Design, Synthesis, Biological Evaluation, and Computational Studies of Novel Ureidopropanamides as Formyl Peptide Receptor 2 (FPR2) Agonists to Target the Resolution of Inflammation in Central Nervous System Disorders.

Formyl peptide receptor 2 (FPR2) agonists can boost the resolution of inflammation and can offer alternative approaches for the treatment of pathologies with underlying chronic neuroinflammation, including neurodegenerative disorders. Starting from the FPR2 agonist 2 previously identified in our laboratory and through fine-tuning of FPR2 potency and metabolic stability, we have identified a new series of ureidopropanamide derivatives endowed with a balanced combination of such properties. Computational studies provided insights into the key interactions of the new compounds for FPR2 activation. In mouse microglial N9 cells and in rat primary microglial cells stimulated with lipopolysaccharide, selected compounds inhibited the production of pro-inflammatory cytokines, counterbalanced the changes in mitochondrial function, and inhibited caspase-3 activity. Among the new agonists, (S)-11l stands out also for the ability to permeate the blood-brain barrier and to accumulate in the mouse brain in vivo, thus representing a valuable pharmacological tool for studies in vivo.

Metabolic Features of Brain Function with Relevance to Clinical Features of Alzheimer and Parkinson Diseases.

Brain metabolism is comprised in Alzheimer's disease (AD) and Parkinson's disease (PD). Since the brain primarily relies on metabolism of glucose, ketone bodies, and amino acids, aspects of these metabolic processes in these disorders-and particularly how these altered metabolic processes are related to oxidative and/or nitrosative stress and the resulting damaged targets-are reviewed in this paper. Greater understanding of the decreased functions in brain metabolism in AD and PD is posited to lead to potentially important therapeutic strategies to address both of these disorders, which cause relatively long-lasting decreased quality of life in patients.

The Non-Gastric H+/K+ ATPase (ATP12A) Is Expressed in Mammalian Spermatozoa.

H+/K+ ATPase Type 2 is an heteromeric membrane protein involved in cation transmembrane transport and consists of two subunits: a specific α subunit (ATP12A) and a non-specific ß subunit. The aim of this study was to demonstrate the presence and establish the localization of ATP12A in spermatozoa from Bubalus bubalis, Bos taurus and Ovis aries. Immunoblotting revealed, in all three species, a major band (100 kDa) corresponding to the expected molecular mass. The ATP12A immunolocalization pattern showed, consistently in the three species, a strong signal at the acrosome. These results, described here for the first time in spermatozoa, are consistent with those observed for the ß1 subunit of Na+/K+ ATPase, suggesting that the latter may assemble with the α subunit to produce a functional ATP12A dimer in sperm cells. The above scenario appeared to be nicely supported by 3D comparative modeling and interaction energy calculations. The expression of ATP12A during different stages of bovine sperm maturation progressively increased, moving from epididymis to deferent ducts. Based on overall results, we hypothesize that ATP12A may play a role in acrosome reactions. Further studies will be required in order to address the functional role of this target protein in sperm physiology.

Small-molecule drugs for cystic fibrosis: Where are we now?

The cystic fibrosis (CF) lung disease is due to the lack/dysfunction of the CF Transmembrane Conductance Regulator (CFTR), a chloride channel expressed by epithelial cells as the main regulator of ion and fluid homeostasis. More than 2000 genetic variation in the CFTR gene are known, among which those with identified pathomechanism have been divided into six mutation classes. A major advancement in the pharmacotherapy of CF has been the development of small-molecule drugs hitting the root of the disease, i.e. the altered ion and fluid transport through the airway epithelium. These drugs, called CFTR modulators, have been advanced to the clinics to treat nearly 90% of CF patients, including the CFTR potentiator ivacaftor, approved for residual function mutations (Classes III and IV), and combinations of correctors (lumacaftor, tezacaftor, elexacaftor) and ivacaftor for patients bearing at least one the F508del mutation, the most frequent mutation belonging to class II. To cover the 10% of CF patients without etiological therapies, other novel small-molecule CFTR modulators are in evaluation of their effectiveness in all the CFTR mutation classes: read-through agents for Class I, correctors, potentiators and amplifiers from different companies for Class II-V, stabilizers for Class VI. In alternative, other solute carriers, such as SLC26A9 and SLC6A14, are the focus of intensive investigation. Finally, other molecular targets are being evaluated for patients with no approved CFTR modulator therapy or as means of enhancing CFTR modulatory therapy, including small molecules forming ion channels, inhibitors of the ENaC sodium channel and potentiators of the calcium-activated chloride channel TMEM16A. This paper aims to give an up-to-date overview of old and novel CFTR modulators as well as of novel strategies based on small-molecule drugs. Further investigations in in-vivo and cell-based models as well as carrying out large prospective studies will be required to determine if novel CFTR modulators, stabilizers, amplifiers, and the ENaC inhibitors or TMEM16A potentiators will further improve the clinical outcomes in CF management.

