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1.
Nat Commun ; 11(1): 5073, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033238

RESUMO

Brain cells continuously produce and release protons into the extracellular space, with the rate of acid production corresponding to the levels of neuronal activity and metabolism. Efficient buffering and removal of excess H+ is essential for brain function, not least because all the electrogenic and biochemical machinery of synaptic transmission is highly sensitive to changes in pH. Here, we describe an astroglial mechanism that contributes to the protection of the brain milieu from acidification. In vivo and in vitro experiments conducted in rodent models show that at least one third of all astrocytes release bicarbonate to buffer extracellular H+ loads associated with increases in neuronal activity. The underlying signalling mechanism involves activity-dependent release of ATP triggering bicarbonate secretion by astrocytes via activation of metabotropic P2Y1 receptors, recruitment of phospholipase C, release of Ca2+ from the internal stores, and facilitated outward HCO3- transport by the electrogenic sodium bicarbonate cotransporter 1, NBCe1. These results show that astrocytes maintain local brain extracellular pH homeostasis via a neuronal activity-dependent release of bicarbonate. The data provide evidence of another important metabolic housekeeping function of these glial cells.


Assuntos
Astrócitos/metabolismo , Bicarbonatos/metabolismo , Encéfalo/metabolismo , Espaço Extracelular/metabolismo , Acetazolamida/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Anidrases Carbônicas/metabolismo , Células Cultivadas , Estimulação Elétrica , Fluorescência , Hipocampo/metabolismo , Concentração de Íons de Hidrogênio , Camundongos Endogâmicos C57BL , Modelos Biológicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Antagonistas Purinérgicos/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Receptores Purinérgicos/metabolismo , Transdução de Sinais , Simportadores de Sódio-Bicarbonato/metabolismo
2.
PLoS One ; 15(10): e0240887, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33119622

RESUMO

The two flagella of Chlamydomonas reinhardtii are of the same size and structure but display functional differences, which are critical for flagellar steering movements. However, biochemical differences between the two flagella have not been identified. Here, we show that fluorescence protein-tagged carbonic anhydrase 6 (CAH6-mNG) preferentially localizes to the trans-flagellum, which is organized by the older of the two flagella-bearing basal bodies. The uneven distribution of CAH6-mNG is established early during flagellar assembly and restored after photobleaching, suggesting that it is based on preferred entry or retention of CAH6-mNG in the trans-flagellum. Since CAH6-mNG moves mostly by diffusion, a role of intraflagellar transport (IFT) in establishing its asymmetric distribution is unlikely. Interestingly, CAH6-mNG is present in both flagella of the non-phototactic bardet-biedl syndrome 1 (bbs1) mutant revealing that the BBSome is involved in establishing CAH6-mNG flagellar asymmetry. Using dikaryon rescue experiments, we show that the de novo assembly of CAH6-mNG in flagella is considerably faster than the removal of ectopic CAH6-mNG from bbs flagella. Thus, different rates of flagellar entry of CAH6-mNG rather than its export from flagella is the likely basis for its asymmetric distribution. The data identify a novel role for the C. reinhardtii BBSome in preventing the entry of CAH6-mNG specifically into the cis-flagellum.


Assuntos
Anidrases Carbônicas/genética , Chlamydomonas reinhardtii/genética , Flagelos/genética , Transporte Proteico/genética , Sequência de Aminoácidos/genética , Corpos Basais/metabolismo , Bicarbonatos/metabolismo , Dióxido de Carbono/metabolismo , Anidrases Carbônicas/metabolismo , Chlamydomonas reinhardtii/enzimologia , Cílios/enzimologia , Cílios/genética , Flagelos/enzimologia , Recuperação de Fluorescência Após Fotodegradação , Humanos , Zigoto/efeitos dos fármacos , Zigoto/crescimento & desenvolvimento
3.
Proc Biol Sci ; 287(1934): 20201506, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32900308

