RESUMO
The compromised viability and function of cardiovascular cells are rescued by small molecules of triazole derivatives (Tzs), identified as 3a and 3b, by preventing mitochondrial dysfunction. The oxidative phosphorylation improves the respiratory control rate in the presence of Tzs independently of the substrates that energize the mitochondria. The F1FO-ATPase, the main candidate in mitochondrial permeability transition pore (mPTP) formation, is the biological target of Tzs and hydrophilic F1 domain of the enzyme is depicted as the binding region of Tzs. The protective effect of Tz molecules on isolated mitochondria was corroborated by immortalized cardiomyocytes results. Indeed, mPTP opening was attenuated in response to ionomycin. Consequently, increased mitochondrial roundness and reduction of both length and interconnections between mitochondria. In in-vitro and ex-vivo models of cardiovascular pathologies (i.e., hypoxia-reoxygenation and hypertension) were used to evaluate the Tzs cardioprotective action. Key parameters of porcine aortic endothelial cells (pAECs) oxidative metabolism and cell viability were not affected by Tzs. However, in the presence of either 1 µM 3a or 0.5 µM 3b the impaired cell metabolism of pAECs injured by hypoxia-reoxygenation was restored to control respiratory profile. Moreover, endothelial cells isolated from SHRSP exposed to high-salt treatment rescued the Complex I activity and the endothelial capability to form vessel-like tubes and vascular function in presence of Tzs. As a result, the specific biochemical mechanism of Tzs to block Ca2+-activated F1FO-ATPase protected cell viability and preserved the pAECs bioenergetic metabolism upon hypoxia-reoxygenation injury. Moreover, SHRSP improved vascular dysfunction in response to a high-salt treatment.
Assuntos
Doenças Cardiovasculares , Proteínas de Transporte da Membrana Mitocondrial , Animais , Suínos , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/farmacologia , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/metabolismo , Células Endoteliais/metabolismo , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Hipóxia/metabolismoRESUMO
This study evaluated the bioenergetic map of mitochondria metabolism in cryopreserved bovine sperm. The detected oligomycin-sensitive basal respiration supported ATP production; frozen-thawed spermatozoa were found to have a coupling efficiency higher than 0.80. Cell respiration, however, was not stimulated by the protonophoric action of FCCP, as its titration with 1, 2, 4 and 6 µM did not stimulate the uncoupling activity on oxidative phosphorylation as highlighted by unresponsive oxygen consumption. The unusual effect on the stimulation of maximal respiration was not related to fibronectin- or PDL-coated plates used for cellular metabolism analysis. Conversely, irradiation of frozen-thawed bovine sperm with the red light improved mitochondrial parameters. In effect, the maximal respiration of red-light-stimulated sperm in PDL-coated plates was higher than the non-irradiated. In spite of this, red-light irradiation had no impact on membrane integrity and mitochondrial activity evaluated by epifluorescence microscopy.
Assuntos
Preservação do Sêmen , Sêmen , Masculino , Animais , Bovinos , Sêmen/metabolismo , Espermatozoides/fisiologia , Metabolismo Energético , Mitocôndrias/fisiologia , Criopreservação/veterinária , Motilidade dos Espermatozoides/fisiologia , Preservação do Sêmen/veterináriaRESUMO
Of the two main sectors of the F-type ATP synthase, the membrane-intrinsic FO domain is the one which, during evolution, has undergone the highest structural variations and changes in subunit composition. The FO complexity in mitochondria is apparently related to additional enzyme functions that lack in bacterial and thylakoid complexes. Indeed, the F-type ATP synthase has the main bioenergetic role to synthesize ATP by exploiting the electrochemical gradient built by respiratory complexes. The FO membrane domain, essential in the enzyme machinery, also participates in the bioenergetic cost of synthesizing ATP and in the formation of the cristae, thus contributing to mitochondrial morphology. The recent enzyme involvement in a high-conductance channel, which forms in the inner mitochondrial membrane and promotes the mitochondrial permeability transition, highlights a new F-type ATP synthase role. Point mutations which cause amino acid substitutions in FO subunits produce mitochondrial dysfunctions and lead to severe pathologies. The FO variability in different species, pointed out by cryo-EM analysis, mirrors the multiple enzyme functions and opens a new scenario in mitochondrial biology.