The J2-Immortalized Murine Macrophage Cell Line Displays Phenotypical and Metabolic Features of Primary BMDMs in Their M1 and M2 Polarization State.

Macrophages are immune cells that are important for the development of the defensive front line of the innate immune system. Following signal recognition, macrophages undergo activation toward specific functional states, consisting not only in the acquisition of specific features but also of peculiar metabolic programs associated with each function. For these reasons, macrophages are often isolated from mice to perform cellular assays to study the mechanisms mediating immune cell activation. This requires expensive and time-consuming breeding and housing of mice strains. To overcome this issue, we analyzed an in-house J2-generated immortalized macrophage cell line from BMDMs, both from a functional and metabolic point of view. By assaying the intracellular and extracellular metabolism coupled with the phenotypic features of immortalized versus primary BMDMs, we concluded that classically and alternatively immortalized macrophages display similar phenotypical, metabolic and functional features compared to primary cells polarized in the same way. Our study validates the use of this immortalized cell line as a suitable model with which to evaluate in vitro how perturbations can influence the phenotypical and functional features of murine macrophages.

Reactive Oxygen Species in Macrophages: Sources and Targets.

Reactive oxygen species (ROS) are fundamental for macrophages to eliminate invasive microorganisms. However, as observed in nonphagocytic cells, ROS play essential roles in processes that are different from pathogen killing, as signal transduction, differentiation, and gene expression. The different outcomes of these events are likely to depend on the specific subcellular site of ROS formation, as well as the duration and extent of ROS production. While excessive accumulation of ROS has long been appreciated for its detrimental effects, there is now a deeper understanding of their roles as signaling molecules. This could explain the failure of the "all or none" pharmacologic approach with global antioxidants to treat several diseases. NADPH oxidase is the first source of ROS that has been identified in macrophages. However, growing evidence highlights mitochondria as a crucial site of ROS formation in these cells, mainly due to electron leakage of the respiratory chain or to enzymes, such as monoamine oxidases. Their role in redox signaling, together with their exact site of formation is only partially elucidated. Hence, it is essential to identify the specific intracellular sources of ROS and how they influence cellular processes in both physiological and pathological conditions to develop therapies targeting oxidative signaling networks. In this review, we will focus on the different sites of ROS formation in macrophages and how they impact on metabolic processes and inflammatory signaling, highlighting the role of mitochondrial as compared to non-mitochondrial ROS sources.

N-acetylaspartate release by glutaminolytic ovarian cancer cells sustains protumoral macrophages.

Glutaminolysis is known to correlate with ovarian cancer aggressiveness and invasion. However, how this affects the tumor microenvironment is elusive. Here, we show that ovarian cancer cells become addicted to extracellular glutamine when silenced for glutamine synthetase (GS), similar to naturally occurring GS-low, glutaminolysis-high ovarian cancer cells. Glutamine addiction elicits a crosstalk mechanism whereby cancer cells release N-acetylaspartate (NAA) which, through the inhibition of the NMDA receptor, and synergistically with IL-10, enforces GS expression in macrophages. In turn, GS-high macrophages acquire M2-like, tumorigenic features. Supporting this in␣vitro model, in silico data and the analysis of ascitic fluid isolated from ovarian cancer patients prove that an M2-like macrophage phenotype, IL-10 release, and NAA levels positively correlate with disease stage. Our study uncovers the unprecedented role of glutamine metabolism in modulating macrophage polarization in highly invasive ovarian cancer and highlights the anti-inflammatory, protumoral function of NAA.

