Carlos Gutiérrez-Merino: Synergy of Theory and Experimentation in Biological Membrane Research.

Professor Carlos Gutiérrez-Merino, a prominent scientist working in the complex realm of biological membranes, has made significant theoretical and experimental contributions to the field. Contemporaneous with the development of the fluid-mosaic model of Singer and Nicolson, the Förster resonance energy transfer (FRET) approach has become an invaluable tool for studying molecular interactions in membranes, providing structural insights on a scale of 1-10 nm and remaining important alongside evolving perspectives on membrane structures. In the last few decades, Gutiérrez-Merino's work has covered multiple facets in the field of FRET, with his contributions producing significant advances in quantitative membrane biology. His more recent experimental work expanded the ground concepts of FRET to high-resolution cell imaging. Commencing in the late 1980s, a series of collaborations between Gutiérrez-Merino and the authors involved research visits and joint investigations focused on the nicotinic acetylcholine receptor and its relation to membrane lipids, fostering a lasting friendship.

Myomerger Induces Membrane Hemifusion and Regulates Fusion Pore Expansion.

Membrane fusion is a crucial mechanism in a wide variety of important events in cell biology from viral infection to exocytosis. However, despite many efforts and much progress, cell-cell fusion has remained elusive to our understanding. Along the life of the fusion pore, large conformational changes take place from the initial lipid bilayer bending, passing through the hemifusion intermediates, and ending with the formation of the first nascent fusion pore. In this sense, computer simulations are an ideal technique for describing such complex lipid remodeling at the molecular level. In this work, we studied the role played by the muscle-specific membrane protein Myomerger during the formation of the fusion pore. We have conducted µs length atomistic and coarse-grained molecular dynamics, together with free-energy calculations using ad hoc collective variables. Our results show that Myomerger favors the hemifusion diaphragm-stalk transition, reduces the nucleation-expansion energy difference, and promotes the formation of nonenlarging fusion pores.

Out-of-plane deformability and its coupling with electrostatics in biomembranes.

Cell membranes are quasi-bidimensional soft systems formed by multipoles in an ordered array that can be polarized in an electric field. Consequently, electrostatic potentials emerge inside membranes, and membranes respond to external electric fields. From a mechanical perspective, membranes can be easily compressed-expanded, laterally deformed, and curved. Bending is particularly easy, and this kind of deformation translates to changes in the relative positions of the negative and positive charges, leading to strain gradient-induced polarization. Conversely, an external electric field gradient will exert a bending stress that translates to mechanical membrane deformation. These phenomena are described through membrane flexoelectricity. Here, we describe this property in lipid bilayers and cell membranes and summarize the studies in the field with emphasis on the effects promoted by membrane asymmetry.

Insights into the equilibrium structure and translocation mechanism of TP1, a spontaneous membrane-translocating peptide.

Crossing the cellular membrane is one of the main barriers during drug discovery; many potential drugs are rejected for their inability to integrate into the intracell fluid. Although many solutions have been proposed to overcome this barrier, arguably the most promising solution is the use of cell-penetrating peptides. Recently, an array of hydrophobic penetrating peptides was discovered via high throughput screening which proved to be able to cross the membrane passively, and although these peptides proved to be effective at penetrating the cell, the details behind the underlying mechanism of this process remain unknown. In this study, we developed a method to find the equilibrium structure at the transmembrane domain of TP1, a hydrophobic penetrating peptide. In this method, we selectively deuterium-label amino acids in the peptidic chain, and employ results of [Formula: see text]H-NMR spectroscopy to find a molecular dynamics simulation of the peptide that reproduces the experimental results. Effectively finding the equilibrium orientation and dynamics of the peptide in the membrane. We employed this equilibrium structure to simulate the entire translocation mechanism and found that after the peptide reaches its equilibrium structure, it must undergo a two-step mechanism in order to completely translocate the membrane, each step involving the flip-flop of each arginine residue in the peptide. This leads us to conclude that the RLLR motif is essential for the translocating activity of the peptide.