Assuntos
Trifosfato de Adenosina , Mitocôndrias/enzimologia , Membranas Mitocondriais/enzimologia , ATPases Mitocondriais Próton-Translocadoras , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Humanos , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/metabolismoRESUMO
LMNA mutation is associated with type-2 familial partial lipodystrophy (FPLD2). The disease causes a disorder characterized by anomalous accumulation of body fat in humans. The dysfunction at the molecular level is triggered by a lamin A/C mutation, impairing the cell metabolism. In human fibroblasts and preadipocytes, a trend for ATP production, mainly supported by mitochondrial oxidative metabolism, is detected. Moreover, primary cell lines with FPLD2 mutation decrease the mitochondrial ATP production if compared with the control, even if no differences are observed in the oxygen consumption rate of bioenergetic parameters (i.e., basal and maximal respiration, spare respiratory capacity, and ATP turnover). Conversely, glycolysis is only inhibited in FPLD2 fibroblast cell lines. We notice that the amount of ATP produced in the fibroblasts is higher than in the preadipocytes, and likewise in the control, with respect to FPLD2, due to a more active oxidative phosphorylation (OXPHOS) and glycolysis. Moreover, the proton leak parameter, which characterizes the transformation of white adipose tissue to brown/beige adipose tissue, is unaffected by FPLD2 mutation. The metabolic profile of fibroblasts and preadipocytes is confirmed by the ability of these cell lines to increase the metabolic potential of both OXPHOS and glycolysis under energy required independently by the FPLD2 mutation.
Assuntos
Lipodistrofia Parcial Familiar , Trifosfato de Adenosina/metabolismo , Tecido Adiposo Marrom/metabolismo , Metabolismo Energético , Fibroblastos/metabolismo , Humanos , Lamina Tipo A/genética , Lipodistrofia Parcial Familiar/genética , Lipodistrofia Parcial Familiar/metabolismoRESUMO
The beneficial effects of bergamot polyphenolic fraction (BPF) on the mitochondrial bioenergetics of porcine aortic endothelial cells (pAECs) were verified under the cardiotoxic action of doxorubicin (DOX). The cell viability of pAECs treated for 24 h with different concentrations of DOX was reduced by 50%, but the negative effect of DOX was reversed in the presence of increasing doses of BPF (100 µg/mL and 200 µg/mL BPF). An analysis of the protective effect of BPF on the toxic action of DOX was also carried out on cell respiration. We observed the inhibition of the mitochondrial activity at 10 µM DOX, which was not restored by 200 µg/mL BPF. Conversely, the decrease in basal respiration and ATP production caused by 0.5 or 1.0 µM DOX were improved in the presence of 100 or 200 µg/mL BPF, respectively. After 24 h of cell recovery with 100 µg/mL or 200 µg/mL BPF on pAECs treated with 0.5 µM or 1.0 µM DOX, respectively, the mitochondrial parameters of oxidative metabolism impaired by DOX were re-boosted.
Assuntos
Doxorrubicina , Células Endoteliais , Animais , Antibióticos Antineoplásicos/farmacologia , Sobrevivência Celular , Doxorrubicina/toxicidade , Coração , Mitocôndrias , SuínosRESUMO
The foetal bovine serum (FBS) concentration could influence functional parameters of IPEC-J2 cells. IPEC-J2 is a non-transformed continuous epithelial cell line that represents an established in vitro model to study porcine gut inflammation and alterations of intestinal integrity. This cell line also represents a good translational model thanks to the high similitudes between pig and human gastrointestinal tract. With the aim to assess if the FBS-dependent functional variations are linked to the bioenergetic aspects, the addition of 5% and 10% FBS in the IPEC-J2 culture medium were tested. Doubling time and TEER measurement indicated that cells cultured at higher FBS dose grow faster and as a more compact monolayer. 10% FBS increases ATP production and mitochondrial oxidative phosphorylation (OxPhos) and does not affect glycolysis. Both at 5% and 10% FBS ATP production mainly comes from OxPhos and FBS concentration does not affect the cell respiration bioenergetic parameters. Noteworthy, IPEC-J2 treated with 5% and 10% FBS have a metabolic potential since both OxPhos and glycolysis increase by > 100% and < 50%, respectively in comparison with baseline metabolism. Moreover, glucose, fatty acids and glutamine constitute the preferred metabolic fuel for mitochondrial respiration at both FBS conditions tested. Accordingly, the cells flexibility to oxidize these substrates shows that IPEC-J2 mitochondria cannot maintain the basal ATP production without oxidizing all the substrates available irrespective of FBS concentration. To sum up, in IPEC-J2 cells OxPhos increases with the FBS-stimulated functional physiological parameters to fulfil ATP requirements.