Cellular Redox State Acts as Switch to Determine the Direction of NNT-Catalyzed Reaction in Cystic Fibrosis Cells.

The redox states of NAD and NADP are linked to each other in the mitochondria thanks to the enzyme nicotinamide nucleotide transhydrogenase (NNT) which, by utilizing the mitochondrial membrane potential (mΔΨ), catalyzes the transfer of redox potential between these two coenzymes, reducing one at the expense of the oxidation of the other. In order to define NNT reaction direction in CF cells, NNT activity under different redox states of cell has been investigated. Using spectrophotometric and western blotting techniques, the presence, abundance and activity level of NNT were determined. In parallel, the levels of NADPH and NADH as well as of mitochondrial and cellular ROS were also quantified. CF cells showed a 70% increase in protein expression compared to the Wt sample; however, regarding NNT activity, it was surprisingly lower in CF cells than healthy cells (about 30%). The cellular redox state, together with the low mΔΨ, pushes to drive NNT reverse reaction, at the expense of its antioxidant potential, thus consuming NADPH to support NADH production. At the same time, the reduced NNT activity prevents the NADH, produced by the reaction, from causing an explosion of ROS by the damaged respiratory chain, in accordance with the reduced level of mitochondrial ROS in NNT-loss cells. This new information on cellular bioenergetics represents an important building block for further understanding the molecular mechanisms responsible for cellular dysfunction in cystic fibrosis.

Mitochondrial Membranes of Human SH-SY5Y Neuroblastoma Cells Express Serotonin 5-HT7 Receptor.

Mitochondria in neurons contribute to energy supply, the regulation of synaptic transmission, Ca2+ homeostasis, neuronal excitability, and stress adaptation. In recent years, several studies have highlighted that the neurotransmitter serotonin (5-HT) plays an important role in mitochondrial biogenesis in cortical neurons, and regulates mitochondrial activity and cellular function in cardiomyocytes. 5-HT exerts its diverse actions by binding to cell surface receptors that are classified into seven distinct families (5-HT1 to 5-HT7). Recently, it was shown that 5-HT3 and 5-HT4 receptors are located on the mitochondrial membrane and participate in the regulation of mitochondrial function. Furthermore, it was observed that activation of brain 5-HT7 receptors rescued mitochondrial dysfunction in female mice from two models of Rett syndrome, a rare neurodevelopmental disorder characterized by severe behavioral and physiological symptoms. Our Western blot analyses performed on cell-lysate and purified mitochondria isolated from neuronal cell line SH-SY5Y showed that 5-HT7 receptors are also expressed into mitochondria. Maximal binding capacity (Bmax) obtained by Scatchard analysis on purified mitochondrial membranes was 0.081 pmol/mg of 5-HT7 receptor protein. Lastly, we evaluated the effect of selective 5-HT7 receptor agonist LP-211 and antagonist (inverse agonist) SB-269970 on mitochondrial respiratory chain (MRC) cytochrome c oxidase activity on mitochondria from SH-SY5Y cells. Our findings provide the first evidence that 5-HT7 receptor is also expressed in mitochondria.

Glufosinate constrains synchronous and metachronous metastasis by promoting anti-tumor macrophages.

Glutamine synthetase (GS) generates glutamine from glutamate and controls the release of inflammatory mediators. In macrophages, GS activity, driven by IL10, associates to the acquisition of M2-like functions. Conditional deletion of GS in macrophages inhibits metastasis by boosting the formation of anti-tumor, M1-like, tumor-associated macrophages (TAMs). From this basis, we evaluated the pharmacological potential of GS inhibitors in targeting metastasis, identifying glufosinate as a specific human GS inhibitor. Glufosinate was tested in both cultured macrophages and on mice bearing metastatic lung, skin and breast cancer. We found that glufosinate rewires macrophages toward an M1-like phenotype both at the primary tumor and metastatic site, countering immunosuppression and promoting vessel sprouting. This was also accompanied to a reduction in cancer cell intravasation and extravasation, leading to synchronous and metachronous metastasis growth inhibition, but no effects on primary tumor growth. Glufosinate treatment was well-tolerated, without liver and brain toxicity, nor hematopoietic defects. These results identify GS as a druggable enzyme to rewire macrophage functions and highlight the potential of targeting metabolic checkpoints in macrophages to treat cancer metastasis.