RESUMO

The sea urchin embryo develops a calcitic endoskeleton through intracellular formation of amorphous calcium carbonate (ACC). Intracellular precipitation of ACC, requires [Formula: see text] concentrating as well as proton export mechanisms to promote calcification. These processes are of fundamental importance in biological mineralization, but remain largely unexplored. Here, we demonstrate that the calcifying primary mesenchyme cells (PMCs) use Na+/H+-exchange (NHE) mechanisms to control cellular pH homeostasis during maintenance of the skeleton. During skeleton re-calcification, pHi of PMCs is increased accompanied by substantial elevation in intracellular [Formula: see text] mediated by the [Formula: see text] cotransporter Sp_Slc4a10. However, PMCs lower their pHi regulatory capacities associated with a reduction in NHE activity. Live-cell imaging using green fluorescent protein reporter constructs in combination with intravesicular pH measurements demonstrated alkaline and acidic populations of vesicles in PMCs and extensive trafficking of large V-type H+-ATPase (VHA)-rich acidic vesicles in blastocoelar filopodial cells. Pharmacological and gene expression analyses underline a central role of the VHA isoforms Sp_ATP6V0a1, Sp_ATP6V01_1 and Sp_ATPa1-4 for the process of skeleton re-calcification. These results highlight novel pH regulatory strategies in calcifying cells of a marine species with important implications for our understanding of the mineralization process in times of rapid changes in oceanic pH.


Assuntos
Bicarbonatos/metabolismo , Ouriços-do-Mar/fisiologia , Animais , Transporte Biológico , Calcificação Fisiológica , Carbonato de Cálcio , Concentração de Íons de Hidrogênio , Oceanos e Mares , Prótons , Água do Mar
4.
Sci Rep ; 10(1): 13732, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792614

RESUMO

This investigation assessed the course of renal compensation of hypoxia-induced respiratory alkalosis by elimination of bicarbonate ions and impairments in anaerobic exercise after different durations of hypoxic exposure. Study A: 16 participants underwent a resting 12-h exposure to normobaric hypoxia (3,000 m). Blood gas analysis was assessed hourly. While blood pH was significantly increased, PO2, PCO2, and SaO2 were decreased within the first hour of hypoxia, and changes remained consistent. A substantial reduction in [HCO3-] levels was observed after 12 h of hypoxic exposure (- 1.35 ± 0.29 mmol/L, p ≤ 0.05). Study B: 24 participants performed in a randomized, cross-over trial portable tethered sprint running (PTSR) tests under normoxia and after either 1 h (n = 12) or 12 h (n = 12) of normobaric hypoxia (3,000 m). No differences occurred for PTSR-related performance parameters, but the reduction in blood lactate levels was greater after 12 h compared with 1 h (- 1.9 ± 2.2 vs 0.0 ± 2.3 mmol/L, p ≤ 0.05). These results indicate uncompensated respiratory alkalosis after 12 h of hypoxia and similar impairment of high-intensity exercise after 1 and 12 h of hypoxic exposure, despite a greater reduction in blood lactate responses after 12 h compared with 1 h of hypoxic exposure.


Assuntos
Equilíbrio Ácido-Base/fisiologia , Exercício Físico/fisiologia , Hipóxia/fisiopatologia , Adulto , Alcalose/metabolismo , Alcalose/fisiopatologia , Altitude , Bicarbonatos/metabolismo , Dióxido de Carbono/metabolismo , Estudos Cross-Over , Feminino , Humanos , Hipóxia/metabolismo , Masculino , Oxigênio/metabolismo , Corrida/fisiologia , Adulto Jovem
5.
Am J Physiol Renal Physiol ; 319(3): F366-F379, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32657159