Assuntos
Trifosfato de Adenosina/biossíntese , Sangue Fetal/metabolismo , Trifosfato de Adenosina/sangue , Animais , Bovinos , Células Cultivadas , SuínosRESUMO
The dithiol reagents phenylarsine oxide (PAO) and dibromobimane (DBrB) have opposite effects on the F1FO-ATPase activity. PAO 20% increases ATP hydrolysis at 50 µM when the enzyme activity is activated by the natural cofactor Mg2+ and at 150 µM when it is activated by Ca2+. The PAO-driven F1FO-ATPase activation is reverted to the basal activity by 50 µM dithiothreitol (DTE). Conversely, 300 µM DBrB decreases the F1FO-ATPase activity by 25% when activated by Mg2+ and by 50% when activated by Ca2+. In both cases, the F1FO-ATPase inhibition by DBrB is insensitive to DTE. The mitochondrial permeability transition pore (mPTP) formation, related to the Ca2+-dependent F1FO-ATPase activity, is stimulated by PAO and desensitized by DBrB. Since PAO and DBrB apparently form adducts with different cysteine couples, the results highlight the crucial role of cross-linking of vicinal dithiols on the F1FO-ATPase, with (ir)reversible redox states, in the mPTP modulation.
Assuntos
Cisteína/química , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Animais , Arsenicais/farmacologia , Compostos Bicíclicos com Pontes/farmacologia , Cálcio/metabolismo , Ditioeritritol/farmacologia , Ativadores de Enzimas/farmacologia , Inibidores Enzimáticos/farmacologia , Magnésio/metabolismo , Mitocôndrias/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/química , SuínosRESUMO
In mammalian cells enzymatic and non-enzymatic pathways produce H2S, a gaseous transmitter which recently emerged as promising therapeutic agent and modulator of mitochondrial bioenergetics. To explore this topic, the H2S donor NaHS, at micromolar concentrations, was tested on swine heart mitochondria. NaHS did not affect the F1FO-ATPase activated by the natural cofactor Mg2, but, when Mg2+ was replaced by Ca2+, a slight 15% enzyme inhibition at 100 µM NaHS was shown. Conversely, both the NADH-O2 and succinate-O2 oxidoreductase activities were totally inhibited by 200 µM NaHS with IC50 values of 61.6 ± 4.1 and 16.5 ± 4.6 µM NaHS, respectively. Since the mitochondrial respiration was equally inhibited by NaHS at both first or second respiratory substrates sites, the H2S generation may prevent the electron transfer from complexes I and II to downhill respiratory chain complexes, probably because H2S competes with O2 in complex IV, thus reducing membrane potential as a consequence of the cytochrome c oxidase activity inhibition. The Complex IV blockage by H2S was consistent with the linear concentration-dependent NADH-O2 oxidoreductase inhibition and exponential succinate-O2 oxidoreductase inhibition by NaHS, whereas the coupling between substrate oxidation and phosphorylation was unaffected by NaHS. Even if H2S is known to cause sulfhydration of cysteine residues, thiol oxidizing (GSSG) or reducing (DTE) agents, did not affect the F1FO-ATPase activities and mitochondrial respiration, thus ruling out any involvement of post-translational modifications of thiols. The permeability transition pore, the lethal channel which forms when the F1FO-ATPase is stimulated by Ca2+, did not open in the presence of NaHS, which showed a similar effect to ruthenium red, thus suggesting a putative Ca2+ transport cycle inhibition.