The hyperchromatic supranuclear stria corresponds to the Golgi apparatus in nasal ciliated cells.

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The preclinical discovery and development of the combination of ivacaftor + tezacaftor used to treat cystic fibrosis.

INTRODUCTION: Cystic Fibrosis (CF) is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. The most common mutation, F508del, induces protein misprocessing and loss of CFTR function. The discovery through in vitro studies of the CFTR correctors (i.e. lumacaftor, tezacaftor) that partially rescue the misprocessing of F508del-CFTR with the potentiator ivacaftor is promising in giving an unprecedented clinical benefit in affected patients. AREAS COVERED: Online databases were searched using key phrases for CF and CFTR modulators. Tezacaftor-ivacaftor treatment has proved to be safer than lumacaftor-ivacaftor, although clinical efficacy is similar. Further clinical efficacy has ensued with the introduction of triple therapy, i.e. applying second-generation correctors, such as VX-569 and VX-445 (elexacaftor) to tezacaftor-ivacaftor. The triple combinations will herald the availability of etiologic therapies for patients for whom no CFTR modulators are currently applied (i.e. F508del/minimal function mutations) and enhance CFTR modulator therapy for patients homozygous for F508del. EXPERT OPINION: CF patient-derived tissue models are being explored to determine donor-specific response to current approved and future novel CFTR modulators for F508del and other rare mutations. The discovery and validation of biomarkers of CFTR modulation will complement these studies in the long term and in real-life world.

Treatment of Cystic Fibrosis Patients Homozygous for F508del with Lumacaftor-Ivacaftor (Orkambi®) Restores Defective CFTR Channel Function in Circulating Mononuclear Cells.

The treatment of cystic fibrosis (CF) patients homozygous for the F508del mutation with Orkambi®, a combination of a corrector (lumacaftor) and a potentiator (ivacaftor) of the mutated CFTR protein, resulted in some amelioration of the respiratory function. However, a great variability in the clinical response was also observed. The aim of this study was to evaluate the response to Orkambi® in a small cohort of F508del/F508del patients (n = 14) in terms of clinical and laboratory parameters, including ex vivo CFTR activity in mononuclear cells (MNCs), during a 12-month treatment. Patients responded with an increase in percent predicted forced expiratory volume in 1 s (FEV1%) and body mass index (BMI) as well as with a decrease in white blood cell (WBC) total counts and serum C-reactive protein (CRP) levels, although not significantly. Sweat chloride and CFTR-dependent chloride efflux were found to decrease and increase, respectively, as compared with pre-therapy values. CFTR and BMI showed a statistically significant correlation during Orkambi® treatment. Clustering analysis showed that CFTR, BMI, sweat chloride, FEV1%, and WBC were strongly associated. These data support the notion that CFTR-dependent chloride efflux in MNCs should be investigated as a sensitive outcome measure of Orkambi® treatment in CF patients.

Pharmacological targets of metabolism in disease: Opportunities from macrophages.

From advances in the knowledge of the immune system, it is emerging that the specialized functions displayed by macrophages during the course of an immune response are supported by specific and dynamically-connected metabolic programs. The study of immunometabolism is demonstrating that metabolic adaptations play a critical role in modulating inflammation and, conversely, inflammation deeply influences the acquisition of specific metabolic settings.This strict connection has been proven to be crucial for the execution of defined immune functional programs and it is now under investigation with respect to several human disorders, such as diabetes, sepsis, cancer, and autoimmunity. The abnormal remodelling of the metabolic pathways in macrophages is now emerging as both marker of disease and potential target of therapeutic intervention. By focusing on key pathological conditions, namely obesity and diabetes, rheumatoid arthritis, atherosclerosis and cancer, we will review the metabolic targets suitable for therapeutic intervention in macrophages. In addition, we will discuss the major obstacles and challenges related to the development of therapeutic strategies for a pharmacological targeting of macrophage's metabolism.