RESUMO

Carbonic anhydrase (CAII) binds to the electrogenic basolateral Na+-[Formula: see text] cotransporter (NBCe1) and facilitates [Formula: see text] reabsorption across the proximal tubule. However, whether the inhibition of CAII with acetazolamide (ACTZ) alters NBCe1 activity and interferes with the ammoniagenesis pathway remains elusive. To address this issue, we compared the renal adaptation of rats treated with ACTZ to NH4Cl loading for up to 2 wk. The results indicated that ACTZ-treated rats exhibited a sustained metabolic acidosis for up to 2 wk, whereas in NH4Cl-loaded rats, metabolic acidosis was corrected within 2 wk of treatment. [Formula: see text] excretion increased by 10-fold in NH4Cl-loaded rats but only slightly (1.7-fold) in ACTZ-treated rats during the first week despite a similar degree of acidosis. Immunoblot experiments showed that the protein abundance of glutaminase (4-fold), glutamate dehydrogenase (6-fold), and SN1 (8-fold) increased significantly in NH4Cl-loaded rats but remained unchanged in ACTZ-treated rats. Na+/H+ exchanger 3 and NBCe1 proteins were upregulated in response to NH4Cl loading but not ACTZ treatment and were rather sharply downregulated after 2 wk of ACTZ treatment. ACTZ causes renal [Formula: see text] wasting and induces metabolic acidosis but inhibits the upregulation of glutamine transporter and ammoniagenic enzymes and thus suppresses ammonia synthesis and secretion in the proximal tubule, which prevented the correction of acidosis. This effect is likely mediated through the inhibition of the CA-NBCe1 metabolon complex, which results in cell alkalinization. During chronic ACTZ treatment, the downregulation of both NBCe1 and Na+/H+ exchanger 3, along with the inhibition of ammoniagenesis and [Formula: see text] generation, contributes to the maintenance of metabolic acidosis.


Assuntos
Acetazolamida/farmacologia , Acidose/metabolismo , Amônia/metabolismo , Bicarbonatos/metabolismo , Diuréticos/farmacologia , Rim/efeitos dos fármacos , Acetazolamida/farmacocinética , Equilíbrio Ácido-Base/efeitos dos fármacos , Adaptação Fisiológica , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Animais , Cloretos/sangue , Diuréticos/farmacocinética , Eletrólitos/sangue , Regulação da Expressão Gênica/efeitos dos fármacos , Meia-Vida , Rim/metabolismo , Rim/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Urinálise
6.
Am J Physiol Cell Physiol ; 319(2): C359-C370, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32520610

RESUMO

SLC4A11 is the only member of the SLC4 family that transports protons rather than bicarbonate. SLC4A11 is expressed in corneal endothelial cells, and its mutation causes corneal endothelial dystrophy, although the mechanism of pathogenesis is unknown. We previously demonstrated that the magnitude of the H+ conductance (Gm) mediated by SLC4A11 is increased by rises in intracellular as well as extracellular pH (pHi and pHe). To better understand this feature and whether it is altered in disease, we studied the pH dependence of wild-type and mutant mouse Slc4a11 expressed in Xenopus oocytes. Using voltage-clamp circuitry in conjunction with a H+-selective microelectrode and a microinjector loaded with NaHCO3, we caused incremental rises in oocyte pHi and measured the effect on Gm. We find that the rise of Gm has a steeper pHi dependence at pHe =8.50 than at pHe =7.50. Data gathered at pHe =8.50 can be fit to the Hill equation enabling the calculation of a pK value that reports pHi dependence. We find that mutation of lysine residues that are close to the first transmembrane span (TM1) causes an alkaline shift in pK. Furthermore, two corneal-dystrophy-causing mutations close to the extracellular end of TM1, E399K and T401K (E368K and T370K in mouse), cause an acidic shift in pK, while a third mutation in the fourth intracellular loop, R804H (R774H in mouse), causes an alkaline shift in pK. This is the first description of determinants of SLC4A11 pH dependence and the first indication that a shift in pH dependence could modify disease expressivity in some cases of corneal dystrophy.