Assuntos
Cálcio/metabolismo , Mitocôndrias Cardíacas/efeitos dos fármacos , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Sulfetos/farmacologia , Animais , Respiração Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Gasotransmissores/metabolismo , Sulfeto de Hidrogênio/metabolismo , Magnésio/metabolismo , Mitocôndrias Cardíacas/metabolismo , Sulfetos/metabolismo , SuínosRESUMO
Phenylglyoxal (PGO), known to cause post-translational modifications of Arg residues, was used to highlight the role of arginine residues of the F1FO-ATPase, which may be crucial to yield the mitochondrial permeability transition pore (mPTP). In swine heart mitochondria PGO inhibits ATP hydrolysis by the F1FO-ATPase either sustained by the natural cofactor Mg2+ or by Ca2+ by a similar uncompetitive inhibition mechanism, namely the tertiary complex (ESI) only forms when the ATP substrate is already bound to the enzyme, and with similar strength, as shown by the similar K'i values (0.82 ± 0.07 mM in presence of Mg2+ and 0.64 ± 0.05 mM in the presence of Ca2+). Multiple inhibitor analysis indicates that features of the F1 catalytic sites and/or the FO proton binding sites are apparently unaffected by PGO. However, PGO and F1 or FO inhibitors can bind the enzyme combine simultaneously. However they mutually hinder to bind the Mg2+-activated F1FO-ATPase, whereas they do not mutually exclude to bind the Ca2+-activated F1FO-ATPase. The putative formation of PGO-arginine adducts, and the consequent spatial rearrangement in the enzyme structure, inhibits the F1FO-ATPase activity but, as shown by the calcium retention capacity evaluation in intact mitochondria, apparently favours the mPTP formation.
Assuntos
Glioxilatos/metabolismo , Ácidos Mandélicos/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Animais , Cálcio/metabolismo , Magnésio/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , SuínosRESUMO
Hydrogen sulfide (H2S) is now considered not only for its toxicity, but also as an endogenously produced gas transmitter with multiple physiological roles, also in maintaining and regulating stem cell physiology. In the present work, we evaluated the effect of a common H2S donor, NaHS, on porcine vascular wall-mesenchymal stem cells (pVW-MSCs). pVW-MSCs were treated for 24 h with increasing doses of NaHS, and the cell viability, cell cycle, and reactive oxygen species (ROS) production were evaluated. Moreover, the long-term effects of NaHS administration on the noteworthy characteristics of pVW-MSCs were analyzed. The MTT test revealed no alteration in cell viability, however, the cell cycle analysis demonstrated that the highest NaHS dose tested (300 µM) determined a block in S phase, which did not depend on the ROS production. Moreover, NaHS (10 µM), continuously administered in culture for 21 days, was able to significantly reduce NG2, Nestin and PDGFR-ß expression. The pro-angiogenic attitude of pVW-MSCs was partially reduced by NaHS: the cells maintained the ability to grow in spheroid and sprouting from that, but endothelial markers (Factor VIII and CD31) were reduced. In conclusion, NaHS can be toxic for pVW-MSCs in high doses, while in low doses, it influences cellular physiology, by affecting the gene expression with a slowing down of the endothelial lineage.
Assuntos
Antígenos de Diferenciação/metabolismo , Vasos Sanguíneos/metabolismo , Células Endoteliais/metabolismo , Células-Tronco Mesenquimais/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Sulfetos/farmacologia , Animais , Vasos Sanguíneos/citologia , Células Endoteliais/citologia , Células-Tronco Mesenquimais/citologia , Espécies Reativas de Oxigênio/metabolismo , SuínosRESUMO
The eukaryotic F1FO-ATP synthase/hydrolase activity is coupled to H+ translocation through the inner mitochondrial membrane. According to a recent model, two asymmetric H+ half-channels in the a subunit translate a transmembrane vertical H+ flux into the rotor rotation required for ATP synthesis/hydrolysis. Along the H+ pathway, conserved aminoacid residues, mainly glutamate, address H+ both in the downhill and uphill transmembrane movements to synthesize or hydrolyze ATP, respectively. Point mutations responsible for these aminoacid changes affect H+ transfer through the membrane and, as a cascade, result in mitochondrial dysfunctions and related pathologies. The involvement of specific aminoacid residues in driving H+ along their transmembrane pathway within a subunit, sustained by the literature and calculated data, leads to depict a model consistent with some mitochondrial disorders.