Differential Expression of ADP/ATP Carriers as a Biomarker of Metabolic Remodeling and Survival in Kidney Cancers.

ADP/ATP carriers (AACs) are mitochondrial transport proteins playing a strategic role in maintaining the respiratory chain activity, fueling the cell with ATP, and also regulating mitochondrial apoptosis. To understand if AACs might represent a new molecular target for cancer treatment, we evaluated AAC expression levels in cancer/normal tissue pairs available on the Tissue Cancer Genome Atlas database (TCGA), observing that AACs are dysregulated in most of the available samples. It was observed that at least two AACs showed a significant differential expression in all the available kidney cancer/normal tissue pairs. Thus, we investigated AAC expression in the corresponding kidney non-cancer (HK2)/cancer (RCC-Shaw and CaKi-1) cell lines, grown in complete medium or serum starvation, for investigating how metabolic alteration induced by different growth conditions might influence AAC expression and resistance to mitochondrial apoptosis initiators, such as "staurosporine" or the AAC highly selective inhibitor "carboxyatractyloside". Our analyses showed that AAC2 and AAC3 transcripts are more expressed than AAC1 in all the investigated kidney cell lines grown in complete medium, whereas serum starvation causes an increase of at least two AAC transcripts in kidney cancer cell lines compared to non-cancer cells. However, the total AAC protein content is decreased in the investigated cancer cell lines, above all in the serum-free medium. The observed decrease in AAC protein content might be responsible for the decrease of OXPHOS activity and for the observed lowered sensitivity to mitochondrial apoptosis induced by staurosporine or carboxyatractyloside. Notably, the cumulative probability of the survival of kidney cancer patients seriously decreases with the decrease of AAC1 expression in KIRC and KIRP tissues making AAC1 a possible new biomarker of metabolic remodeling and survival in kidney cancers.

An Intriguing Involvement of Mitochondria in Cystic Fibrosis.

Cystic fibrosis (CF) occurs when the cystic fibrosis transmembrane conductance regulator (CFTR) protein is not synthetized and folded correctly. The CFTR protein helps to maintain the balance of salt and water on many body surfaces, such as the lung surface. When the protein is not working correctly, chloride becomes trapped in cells, then water cannot hydrate the cellular surface and the mucus covering the cells becomes thick and sticky. Furthermore, a defective CFTR appears to produce a redox imbalance in epithelial cells and extracellular fluids and to cause an abnormal generation of reactive oxygen species: as a consequence, oxidative stress has been implicated as a causative factor in the aetiology of the process. Moreover, massive evidences show that defective CFTR gives rise to extracellular GSH level decrease and elevated glucose concentrations in airway surface liquid (ASL), thus encouraging lung infection by pathogens in the CF advancement. Recent research in progress aims to rediscover a possible role of mitochondria in CF. Here the latest new and recent studies on mitochondrial bioenergetics are collected. Surprisingly, they have enabled us to ascertain that mitochondria have a leading role in opposing the high ASL glucose level as well as oxidative stress in CF.

G-CSF and GM-CSF Modify Neutrophil Functions at Concentrations found in Cystic Fibrosis.

The role of colony stimulating factors (CSFs) in cystic fibrosis (CF) circulating neutrophils has not been thoroughly evaluated, considering that the neutrophil burden of lung inflammation in these subjects is very high. The aim of this study was to assess granulocyte-CSF (G-CSF) and granulocyte-macrophage-CSF (GM-CSF) levels in CF patients in various clinical conditions and how these cytokines impact on activation and priming of neutrophils. G-CSF and GM-CSF levels were measured in sputum and serum samples of stable CF patients (n = 21) and in CF patients with acute exacerbation before and after a course of antibiotic therapy (n = 19). CSFs were tested on non CF neutrophils to investigate their effects on reactive oxygen species (ROS) production, degranulation (CD66b, elastase, lactoferrin, MMP-9), and chemotaxis. At very low concentrations found in CF patients (0.005-0.1 ng/ml), both cytokines inhibited ROS production, while higher concentrations (1-5 ng/ml) exerted a stimulatory effect. While either CSF induced elastase and MMP-9 secretion, lactoferrin levels were increased only by G-CSF. Chemotaxis was inhibited by GM-CSF, but was increased by G-CSF. However, when present together at low concentrations, CSFs increased basal and fMLP-stimulated ROS production and chemotaxis. These results suggest the CSF levels that circulating neutrophils face before extravasating into the lungs of CF patients may enhance their function contributing to the airway damage.