Aromaticity at position 39 in α-synuclein: A modulator of amyloid fibril assembly and membrane-bound conformations.

Recent studies revealed that molecular events related with the physiology and pathology of αS might be regulated by specific sequence motifs in the primary sequence of αS. The importance of individual residues in these motifs remains an important open avenue of investigation. In this work, we have addressed the structural details related to the amyloid fibril assembly and lipid-binding features of αS through the design of site-directed mutants at position 39 of the protein and their study by in vitro and in vivo assays. We demonstrated that aromaticity at position 39 of αS primary sequence influences strongly the aggregation properties and the membrane-bound conformations of the protein, molecular features that might have important repercussions for the function and dysfunction of αS. Considering that aggregation and membrane damage is an important driver of cellular toxicity in amyloid diseases, future work is needed to link our findings with studies based on toxicity and neuronal cell death. BRIEF STATEMENT OUTLINING SIGNIFICANCE: Modulation by distinct sequential motifs and specific residues of αS on its physiological and pathological states is an active area of research. Here, we demonstrated that aromaticity at position 39 of αS modulates the membrane-bound conformations of the protein, whereas removal of aromatic functionality at position 39 reduces strongly the amyloid assembly in vitro and in vivo. Our study provides new evidence for the modulation of molecular events related with the physiology and pathology of αS.

Membrane rigidity regulates E. coli proliferation rates.

Combining single cell experiments, population dynamics and theoretical methods of membrane mechanics, we put forward that the rate of cell proliferation in E. coli colonies can be regulated by modifiers of the mechanical properties of the bacterial membrane. Bacterial proliferation was modelled as mediated by cell division through a membrane constriction divisome based on FtsZ, a mechanically competent protein at elastic interaction against membrane rigidity. Using membrane fluctuation spectroscopy in the single cells, we revealed either membrane stiffening when considering hydrophobic long chain fatty substances, or membrane softening if short-chained hydrophilic molecules are used. Membrane stiffeners caused hindered growth under normal division in the microbial cultures, as expected for membrane rigidification. Membrane softeners, however, altered regular cell division causing persistent microbes that abnormally grow as long filamentous cells proliferating apparently faster. We invoke the concept of effective growth rate under the assumption of a heterogeneous population structure composed by distinguishable individuals with different FtsZ-content leading the possible forms of cell proliferation, from regular division in two normal daughters to continuous growing filamentation and budding. The results settle altogether into a master plot that captures a universal scaling between membrane rigidity and the divisional instability mediated by FtsZ at the onset of membrane constriction.

Analysis of RNA yield in extracellular vesicles isolated by membrane affinity column and differential ultracentrifugation.

Extracellular vesicles (EV) have attracted much attention as potential biomarkers due to their protein, RNA and other nucleic acid content. The most common method used for EV isolation is differential ultracentrifugation (DU), however given the DU technical difficulties, other more practical methods have surged, such as membrane-affinity column commercial kits. Here, we assessed one commercial kit in terms of EV recovery and EV-derived RNA yield and compared it with a DU protocol. Our data shows that the commercial kit preparation results in a lower count of EV-like structures and a reduced expression of EV markers when compared to DU samples. Thus, apparently suggesting that the commercial kit had a lower EV yield. However, these findings did not reflect on RNA yield, which was greater with the commercial kit, even after an enzymatic treatment with proteinase K and RNAse A. We conclude that the kit has a higher EV-derived RNA yield in comparison to our DU protocol, suggesting that it may be the method of choice for RNA sequencing purposes.

OxLDL alterations in endothelial cell membrane dynamics leads to changes in vesicle trafficking and increases cell susceptibility to injury.

