA adducts. In patients, it exhibits a range of short-term side effects that are unlikely to be related to its genomic action. As cisplatin has been shown to modify membrane properties in different cell systems, we investigated its effects on mechanosensitive ion transporters and channels. We show here that cisplatin is a noncompetitive inhibitor of the mechanosensitive Na(+)/H(+) exchanger NHE-1, with a half-inhibition concentration of 30 μg/mL associated with a decrease in V(max) and Hill coefficient. We also showed that it blocks the Cl(-) and K(+) mechanosensitive channels VSORC and TREK-1 at similar concentrations. In contrast, the nonmechanosensitive Cl(-) and K(+) channels CFTR and TASK-1 and the Na(+)-coupled glucose transport, which share functional features with VSORC, TREK-1, and NHE-1, respectively, were insensitive to cisplatin. We next investigated whether cisplatin action was due to a direct effect on membrane or to cortical actin remodeling that would affect mechanosensors. Using scanning electron microscopy, in vivo actin labeling, and atomic force microscopy, we did not observe any modification of the Youngs modulus and actin cytoskeleton for up to 60 and 120 μg/mL cisplatin, whereas these concentrations modified membrane morphology. Our results reveal a novel mechanism for cisplatin, which affects mechanosensitive channels and transporters involved in cell fate programs and/or expressed in mechanosensitive organs in which cisplatin elicits strong secondary effects, such as the inner ear or the peripheral nervous system. These results might constitute a common denominator to previously unrelated effects of this drug.

Characterization of sabiporide, a new specific NHE-1 inhibitor exhibiting slow dissociation kinetics and cardioprotective effects.

Sabiporide, a new benzoguanidine, was characterized on fibroblasts stably expressing the Na(+)/H(+) exchanger isoforms NHE-1, NHE-2 and NHE-3. 22Na(+) uptake experiments show that this compound possesses a K(i) of 5+/-1.2 x 10(-8) M for NHE-1, and discriminates efficiently between the NHE-1, -2 and -3 isoforms (K(i) for NHE-2: 3+/-0.9 x 10(-6) M, and K(i)>1 mM for NHE-3). Similar K(i) values are obtained on rat cardiomyocytes and human platelets expressing NHE-1 (K(i)s of 7+/-1 x 10(-9) and 2.7+/-0.4 x 10(-8) M respectively). Interestingly, when compared with amiloride and cariporide, sabiporide inhibition persists even after this molecule had been rinsed out (half time of 7 h for sabiporide, and of 1 and 2.5 min for amiloride and cariporide, respectively), the decay of all these molecules exhibiting a complex multiexponential behavior. Thus, sabiporide, which possesses remarkable cardioprotective properties, is a specific NHE-1 inhibitor possessing unique binding kinetics.

Hypoxia promotes tumor cell survival in acidic conditions by preserving ATP levels.

The efficacy of targeting pH disruption to induce cell death in the acidic and hypoxic tumor microenvironment continues to be assessed. Here we analyzed the impact of varying levels of hypoxia in acidic conditions on fibroblast and tumor cell survival. Across all cell lines tested, hypoxia (1% O2) provided protection against acidosis induced cell death compared to normoxia. Meanwhile severe hypoxia (0.1% O2) removed this protection and in some cases exacerbated acidosis‐induced cell death. Differential survival between cell types during external acidosis correlated with their respective intracellular pH regulating capabilities. Cellular ATP measurements were conducted to determine their contribution to cell survival under these combined stresses. In general, hypoxia (1% O2) maintained elevated ATP levels in acidic conditions while severe hypoxia did not. To further explore this interaction we combined acidosis with ATP depletion using 2‐deoxyglucose and observed an enhanced rate of cell mortality. Striking results were also observed with hypoxia providing protection against cell death in spite of a severe metabolic stress induced by a combination of acidosis and oligomycin. Finally, we demonstrated that both HIF1α and HIF2α expression were drastically reduced in hypoxic and acidic conditions indicating a sensitivity of this protein to cellular pH conditions. This knockdown of HIF expression by acidosis has implications for the development of therapies targeting the disruption of cellular pH regulation. Our results reinforce the proof of concept that acidosis and metabolic disruption affecting ATP levels could be exploited as a tumor cell killing strategy. J. Cell. Physiol. 228: 1854–1862, 2013.

Na/H exchange activities in NHE1-transfected OK-cells : cell polarity and regulation

The human fibroblast, “amiloride-sensitive” Na/H exchanger (NHE1) was transfected into opossum kidney cells (OK cells) (OK/NHE1 cells). Northern blot analysis confirmed that the NHE1 message is expressed in OK/NHE1 cells. In immunoblot analysis, an anti-human NHE1 antibody labelled a membrane protein only present in OK/NHE1 cells. In contrast to the parental cell line containing only an apically located, “amilorideresistant” Na/H exchange activity, OK/NHE1 cells contain apically and basolaterally located Na/H exchange activities, the apical activity being “amiloride resistant” and the basolateral being “amiloride sensitive”. Parathyroid hormone (PTH) inhibited apical transport activity (OK and OK/NHE1 cells) but had no effect on basolateral transport activity (OK/NHE1 cells). Pharmacological activation of protein kinase A (forskolin) decreased both apical and basolateral Na/H exchange activity. Incubation with phorbol ester (exogenous activation of protein kinase C) reduced apical Na/H exchange activity (OK and OK/NHE1 cells) but had only a moderate, inhibitory effect on basolateral Na/H exchange activity (OK/NHE1 cells). These results indicate that transfection of OK cells with human fibroblast NHE1 cDNA encoding an “amiloride-sensitive” form of the Na/H exchanger results in expression of basolaterally located “NHE1-related” transport activity. Regulatory control of intracellular Na/H exchange activities (apically versus basolaterally located) and intercellular Na/H exchange activities (NHE1-related) differs. This may relate to cell-specific properties as well as to exchanger-specific properties.