Beyond the Big Five: Investigating Myostatin Structure, Polymorphism and Expression in Camelus dromedarius.

Myostatin, a negative regulator of skeletal muscle mass in animals, has been shown to play a role in determining muscular hypertrophy in several livestock species, and a high degree of polymorphism has been previously reported for this gene in humans and cattle. In this study, we provide a characterization of the myostatin gene in the dromedary (Camelus dromedarius) at the genomic, transcript and protein level. The gene was found to share high structural and sequence similarity with other mammals, notably Old World camelids. 3D modeling highlighted several non-conservative SNP variants compared to the bovine, as well as putative functional variants involved in the stability of the myostatin dimer. NGS data for nine dromedaries from various countries revealed 66 novel SNPs, all of them falling either upstream or downstream the coding region. The analysis also confirmed the presence of three previously described SNPs in intron 1, predicted here to alter both splicing and transcription factor binding sites (TFBS), thus possibly impacting myostatin processing and/or regulation. Several putative TFBS were identified in the myostatin upstream region, some of them belonging to the myogenic regulatory factor family. Patterns of SNP distribution across countries, as suggested by Bayesian clustering of the nine dromedaries using the 69 SNPs, pointed to weak geographic differentiation, in line with known recurrent gene flow at ancient trading centers along caravan routes. Myostatin expression was investigated in a set of 8 skeletal muscles, both at transcript and protein level, via Digital Droplet PCR and Western Blotting, respectively. No significant differences were observed at the transcript level, while, at the protein level, the only significant differences concerned the promyostatin dimer (75 kDa), in four pair-wise comparisons, all involving the tensor fasciae latae muscle. Beside the mentioned band at 75 kDa, additional bands were observed at around 40 and 25 kDa, corresponding to the promyostatin monomer and the active C-terminal myostatin dimer, respectively. Their weaker intensity suggests that the unprocessed myostatin dimers could act as important reservoirs of slowly available myostatin forms. Under this assumption, the sequential cleavage steps may contribute additional layers of control within an already complex regulatory framework.

Gap Junctions Are Involved in the Rescue of CFTR-Dependent Chloride Efflux by Amniotic Mesenchymal Stem Cells in Coculture with Cystic Fibrosis CFBE41o- Cells.

We previously found that human amniotic mesenchymal stem cells (hAMSCs) in coculture with CF immortalised airway epithelial cells (CFBE41o- line, CFBE) on Transwell® filters acquired an epithelial phenotype and led to the expression of a mature and functional CFTR protein. In order to explore the role of gap junction- (GJ-) mediated intercellular communication (GJIC) in this rescue, cocultures (hAMSC : CFBE, 1 : 5 ratio) were studied for the formation of GJIC, before and after silencing connexin 43 (Cx43), a major component of GJs. Functional GJs in cocultures were inhibited when the expression of the Cx43 protein was downregulated. Transfection of cocultures with siRNA against Cx43 resulted in the absence of specific CFTR signal on the apical membrane and reduction in the mature form of CFTR (band C), and in parallel, the CFTR-dependent chloride channel activity was significantly decreased. Cx43 downregulation determined also a decrease in transepithelial resistance and an increase in paracellular permeability as compared with control cocultures, implying that GJIC may regulate CFTR expression and function that in turn modulate airway epithelium tightness. These results indicate that GJIC is involved in the correction of CFTR chloride channel activity upon the acquisition of an epithelial phenotype by hAMSCs in coculture with CF cells.

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

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