Plasma membrane repair (PMR) is an important process for cell homeostasis, especially for cells under constant physical stress. Repair involves a sequence of Ca2+-dependent events, including lysosomal exocytosis and subsequent compensatory endocytosis. Cholesterol sequestration from plasma membrane causes actin cytoskeleton reorganization and polymerization, increasing cell stiffness, which leads to exocytosis and reduction of a peripheral pool of lysosomes involved in PMR. These changes in mechanical properties are similar to those observed in cells exposed to oxidized Low Density Lipoprotein (oxLDL), a key molecule during atherosclerosis development. Using a human umbilical vein endothelial cell line (EAhY926) we evaluated the influence of mechanical modulation induced by oxLDL in PMR and its effect in endothelial fragility. Similar to MßCD (a drug capable of sequestering cholesterol) treatment, oxLDL exposure led to actin reorganization and de novo polymerization, as well as an increase in cell rigidity and lysosomal exocytosis. Additionally, for both MßCD and oxLDL treated cells, there was an initial increase in endocytic events, likely triggered by the peak of exocytosis induced by both treatments. However, no further endocytic events were observed, suggesting that constitutive endocytosis is blocked upon treatment and that the reorganized cytoskeleton function as a mechanical barrier to membrane traffic. Finally, the increase in cell rigidity renders cells more prone to mechanical injury. Together, these data show that mechanical modulation induced by oxLDL exposure not only alters membrane traffic in cells, but also makes them more susceptible to mechanical injury, which may likely contribute to the initial steps of atherosclerosis development.

Production and evaluation of leukocyte- and thrombocyte-rich fibrin membranes in birds.

The aim of this study was the preparation and histological evaluation of Leukocyte- and Thrombocyte-Rich Fibrin (L-TRF) membranes obtained from the blood of four bird species. Forty adult healthy birds were divided into four groups of equal size: G1 - macaws, G2 - domestic chickens, G3 - parrots, G4 - toco toucans. A total of 0.5 mL of blood was collected from each bird, put into a glass tube without anticoagulant and centrifuged at 3000 rpm for 10 min. L-TRF membranes produced after compression of the clot were processed for histological analysis. The ratio of thrombocytes/area was not significantly different among Groups G2, G3 and G4, but a significant difference was found between Groups G1 and G2 with the highest thrombocyte concentration/area in G1. The groups did not differ statistically in the number of leukocytes/area. The fibrin-to-cells ratio did not vary statistically among Groups G1, G2 and G3, but this ratio was significantly higher in Group G4 than in the other groups. The thrombocyte-to-leukocyte ratio was the highest in Group G1, but it did not differ among Groups G2, G3 and G4. In conclusion, the centrifugation protocol allowed the production of L-TRF membranes in the four bird species studied. Histologically, cell ratios were analogous in domestic chickens and parrots, and macaws had the highest ratio of thrombocytes.

Transmittance and Autofluorescence of Neonatal Rat Stratum Corneum: Nerolidol Increases the Dynamics and Partitioning of Protoporphyrin IX into Intercellular Membranes.

In this work, we developed an experimental apparatus to directly measure transmittance and fluorescence in the stratum corneum (SC) ex vivo. The SC transmittance varied from ~6 to ~52 % in the wavelength range of 280-850 nm. For 260-300 nm excitation, the SC autofluorescence showed a strong emission band between 290 and 425 nm, which is associated with tryptophan, and another in the 600-670 nm range, which we attributed to a process involving resonance energy transfer to very hydrophobic keratin filaments. Weaker emission associated with less hydrophobic keratin filaments was also observed in the wavelength range of 350-480 nm. Protoporphyrin IX (PpIX) was incorporated into SC membranes, and its penetration was further increased by the addition of nerolidol to the treatment suspension. Both PpIX and the endogenous porphyrins showed fluorescence anisotropy consistent with their localization in SC membranes, and their molecular dynamics increased significantly in the presence of 1 % nerolidol. The emission and excitation spectra of PpIX and the endogenous SC porphyrins showed similar alterations during the photobleaching induced by 405-nm irradiation. This work also highlights the SC contribution to skin autofluorescence, which could be useful for fluorescence spectroscopy applications in the early diagnosis of skin diseases.