Assuntos
Proteínas de Transporte de Ânions/genética , Transporte Biológico/genética , Distrofias Hereditárias da Córnea/genética , Lisina/genética , Simportadores/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Bicarbonatos/metabolismo , Distrofias Hereditárias da Córnea/metabolismo , Distrofias Hereditárias da Córnea/patologia , Modelos Animais de Doenças , Epitélio Posterior/metabolismo , Epitélio Posterior/patologia , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Transporte de Íons/genética , Lisina/metabolismo , Camundongos , Mutação/genética , Oócitos/metabolismo , Oócitos/patologia , Sódio , Xenopus/genética
7.
Biochemistry (Mosc) ; 85(6): 697-708, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32586233

RESUMO

The structure and functional organization of the photosystem I (PSI) reaction center (RC) donor side has a significant similarity to the reaction centers of purple bacteria (bRCs), despite the fact that they belong to different types of RCs. Moreover, the redox potential values of their primary electron donors are identical (~0.5 V). In our earlier reports [Khorobrykh et al. (2008) Phylos. Trans. R. Soc. B., 363, 1245-1251; Terentyev et al. (2011) Biochemistry (Moscow), 76, 1360-1366; Khorobrykh et al. (2018) ChemBioChem, 14, 1725-1731], we have demonstrated redox interaction of low-potential Mn2+-bicarbonate complexes with bRCs, which might have been one of the first steps in the evolutionary origin of Mn-cluster of the photosystem II water-oxidizing complex that occurred in the Archean (over 3 billion years ago). In this study, we investigated redox interactions between Mn2+-bicarbonate complexes and PSI. Such interactions were almost absent in the original PSI preparations and emerged only in preoxidized PSI preparations containing ~50% oxidized RCs. The interaction between Mn2+-bicarbonate complexes and PSI required increased Mn2+ concentrations, while its dependence on the HCO3- concentration indicated involvement of electroneutral low-potential [Mn(HCO3)2] complex in the process. Analysis of the PSI crystal structure revealed steric hindrances on the RC donor side, which could block the redox interaction between Mn2+-bicarbonate complexes and oxidized primary electron donor. Comparison of structures of RCs from the PSI and ancient RCs from heliobacteria belonging to the same type of RCs suggested that such hindrances should be absent in the primitive PSI in the Archean and allowed to explain their evolutionary origin as a consequence of PSI RCs into the united electron transport chain (ETC) of the photosynthetic membrane that was accompanied by the evolutionary loss of PSI capacity for the redox interaction with Mn2+-bicarbonate complexes.


Assuntos
Bicarbonatos/química , Evolução Biológica , Transporte de Elétrons , Manganês/química , Oxirredução , Fotossíntese , Complexo de Proteína do Fotossistema I/metabolismo , Bicarbonatos/metabolismo , Elétrons , Manganês/metabolismo , Água/química
8.
Biochem J ; 477(11): 2027-2038, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32497192

RESUMO

Alkenes and ketones are two classes of ubiquitous, toxic organic compounds in natural environments produced in several biological and anthropogenic processes. In spite of their toxicity, these compounds are utilized as primary carbon and energy sources or are generated as intermediate metabolites in the metabolism of other compounds by many diverse bacteria. The aerobic metabolism of some of the smallest and most volatile of these compounds (propylene, acetone, isopropanol) involves novel carboxylation reactions resulting in a common product acetoacetate. Propylene is metabolized in a four-step pathway involving five enzymes where the penultimate step is a carboxylation reaction catalyzed by a unique disulfide oxidoreductase that couples reductive cleavage of a thioether linkage with carboxylation to produce acetoacetate. The carboxylation of isopropanol begins with conversion to acetone via an alcohol dehydrogenase. Acetone is converted to acetoacetate in a single step by an acetone carboxylase which couples the hydrolysis of MgATP to the activation of both acetone and bicarbonate, generating highly reactive intermediates that are condensed into acetoacetate at a Mn2+ containing the active site. Acetoacetate is then utilized in central metabolism where it is readily converted to acetyl-coenzyme A and subsequently converted into biomass or utilized in energy metabolism via the tricarboxylic acid cycle. This review summarizes recent structural and biochemical findings that have contributed significant insights into the mechanism of these two unique carboxylating enzymes.