Assuntos
Aminoácidos/metabolismo , Hidrogênio/metabolismo , Mitocôndrias/patologia , Membranas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Animais , Humanos , Hidrólise , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/química , Conformação ProteicaRESUMO
Selenite as an inorganic form of selenium can affect the redox state of mitochondria by modifying the thiol groups of cysteines. The F1FO-ATPase has been identified as a mitochondrial target of this compound. Indeed, the bifunctional mechanism of ATP turnover of F1FO-ATPase was differently modified by selenite. The activity of ATP hydrolysis was stimulated, whereas the ADP phosphorylation was inhibited. We ascertain that a possible new protein adduct identified as seleno-dithiol (-S-Se-S-) mercaptoethanol-sensitive caused the activation of F-ATPase activity and the oxidation of free -SH groups in mitochondria. Conversely, the inhibition of ATP synthesis by selenite might be irreversible. The kinetic analysis of the activation mechanism was an uncompetitive mixed type with respect to the ATP substrate. Selenite bound more selectively to the F1FO-ATPase loaded with the substrate by preferentially forming a tertiary (enzyme-ATP-selenite) complex. Otherwise, the selenite was a competitive mixed-type activator with respect to the Mg2+ cofactor. Thus, selenite more specifically bound to the free enzyme forming the complex enzyme-selenite. However, even if the selenite impaired the catalysis of F1FO-ATPase, the mitochondrial permeability transition pore phenomenon was unaffected. Therefore, the reversible energy transduction mechanism of F1FO-ATPase can be oppositely regulated by selenite.
Assuntos
Adenosina Trifosfatases , Compostos de Sulfidrila , Adenosina Trifosfatases/metabolismo , Fosforilação , Compostos de Sulfidrila/metabolismo , Cinética , Hidrólise , Mitocôndrias/metabolismo , Oxirredução , Trifosfato de Adenosina/metabolismoRESUMO
Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEVs) that modulate several reproductive-related processes. This study investigated the effect of two sEV subsets, small (S-sEVs) and large (L-sEVs), on porcine in vitro fertilization (IVF). The sEVs were isolated from nine SP pools (five ejaculates/pool) using a size-exclusion chromatography-based procedure and characterized for quantity (total protein), morphology (cryogenic electron microscopy), size distribution (dynamic light scattering), purity and EV-protein markers (flow cytometry; albumin, CD81, HSP90ß). The characterization confirmed the existence of two subsets of high purity (low albumin content) sEVs that differed in size (S- and L-sEVs). In vitro fertilization was performed with in vitro matured oocytes and frozen-thawed spermatozoa and the IVF medium was supplemented during gamete coincubation (1 h at 38.5 °C, 5 % CO2 in a humidified atmosphere) with three different concentrations of each sEV subset: 0 (control, without sEVs), 0.1, and 0.2 mg/mL. The first experiment showed that sEVs, regardless of subset and concentration, decreased penetration rates and total IVF efficiency (P < 0.0001). In a subsequent experiment, it was shown that sEVs, regardless of subset and concentration, impaired the ability of spermatozoa to bind to the zona pellucida of oocytes (P < 0.0001). The following experiment showed that sEVs, regardless of the subset, bound to frozen-thawed sperm but not to in vitro matured oocytes, indicating that sEVs would affect sperm functionality but not oocyte functionality. The lack of effect on oocytes was confirmed by incubating sEVs with oocytes prior to IVF, achieving sperm-zona pellucida binding results similar to those of control. In the last experiment, conducted under IVF conditions, sperm functionality was analyzed in terms of tyrosine phosphorylation, acrosome integrity and metabolism. The sEVs, regardless of the subset, did not affect sperm tyrosine phosphorylation or acrosome integrity, but did influence sperm metabolism by decreasing sperm ATP production under capacitating conditions. In conclusion, this study demonstrated that the presence of sEVs on IVF medium impairs IVF outcomes, most likely by altering sperm metabolism.