Sensing membrane thickness: Lessons learned from cold stress.

The lipid bilayer component of biological membranes is important for the distribution, organization, and function of bilayer spanning proteins. These physical barriers are subjected to bilayer perturbations. As a consequence, nature has evolved proteins that are able to sense changes in the bilayer properties and transform these lipid-mediated stimuli into intracellular signals. A structural feature that most signal-transducing membrane-embedded proteins have in common is one or more α-helices that traverse the lipid bilayer. Because of the interaction with the surrounding lipids, the organization of these transmembrane helices will be sensitive to membrane properties, like hydrophobic thickness. The helices may adapt to the lipids in different ways, which in turn can influence the structure and function of the intact membrane proteins. We review recent insights into the molecular basis of thermosensing via changes in membrane thickness and consider examples in which the hydrophobic matching can be demonstrated using reconstituted membrane systems. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

Lifelong consumption of trans fatty acids promotes striatal impairments on Na(+)/K(+) ATPase activity and BDNF mRNA expression in an animal model of mania.

PURPOSE: To evaluate the toxicity of chronic consumption of processed foods that are rich in trans fat on the lipid composition of brain membranes, as well as its functional repercussions. METHODS: A second generation of male rats born from mothers and grandmothers supplemented with soybean oil (SOC, an isocaloric control group) or hydrogenated vegetable fat (HVF, rich in TFA) (3g/kg; p.o.) were kept under oral treatment until 90 days of age, when they were exposed to an AMPH-induced model of mania. RESULTS: The HVF group presented 0.38% of TFA incorporation in the striatum, affecting Na(+)/K(+) ATPase activity, which was decreased per se and following AMPH-exposure. The HVF group also showed increased protein carbonyl (PC) and brain-derived neurotrophic factor (BDNF) mRNA levels after AMPH administration, while these oxidative and molecular changes were not observed in the other experimental groups. Additionally, a negative correlation between striatal Na(+)/K(+) ATPase activity and PC levels (r(2)=0.49) was observed. CONCLUSION: The prolonged consumption of trans fat allows TFA incorporation and increases striatal oxidative status, thus impairing the functionality of Na(+)/K(+)-ATPase and affecting molecular targets as BDNF mRNA. We hypothesized that the chronic intake of processed foods (rich in TFA) facilitates the development of neuropsychiatric diseases, particularly bipolar disorder.

Lipid-free antigen B subunits from echinococcus granulosus: oligomerization, ligand binding, and membrane interaction properties.

BACKGROUND: The hydatid disease parasite Echinococcus granulosus has a restricted lipid metabolism, and needs to harvest essential lipids from the host. Antigen B (EgAgB), an abundant lipoprotein of the larval stage (hydatid cyst), is thought to be important in lipid storage and transport. It contains a wide variety of lipid classes, from highly hydrophobic compounds to phospholipids. Its protein component belongs to the cestode-specific Hydrophobic Ligand Binding Protein family, which includes five 8-kDa isoforms encoded by a multigene family (EgAgB1-EgAgB5). How lipid and protein components are assembled into EgAgB particles remains unknown. EgAgB apolipoproteins self-associate into large oligomers, but the functional contribution of lipids to oligomerization is uncertain. Furthermore, binding of fatty acids to some EgAgB subunits has been reported, but their ability to bind other lipids and transfer them to acceptor membranes has not been studied. METHODOLOGY/PRINCIPAL FINDINGS: Lipid-free EgAgB subunits obtained by reverse-phase HPLC were used to analyse their oligomerization, ligand binding and membrane interaction properties. Size exclusion chromatography and cross-linking experiments showed that EgAgB8/2 and EgAgB8/3 can self-associate, suggesting that lipids are not required for oligomerization. Furthermore, using fluorescent probes, both subunits were found to bind fatty acids, but not cholesterol analogues. Analysis of fatty acid transfer to phospholipid vesicles demonstrated that EgAgB8/2 and EgAgB8/3 are potentially capable of transferring fatty acids to membranes, and that the efficiency of transfer is dependent on the surface charge of the vesicles. CONCLUSIONS/SIGNIFICANCE: We show that EgAgB apolipoproteins can oligomerize in the absence of lipids, and can bind and transfer fatty acids to phospholipid membranes. Since imported fatty acids are essential for Echinococcus granulosus, these findings provide a mechanism whereby EgAgB could engage in lipid acquisition and/or transport between parasite tissues. These results may therefore indicate vulnerabilities open to targeting by new types of drugs for hydatidosis therapy.