Assuntos
Acetona/metabolismo , Alcenos/metabolismo , Bactérias/metabolismo , 2-Propanol/metabolismo , Acetoacetatos/metabolismo , Acetilcoenzima A/metabolismo , Bicarbonatos/metabolismo , Catálise , Ciclo do Ácido Cítrico/fisiologia
9.
Am J Respir Crit Care Med ; 202(7): 996-1004, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32551817

RESUMO

Rationale: Two distinct phenotypes of acute respiratory distress syndrome (ARDS) with differential clinical outcomes and responses to randomly assigned treatment have consistently been identified in randomized controlled trial cohorts using latent class analysis. Plasma biomarkers, key components in phenotype identification, currently lack point-of-care assays and represent a barrier to the clinical implementation of phenotypes.Objectives: The objective of this study was to develop models to classify ARDS phenotypes using readily available clinical data only.Methods: Three randomized controlled trial cohorts served as the training data set (ARMA [High vs. Low Vt], ALVEOLI [Assessment of Low Vt and Elevated End-Expiratory Pressure to Obviate Lung Injury], and FACTT [Fluids and Catheter Treatment Trial]; n = 2,022), and a fourth served as the validation data set (SAILS [Statins for Acutely Injured Lungs from Sepsis]; n = 745). A gradient-boosted machine algorithm was used to develop classifier models using 24 variables (demographics, vital signs, laboratory, and respiratory variables) at enrollment. In two secondary analyses, the ALVEOLI and FACTT cohorts each, individually, served as the validation data set, and the remaining combined cohorts formed the training data set for each analysis. Model performance was evaluated against the latent class analysis-derived phenotype.Measurements and Main Results: For the primary analysis, the model accurately classified the phenotypes in the validation cohort (area under the receiver operating characteristic curve [AUC], 0.95; 95% confidence interval [CI], 0.94-0.96). Using a probability cutoff of 0.5 to assign class, inflammatory biomarkers (IL-6, IL-8, and sTNFR-1; P < 0.0001) and 90-day mortality (38% vs. 24%; P = 0.0002) were significantly higher in the hyperinflammatory phenotype as classified by the model. Model accuracy was similar when ALVEOLI (AUC, 0.94; 95% CI, 0.92-0.96) and FACTT (AUC, 0.94; 95% CI, 0.92-0.95) were used as the validation cohorts. Significant treatment interactions were observed with the clinical classifier model-assigned phenotypes in both ALVEOLI (P = 0.0113) and FACTT (P = 0.0072) cohorts.Conclusions: ARDS phenotypes can be accurately identified using machine learning models based on readily available clinical data and may enable rapid phenotype identification at the bedside.


Assuntos
Aprendizado de Máquina , Síndrome do Desconforto Respiratório do Adulto/classificação , Fatores Etários , Área Sob a Curva , Bicarbonatos/metabolismo , Bilirrubina/metabolismo , Biomarcadores Tumorais , Pressão Sanguínea , Dióxido de Carbono/metabolismo , Creatinina/metabolismo , Humanos , Inflamação , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Análise de Classes Latentes , Contagem de Leucócitos , Mortalidade , Oxigênio/metabolismo , Pressão Parcial , Fenótipo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Contagem de Plaquetas , Prognóstico , Proteína C/metabolismo , Ventilação Pulmonar , Ensaios Clínicos Controlados Aleatórios como Assunto , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Síndrome do Desconforto Respiratório do Adulto/imunologia , Síndrome do Desconforto Respiratório do Adulto/fisiopatologia , Síndrome do Desconforto Respiratório do Adulto/terapia , Albumina Sérica/metabolismo , Volume de Ventilação Pulmonar , Vasoconstritores/uso terapêutico , Sinais Vitais
10.
Am J Physiol Cell Physiol ; 319(2): C331-C344, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32432926