Assuntos
Sêmen , Interações Espermatozoide-Óvulo , Masculino , Suínos , Animais , Fertilização in vitro/veterinária , Fertilização in vitro/métodos , Espermatozoides/metabolismo , Oócitos , Zona Pelúcida/metabolismo , Albuminas/metabolismo , Tirosina/metabolismoRESUMO
Naringenin (NRG) was characterized for its ability to counteract mitochondrial dysfunction which is linked to cardiovascular diseases. The F1FO-ATPase can act as a molecular target of NRG. The interaction of NRG with this enzyme can avoid the energy transmission mechanism of ATP hydrolysis, especially in the presence of Ca2+ cation used as cofactor. Indeed, NRG was a selective inhibitor of the hydrophilic F1 domain displaying a binding site overlapped with quercetin in the inside surface of an annulus made by the three α and the three ß subunits arranged alternatively in a hexamer. The kinetic constant of inhibition suggested that NRG preferred the enzyme activated by Ca2+ rather than the F1FO-ATPase activated by the natural cofactor Mg2+. From the inhibition type mechanism of NRG stemmed the possibility to speculate that NRG can prevent the activation of F1FO-ATPase by Ca2+. The event correlated to the protective role in the mitochondrial permeability transition pore opening by NRG as well as to the reduction of ROS production probably linked to the NRG chemical structure with antioxidant action. Moreover, in primary cerebral endothelial cells (ECs) obtained from stroke prone spontaneously hypertensive rats NRG had a protective effect on salt-induced injury by restoring cell viability and endothelial cell tube formation while also rescuing complex I activity.
Assuntos
Células Endoteliais , Flavanonas , Poro de Transição de Permeabilidade Mitocondrial , Flavanonas/farmacologia , Animais , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Ratos , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Ratos Endogâmicos SHR , Cloreto de Sódio/farmacologia , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Cálcio/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismoRESUMO
We have investigated NADH and succinate aerobic oxidation in frozen and thawed swine heart mitochondria. Simultaneous oxidation of NADH and succinate showed complete additivity under a variety of experimental conditions, suggesting that the electron fluxes originating from NADH and succinate are completely independent and do not mix at the level of the so-called mobile diffusible components. We ascribe the results to mixing of the fluxes at the level of cytochrome c in bovine mitochondria: the Complex IV flux control coefficient in NADH oxidation was high in swine mitochondria but very low in bovine mitochondria, suggesting a stronger interaction of cytochrome c with the supercomplex in the former. This was not the case in succinate oxidation, in which Complex IV exerted little control also in swine mitochondria. We interpret the data in swine mitochondria as restriction of the NADH flux by channelling within the I-III2-IV supercomplex, whereas the flux from succinate shows pool mixing for both Coenzyme Q and probably cytochrome c. The difference between the two types of mitochondria may be ascribed to different lipid composition affecting the cytochrome c binding properties, as suggested by breaks in Arrhenius plots of Complex IV activity occurring at higher temperatures in bovine mitochondria.
Assuntos
Mitocôndrias Cardíacas , Ácido Succínico , Animais , Bovinos , Suínos , Mitocôndrias Cardíacas/metabolismo , NAD/metabolismo , Citocromos c/metabolismo , Elétrons , Succinatos/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismoRESUMO
Cellular metabolism is an important feature of spermatozoa that deserves more insights to be fully understood, in particular in porcine semen physiology. The present study aims to characterize the balance between glycolytic and oxidative metabolism in boar sperm cells. Agilent Seahorse technology was used to assess both oxygen consumption rate (OCR), as an oxidative metabolism index, and extracellular acidification rate (ECAR), as a glycolytic index. Different metabolic parameters were studied on freshly ejaculated sperm cells (identified as day zero sample, d0) and after one day of storage at 17 °C in Androhep extender (d1). Mitochondrial ATP production rate (MitoATP) was higher than the glycolytic ATP production rate (glycoATP) at both d0 and d1 while at d1 the amount of ATP production decreased, in particular, due to OXPHOS reduction. Conversely, glycoATP was not significantly different between d0 and d1. Interestingly, OCR profile showed no different bioenergetic parameters (i.e. ATP turnover, basal or maximal respiration, and spare respiration) between d0 and d1, thus indicating that sperm cell metabolism was reversibly decreased by preservation conditions. Other metabolic parameters showed the same trend, irrespective of the storage time: under stressed conditions (oligomycin plus FCCP), spermatozoa showed an increase in mitochondrial respiration while the metabolic potential of glycolysis did not undergo variations when compared to baseline metabolism. The rate of oxidation of fuel substrates - glucose, fatty acids, and glutamine - showed that sperm reliance on glucose oxidation to maintain baseline respiration was higher than fatty acids or glutamine. Interestingly spermatozoa demonstrated to have a low "capacity" parameter, which indicates that they cannot use only a single fuel substrate to produce energy. This feature of sperm metabolism to be unable to increase oxidation of a particular fuel to compensate for inhibition of alternative fuel pathway(s) was demonstrated by the negative value of "flexibility". Our results showed that ATP production in boar sperm cells relied on mitochondrial oxidative metabolism in freshly ejaculated cells, while, under liquid storage conditions, their oxidative metabolism decreased while the glycolysis remained constant. These results open new fields of research in the preservation techniques of boar sperm cells.