Sticholysin I-membrane interaction: an interplay between the presence of sphingomyelin and membrane fluidity.

Sticholysin I (St I) is a pore-forming toxin (PFT) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin protein family, a unique class of eukaryotic PFT exclusively found in sea anemones. As for actinoporins, it has been proposed that the presence of sphingomyelin (SM) and the coexistence of lipid phases increase binding to the target membrane. However, little is known about the role of membrane structure and dynamics (phase state, fluidity, presence of lipid domains) on actinoporins' activity or which regions of the membrane are the most favorable platforms for protein insertion. To gain insight into the role of SM on the interaction of St I to lipid membranes we studied their binding to monolayers of phosphatidylcholine (PC) and SM in different proportions. Additionally, the effect of acyl chain length and unsaturation, two features related to membrane fluidity, was evaluated on St I binding to monolayers. This study revealed that St I binds and penetrates preferentially and with a faster kinetic to liquid-expanded films with high lateral mobility and moderately enriched in SM. A high content of SM induces a lower lateral diffusion and/or liquid-condensed phases, which hinder St I binding and penetration to the lipid monolayer. Furthermore, the presence of lipid domain borders does not appear as an important factor for St I binding to the lipid monolayer.

Organic anion transporter 5 (Oat5) renal expression and urinary excretion in rats treated with cisplatin: a potential biomarker of cisplatin-induced nephrotoxicity.

Cisplatin is one of the most potent chemotherapeutic antitumor drugs used in the treatment of a wide range of solid tumors. Its primary dose-limiting side effect is nephrotoxicity. The organic anion transporter 5 (Oat5) is exclusively localized in the kidney. Oat5 urinary excretion was recently proposed as a potential early biomarker of acute kidney injury (AKI). The aim of this study was to evaluate Oat5 renal expression and its urinary excretion in rats exposed to different doses of cisplatin, in comparison with traditional markers of renal injury, like renal histology, creatinine and urea plasma levels, creatinine clearance, protein and glucose urinary levels and urinary alkaline phosphatase (AP) activity. Male Wistar rats were treated with a single injection of cisplatin at different doses of 1, 2, 5 and 10 mg/kg b.w., i.p. (Cis1, Cis2, Cis5 and Cis10, n = 4, respectively) and experiments were carried out 48 h after cisplatin administration. The renal expression of Oat5 was evaluated by immunohistochemistry and Western blotting. Oat5 abundance, AP activity, creatinine, glucose and proteins were assayed in urine. Creatinine clearance and creatinine and urea plasma levels were also evaluated. In this experimental model, plasma urea and creatinine levels, creatinine clearance, AP urinary activity and protein and glucose urinary levels were significantly modified only at the highest cisplatin dose of 10 mg/kg b.w., i.p., as compared to control rats. In contrast, Oat5 urinary abundance was increased in a dose-related manner after the administration of cisplatin. Oat5 urinary abundance was elevated at a dose as low as 1 mg/kg b.w., i.p., implying renal perturbation, when no modifications of traditional markers of renal injury are yet observed. Oat5 renal expression was decreased in a dose-related manner, both in homogenates and apical membranes from cisplatin-treated kidneys. The increase in urinary Oat5 excretion might explain the decrease in the amount of Oat5 molecules in the renal tubule cells. Hence, the preclinical animal results showed in this work propose that Oat5 urinary excretion might potentially serve as a non-invasive early biomarker of cisplatin-induced AKI.