RESUMO

The pH of airway surface liquid (ASL) is a key factor that determines respiratory host defense; ASL acidification impairs and alkalinization enhances key defense mechanisms. Under healthy conditions, airway epithelia secrete base ([Formula: see text]) and acid (H+) to control ASL pH (pHASL). Neutrophil-predominant inflammation is a hallmark of several airway diseases, and TNFα and IL-17 are key drivers. However, how these cytokines perturb pHASL regulation is uncertain. In primary cultures of differentiated human airway epithelia, TNFα decreased and IL-17 did not change pHASL. However, the combination (TNFα+IL-17) markedly increased pHASL by increasing [Formula: see text] secretion. TNFα+IL-17 increased expression and function of two apical [Formula: see text] transporters, CFTR anion channels and pendrin Cl-/[Formula: see text] exchangers. Both were required for maximal alkalinization. TNFα+IL-17 induced pendrin expression primarily in secretory cells where it was coexpressed with CFTR. Interestingly, significant pendrin expression was not detected in CFTR-rich ionocytes. These results indicate that TNFα+IL-17 stimulate [Formula: see text] secretion via CFTR and pendrin to alkalinize ASL, which may represent an important defense mechanism in inflamed airways.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Interleucina-17/genética , Mucosa Respiratória/metabolismo , Transportadores de Sulfato/genética , Fator de Necrose Tumoral alfa/genética , Álcalis/metabolismo , Bicarbonatos/metabolismo , Antiportadores de Cloreto-Bicarbonato/metabolismo , Citocinas/genética , Citocinas/metabolismo , Células Epiteliais/metabolismo , Humanos , Interleucina-17/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
11.
Sci Rep ; 10(1): 8440, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32439937

RESUMO

One of the most common mutations in Cystic Fibrosis (CF) patients is the deletion of the amino acid phenylalanine at position 508. This mutation causes both the protein trafficking defect and an early degradation. Over time, small molecules, called correctors, capable of increasing the amount of mutated channel in the plasma membrane and causing an increase in its transport activity have been developed. This study shows that incubating in vitro cells permanently transfected with the mutated channel with the correctors VX809, VX661 and Corr4a, and the combination of VX809 and Corr4a, a recovery of anion transport activity is observed. Interestingly, the permeability of bicarbonate increases in the cells containing corrected p.F508del CFTR channels is greater than the increase of the halide permeability. These different increases of the permeability of bicarbonate and halides are consistent with the concept that the structural conformation of the pore of the corrector-rescued p.F508del channels would be different than the normal wild type CFTR protein.


Assuntos
Aminopiridinas/farmacologia , Benzodioxóis/farmacologia , Bicarbonatos/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Mutação , Animais , Membrana Celular , Células Cultivadas , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Transporte Proteico , Ratos , Glândula Tireoide/citologia , Glândula Tireoide/efeitos dos fármacos , Glândula Tireoide/metabolismo
12.
Ecotoxicol Environ Saf ; 196: 110512, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32244115

RESUMO

Although there are numerous studies on bisphenol A (BPA) on the testis and spermatozoa, the effect of BPA on the physiological link between the testis and maturation of spermatozoa has not been studied. To provide an optimal environment (acidic pH) for sperm maturation in the epididymis, clear cells secrete protons and principal cells reabsorb bicarbonate and the secreted proton. Because of its crucial role in sperm maturation and fertility, functional changes in the epididymis following BPA exposure must be considered to fully understand the mechanisms of BPA on male fertility. Here, we identified the adverse effects of BPA exposure during puberty in male mice. CD-1 male mice were gavaged daily with vehicle (corn oil) and 50 mg BPA/kg-BW for 6 weeks. We determined the changes in epididymis, functional sperm parameters including motility, capacitation status, tyrosine phosphorylation, and fertility-related protein expression and in vitro and in vivo fertility rate following BPA exposure. Expression of vacuolar-type H + -ATPase is necessary for the secretion of protons by clear cells of the caput epididymis and was directly down-regulated following BPA exposure, while there were no changes in the other epithelial cell types in the epididymis. Also, pERK 1/2 signaling pathway was increased significantly in the caput epididymis following BPA exposure. Consequently, the luminal pH slightly increased, resulting in premature capacitation of spermatozoa. Moreover, there was a significant loss of the acrosomal membrane following an increase of protein tyrosine phosphorylation, while PKA activity decreased during sperm capacitation. Fertility-related proteins also showed aberrant expression upon BPA exposure. These modifications resulted in decreased male fertility in vitro and in vivo.