Assuntos
Glutamina , Sêmen , Masculino , Animais , Suínos , Sêmen/metabolismo , Metabolismo Energético , Espermatozoides/fisiologia , Glucose/metabolismo , Trifosfato de Adenosina/metabolismoRESUMO
An efficient, eco-compatible, and very cheap method for the construction of fully substituted pyrazoles (Pzs) via eliminative nitrilimine-alkene 1,3-dipolar cycloaddition (ENAC) reaction was developed in excellent yield and high regioselectivity. Enaminones and nitrilimines generated in situ were selected as dipolarophiles and dipoles, respectively. A deep screening of the employed base, solvent, and temperature was carried out to optimize reaction conditions. Recycling tests of ionic liquid were performed, furnishing efficient performance until six cycles. Finally, a plausible mechanism of cycloaddition was proposed. Then, the effect of three different structures of Pzs was evaluated on the F1FO-ATPase activity and mitochondrial permeability transition pore (mPTP) opening. The Pz derivatives' titration curves of 6a, 6h, and 6o on the F1FO-ATPase showed a reduced activity of 86%, 35%, and 31%, respectively. Enzyme inhibition analysis depicted an uncompetitive mechanism with the typical formation of the tertiary complex enzyme-substrate-inhibitor (ESI). The dissociation constant of the ESI complex (Ki') in the presence of the 6a had a lower order of magnitude than other Pzs. The pyrazole core might set the specific mechanism of inhibition with the F1FO-ATPase, whereas specific functional groups of Pzs might modulate the binding affinity. The mPTP opening decreased in Pz-treated mitochondria and the Pzs' inhibitory effect on the mPTP was concentration-dependent with 6a and 6o. Indeed, the mPTP was more efficiently blocked with 0.1 mM 6a than with 1 mM 6a. On the contrary, 1 mM 6o had stronger desensitization of mPTP formation than 0.1 mM 6o. The F1FO-ATPase is a target of Pzs blocking mPTP formation.
RESUMO
The bergamot polyphenolic fraction (BPF) was evaluated in the F1FO-ATPase activity of swine heart mitochondria. In the presence of a concentration higher than 50 µg/mL BPF, the ATPase activity of F1FO-ATPase, dependent on the natural cofactor Mg2+, increased by 15%, whereas the enzyme activity in the presence of Ca2+ was inhibited by 10%. By considering this opposite BPF effect, the F1FO-ATPase activity involved in providing ATP synthesis in oxidative phosphorylation and triggering mitochondrial permeability transition pore (mPTP) formation has been evaluated. The BPF improved the catalytic coupling of oxidative phosphorylation in the presence of a substrate at the first phosphorylation site, boosting the respiratory control ratios (state 3/state 4) by 25% and 85% with 50 µg/mL and 100 µg/mL BPF, respectively. Conversely, the substrate at the second phosphorylation site led to the improvement of the state 3/state 4 ratios by 15% only with 100 µg/mL BPF. Moreover, the BPF carried out its beneficial effect on the mPTP phenomenon by desensitizing the pore opening. The acute effect of the BPF on the metabolism of porcine aortica endothelial cells (pAECs) showed an ATP rate index greater than one, which points out a prevailing mitochondrial oxidative metabolism with respect to the glycolytic pathway, and this ratio rose by about three times with 100 µg/mL BPF. Consistently, the mitochondrial ATP turnover, in addition to the basal and maximal respiration, were higher in the presence of the BPF than in the controls, and the MTT test revealed an increase in cell viability with a BPF concentration above 200 µg/mL. Therefore, the molecule mixture of the BPF aims to ensure good performance of the mitochondrial bioenergetic parameters.