Evaluation of the Defay-Prigogine model for the membrane interphase in relation to biological response in membrane-protein interactions.

Surface water activity appears as a common factor when the interaction of several aqueous soluble and surface active proteins with lipid membranes of different compositions is measured by the changes in surface pressure of a lipid monolayer. The perturbation of the lipid surface caused by aqueous soluble proteins depends on the composition of the hydrocarbon phases, either modified by unsaturated bonds in the acyl chains or by inclusion of cholesterol. The cut-off (critical) surface pressure in monolayers, at which no effect of the proteins is found, is related to the composition of the head group region. The perturbation of surface pressure is produced by proteins when the area per lipid is above just 4% larger than that corresponding to the hydration shell of the phospholipid head groups found in the cut-off. This area excess gives place to regions in which the chemical potential of water changes with respect to bulk water. According to the Defay-Prigogine relation this interfacial water activity is the reason of the surface pressure increase induced by aqueous soluble proteins injected in the subphase. As predicted by solution chemistry, the increase of surface pressure is independent of the protein nature but depends on the water surface state determined by the lipid composition.

Direct interaction between EgFABP1, a fatty acid binding protein from Echinococcus granulosus, and phospholipid membranes.

BACKGROUND: Growth and maintenance of hydatid cysts produced by Echinococcus granulosus have a high requirement for host lipids for biosynthetic processes, membrane building and possibly cellular and developmental signalling. This requires a high degree of lipid trafficking facilitated by lipid transporter proteins. Members of the fatty acid binding protein (FABP) family have been identified in Echinococcus granulosus, one of which, EgFABP1 is expressed at the tegumental level in the protoscoleces, but it has also been described in both hydatid cyst fluid and secretions of protoscoleces. In spite of a considerable amount of structural and biophysical information on the FABPs in general, their specific functions remain mysterious. METHODOLOGY/PRINCIPAL FINDINGS: We have investigated the way in which EgFABP1 may interact with membranes using a variety of fluorescence-based techniques and artificial small unilamellar vesicles. We first found that bacterial recombinant EgFABP1 is loaded with fatty acids from the synthesising bacteria, and that fatty acid binding increases its resistance to proteinases, possibly due to subtle conformational changes induced on EgFABP1. By manipulating the composition of lipid vesicles and the ionic environment, we found that EgFABP1 interacts with membranes in a direct contact, collisional, manner to exchange ligand, involving both ionic and hydrophobic interactions. Moreover, we observed that the protein can compete with cytochrome c for association with the surface of small unilamellar vesicles (SUVs). CONCLUSIONS/SIGNIFICANCE: This work constitutes a first approach to the understanding of protein-membrane interactions of EgFABP1. The results suggest that this protein may be actively involved in the exchange and transport of fatty acids between different membranes and cellular compartments within the parasite.

Involvement of the glutamatergic system in the nociception induced intrathecally for a TRPA1 agonist in rats.