Assuntos
Compostos Benzidrílicos/toxicidade , Poluentes Ambientais/toxicidade , Fertilidade/efeitos dos fármacos , Fenóis/toxicidade , Maturação do Esperma/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Animais , Bicarbonatos/metabolismo , Epididimo/efeitos dos fármacos , Masculino , Camundongos , Fosforilação , Transdução de Sinais , Capacitação Espermática/efeitos dos fármacos , Motilidade Espermática/efeitos dos fármacos , Espermatozoides/metabolismo , Testículo/efeitos dos fármacos
13.
J Microbiol Biotechnol ; 30(6): 868-877, 2020 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-32238762

RESUMO

Bicarbonate has been considered as a better approach for supplying CO2 to microalgae cells microenvironments than gas bubbling owing t°Cost-effectiveness and easy operation. However, the ß-carotene production was too low in Dunaliella salina cultivated with bicarbonate in previous studies. Also, the difference in photosynthetic efficiency between these tw°Carbon sources (bicarbonate and CO2) has seldom been discussed. In this study, the culture conditions, including NaHCO3, Ca2+, Mg2+ and microelement concentrations, were optimized when bicarbonate was used as carbon source. Under optimized condition, a maximum biomass concentration of 0.71 g/l and corresponding ß-carotene content of 4.76% were obtained, with ß-carotene yield of 32.0 mg/l, much higher than previous studies with NaHCO3. Finally, these optimized conditions with bicarbonate were compared with CO2 bubbling by online monitoring. There was a notable difference in Fv/Fm value between cultivations with bicarbonate and CO2, but there was no difference in the Fv/Fm periodic changing patterns. This indicates that the high concentration of NaHCO3 used in this study served as a stress factor for ß-carotene accumulation, although high productivity of biomass was still obtained.


Assuntos
Bicarbonatos/metabolismo , Clorofíceas/metabolismo , Microalgas/metabolismo , beta Caroteno/metabolismo , Bicarbonatos/química , Cálcio/metabolismo , Carbono/metabolismo , Meios de Cultura/química , Magnésio/metabolismo , beta Caroteno/análise , beta Caroteno/química
14.
Am J Physiol Renal Physiol ; 318(6): F1418-F1429, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32308019

RESUMO

The use of high dialysate bicarbonate for hemodialysis in end-stage renal disease is associated with increased mortality, but potential physiological mediators are poorly understood. Alkalinization due to high dialysate bicarbonate may stimulate organic acid generation, which could lead to poor outcomes. Using measurements of ß-hydroxybutyrate (BHB) and lactate, we quantified organic anion (OA) balance in two single-arm studies comparing high and low bicarbonate prescriptions. In study 1 (n = 10), patients became alkalemic using 37 meq/L dialysate bicarbonate; in contrast, with the use of 27 meq/L dialysate, net bicarbonate loss occurred and blood bicarbonate decreased. Total OA losses were not higher with 37 meq/L dialysate bicarbonate (50.9 vs. 49.1 meq using 27 meq/L, P = 0.66); serum BHB increased in both treatments similarly (P = 0.27); and blood lactate was only slightly higher with the use of 37 meq/L dialysate (P = 0.048), differing by 0.2 meq/L at the end of hemodialysis. In study 2 (n = 7), patients achieved steady state on two bicarbonate prescriptions: they were significantly more acidemic when dialyzed against a 30 meq/L bicarbonate dialysate compared with 35 meq/L and, as in study 1, became alkalemic when dialyzed against the higher bicarbonate dialysate. OA losses were similar to those in study 1 and again did not differ between treatments (38.9 vs. 43.5 meq, P = 0.42). Finally, free fatty acid levels increased throughout hemodialysis and correlated with the change in serum BHB (r = 0.81, P < 0.001), implicating upregulation of lipolysis as the mechanism for increased ketone production. In conclusion, lowering dialysate bicarbonate does not meaningfully reduce organic acid generation during hemodialysis or modify organic anion losses into dialysate.