Assuntos
Cálcio , Células Endoteliais , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Células Endoteliais/metabolismo , Metabolismo Energético , Mitocôndrias Cardíacas/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , SuínosRESUMO
The mitochondrial permeability transition pore (PTP), which drives regulated cell death when Ca2+ concentration suddenly increases in mitochondria, was related to changes in the Ca2+-activated F1FO-ATPase. The effects of the gadolinium cation (Gd3+), widely used for diagnosis and therapy, and reported as PTP blocker, were evaluated on the F1FO-ATPase activated by Mg2+ or Ca2+ and on the PTP. Gd3+ more effectively inhibits the Ca2+-activated F1FO-ATPase than the Mg2+-activated F1FO-ATPase by a mixed-type inhibition on the former and by uncompetitive mechanism on the latter. Most likely Gd3+ binding to F1, is favoured by Ca2+ insertion. The maximal inactivation rates (kinact) of pseudo-first order inactivation are similar either when the F1FO-ATPase is activated by Ca2+ or by Mg2+. The half-maximal inactivator concentrations (KI) are 2.35 ± 0.35 mM and 0.72 ± 0.11 mM, respectively. The potency of a mechanism-based inhibitor (kinact/KI) also highlights a higher inhibition efficiency of Gd3+ on the Ca2+-activated F1FO-ATPase (0.59 ± 0.09 mM-1âs-1) than on the Mg2+-activated F1FO-ATPase (0.13 ± 0.02 mM-1âs-1). Consistently, the PTP is desensitized in presence of Gd3+. The Gd3+ inhibition on both the mitochondrial Ca2+-activated F1FO-ATPase and the PTP strengthens the link between the PTP and the F1FO-ATPase when activated by Ca2+ and provides insights on the biological effects of Gd3+.
Assuntos
Inibidores Enzimáticos/farmacologia , Gadolínio/farmacologia , Mitocôndrias/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/farmacologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Animais , Cálcio/metabolismo , Cátions , Ativação Enzimática/efeitos dos fármacos , Cinética , Magnésio/metabolismo , Mitocôndrias/efeitos dos fármacos , ATPases Mitocondriais Próton-Translocadoras/química , Modelos Moleculares , Conformação Proteica , Sus scrofaRESUMO
The fat-soluble vitamin K (VK) has long been known as a requirement for blood coagulation, but like other vitamins, has been recently recognized to play further physiological roles, particularly in cell development and homeostasis. Vertebrates cannot de novo synthesize VK, which is essential, and it can only be obtained from the diet or by the activity of the gut microbiota. The IPEC-J2 cell line, obtained from porcine small intestine, which shows strong similarities to the human one, represents an excellent functional model to in vitro study the effect of compounds at the intestinal level. The acute VK treatments on the bioenergetic features of IPEC-J2 cells were evaluated by Seahorse XP Agilent technology. VK exists in different structurally related forms (vitamers), all featured by a naphtoquinone moiety, but with distinct effects on IPEC-J2 energy metabolism. The VK1, which has a long hydrocarbon chain, at both concentrations (5 and 10 µM), increases the cellular ATP production due to oxidative phosphorylation (OXPHOS) by 5% and by 30% through glycolysis. The VK2 at 5 µM only stimulates ATP production by OXPHOS. Conversely, 10 µM VK3, which lacks the long side chain, inhibits OXPHOS by 30% and glycolysis by 45%. However, even if IPEC-J2 cells mainly prefer OXPHOS to glycolysis to produce ATP, the OXPHOS/glycolysis ratio significantly decreases in VK1-treated cells, is unaffected by VK2, and only significantly increased by 10 µM VK3. VK1, at the two concentrations tested, does not affect the mitochondrial bioenergetic parameters, while 5 µM VK2 increases and 5 µM VK3 reduces the mitochondrial respiration (i.e., maximal respiration and spare respiratory capacity). Moreover, 10 µM VK3 impairs OXPHOS, as shown by the increase in the proton leak, namely the proton backward entry to the matrix space, thus pointing out mitochondrial toxicity. Furthermore, in the presence of both VK1 and VK2 concentrations, the glycolytic parameters, namely the glycolytic capacity and the glycolytic reserve, are unaltered. In contrast, the inhibition of glycoATP production by VK3 is linked to the 80% inhibition of glycolysis, resulting in a reduced glycolytic capacity and reserve. These data, which demonstrate the VK ability to differently modulate IPEC-J2 cell energy metabolism according to the different structural features of the vitamers, can mirror VK modulatory effects on the cell membrane features and, as a cascade, on the epithelial cell properties and gut functions: balance of salt and water, macromolecule cleavage, detoxification of harmful compounds, and nitrogen recycling.