The transient receptor potential ankyrin 1 (TRPA1) is expressed in peripheral and spinal terminals of sensory neurons, jointly to the vanilloid receptor (TRPV1). A relevant peripheral role of TRPA1 receptor has been implicated in a variety of processes, including the detection of noxious cold, and diverse painful stimulus, but the functional role of TRPA1 receptor in nociceptive transmission at spinal cord in vivo is poorly known. Therefore, the aim of this study was to evaluate whether the glutamatergic system is involved in the transmission of nociceptive stimulus induced for a TRPA1 agonist in the rat spinal cord. We observed that cinnamaldehyde, a TRPA1 agonist, on spinal cord synaptosomes leads to an increase in [Ca(2+)](i) and a rapid release of glutamate, but was not able to change the specific [(3)H]-glutamate binding. In addition, spinally administered cinnamaldehyde produced heat hyperalgesia and mechanical allodynia in rats. This behavior was reduced by the co-injection (i.t.) of camphor (TRPA1 antagonist) or MK-801 (N-methyl-D-aspartate (NMDA) receptor antagonist) to cinnamaldehyde. Besides, the pretreatment with resiniferatoxin (RTX), a potent TRPV1 agonist, abolished the cinnamaldehyde-induced heat hyperalgesia. Here, we showed that intrathecal RTX results in a decrease in TRPA1 and TRPV1 immunoreactivity in dorsal root ganglion. Collectively, our results demonstrate the pertinent participation of spinal TRPA1 in the possible enhancement of glutamatergic transmission of nociceptive signals leading to increase of the hypersensitivity, here observed as heat hyperalgesia. Then the modulation of spinal TRPA1 might be a valuable target in painful conditions associated with central pain hypersensitivity.

Long-term proline exposure alters nucleotide catabolism and ectonucleotidase gene expression in zebrafish brain.

Hyperprolinemia is an inherited disorder of proline metabolism and hyperprolinemic patients can present neurological manifestations, such as seizures cognitive dysfunctions, and psychotic disorders. However, the underlying mechanisms of these symptoms are still unclear. Since adenine nucleotides play crucial roles in neurotransmission and neuromodulation, we evaluated the in vivo and in vitro effects of proline on ectonucleotidase activities and gene expression in zebrafish brain. For the in vivo studies, animals were exposed at two proline concentrations (1.5 and 3.0 mM) during 1 h or 7 days (short- or long-term treatments, respectively). For the in vitro assays, different proline concentrations (ranging from 3.0 to 1000 µM) were tested. Short-term proline exposure did not promote significant changes on the ectonucleotidase activities and gene expression. Long-term proline exposure significantly increased ATP catabolism in both concentrations tested (14 % and 22 %, respectively), whereas ADP and AMP hydrolysis were increased only at 3.0 mM proline (21 % and 17 %, respectively) when compared to control. Moreover, the relative gene expression of enpd3 increased in both treated groups after long-term proline, whereas enptd1 increased only at 3.0 mM proline. Proline in vitro did not promote significant changes on ectonucleotidase activities. Altogether, these data indicate that the enzymes responsible for the control of extracellular nucleotides levels might be altered after proline exposure in zebrafish, contributing to better understand the pathophysiology of this disease. Moreover, such findings might facilitate the use of the zebrafish as a complementary vertebrate model for studying inborn errors of amino acid metabolism.

MK-801 alters Na+, K+-ATPase activity and oxidative status in zebrafish brain: reversal by antipsychotic drugs.

Schizophrenia is a debilitating mental disorder with a global prevalence of 1% and its etiology remains poorly understood. In the current study we investigated the influence of antipsychotic drugs on the effects of MK-801 administration, which is a drug that mimics biochemical changes observed in schizophrenia, on Na(+), K(+)-ATPase activity and some parameters of oxidative stress in zebrafish brain. Our results showed that MK-801 treatment significantly decreased Na(+), K(+)-ATPase activity, and all antipsychotics tested prevented such effects. Acute MK-801 treatment did not alter reactive oxygen/nitrogen species by 2'7'-dichlorofluorscein (H2DCF) oxidation assay, but increased the levels of thiobarbituric acid reactive substances (TBARS), when compared with controls. Some antipsychotics such as sulpiride, olanzapine, and haloperidol prevented the increase of TBARS caused by MK-801. These findings indicate oxidative damage might be a mechanism involved in the decrease of Na(+), K(+)-ATPase activity induced by MK-801. The parameters evaluated in this study had not yet been tested in this animal model using the MK-801, suggesting that zebrafish is an animal model that can contribute for providing information on potential treatments and disease characteristics.