Assuntos
Ácido 3-Hidroxibutírico/sangue , Equilíbrio Ácido-Base , Alcalose/sangue , Bicarbonatos/administração & dosagem , Soluções para Hemodiálise/administração & dosagem , Falência Renal Crônica/terapia , Ácido Láctico/sangue , Diálise Renal , Adulto , Idoso , Idoso de 80 Anos ou mais , Alcalose/diagnóstico , Alcalose/etiologia , Alcalose/fisiopatologia , Bicarbonatos/efeitos adversos , Bicarbonatos/metabolismo , Biomarcadores/sangue , Ácidos Graxos não Esterificados/sangue , Feminino , Soluções para Hemodiálise/efeitos adversos , Soluções para Hemodiálise/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Falência Renal Crônica/sangue , Falência Renal Crônica/diagnóstico , Falência Renal Crônica/fisiopatologia , Lipólise , Masculino , Pessoa de Meia-Idade , Diálise Renal/efeitos adversos , Fatores de Tempo , Resultado do Tratamento
15.
Int J Mol Sci ; 21(7)2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32244610

RESUMO

Carbonic anhydrase is a ubiquitous metalloenzyme that catalyzes the reversible interconversion of CO2/HCO3-. Equilibrium of these species is maintained by the action of carbonic anhydrase. Recent advances in magnetic resonance spectroscopy have allowed, for the first time, in vivo characterization of carbonic anhydrase in the human brain. In this article, we review the theories and techniques of in vivo 13C magnetization (saturation) transfer magnetic resonance spectroscopy as they are applied to measuring the rate of exchange between CO2 and HCO3- catalyzed by carbonic anhydrase. Inhibitors of carbonic anhydrase have a wide range of therapeutic applications. Role of carbonic anhydrases and their inhibitors in many diseases are also reviewed to illustrate future applications of in vivo carbonic anhydrase assessment by magnetic resonance spectroscopy.


Assuntos
Bicarbonatos/metabolismo , Encéfalo/metabolismo , Dióxido de Carbono/metabolismo , Anidrases Carbônicas/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Algoritmos , Biocatálise , Isótopos de Carbono/metabolismo , Humanos , Isoenzimas/metabolismo
16.
Am J Physiol Lung Cell Mol Physiol ; 318(5): L873-L887, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32160007

RESUMO

Tenacious mucus produced by tracheal and bronchial submucosal glands is a defining feature of several airway diseases, including cystic fibrosis (CF). Airway acidification as a driving force of CF airway pathology has been controversial. Here we tested the hypothesis that transient airway acidification produces pathologic mucus and impairs mucociliary transport. We studied pigs challenged with intra-airway acid. Acid had a minimal effect on mucus properties under basal conditions. However, cholinergic stimulation in acid-challenged pigs revealed retention of mucin 5B (MUC5B) in the submucosal glands, decreased concentrations of MUC5B in the lung lavage fluid, and airway obstruction. To more closely mimic a CF-like environment, we also examined mucus secretion and transport following cholinergic stimulation under diminished bicarbonate and chloride transport conditions ex vivo. Under these conditions, airways from acid-challenged pigs displayed extensive mucus films and decreased mucociliary transport. Pretreatment with diminazene aceturate, a small molecule with ability to inhibit acid detection through blockade of the acid-sensing ion channel (ASIC) at the doses provided, did not prevent acid-induced pathologic mucus or transport defects but did mitigate airway obstruction. These findings suggest that transient airway acidification early in life has significant impacts on mucus secretion and transport properties. Furthermore, they highlight diminazene aceturate as an agent that might be beneficial in alleviating airway obstruction.


Assuntos
Ácido Acético/administração & dosagem , Bloqueadores do Canal Iônico Sensível a Ácido/farmacologia , Canais Iônicos Sensíveis a Ácido/genética , Obstrução das Vias Respiratórias/induzido quimicamente , Fibrose Cística/induzido quimicamente , Diminazena/análogos & derivados , Canais Iônicos Sensíveis a Ácido/metabolismo , Obstrução das Vias Respiratórias/tratamento farmacológico , Obstrução das Vias Respiratórias/metabolismo , Obstrução das Vias Respiratórias/patologia , Animais , Animais Recém-Nascidos , Bicarbonatos/metabolismo , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Brônquios/patologia , Líquido da Lavagem Broncoalveolar/química , Cloretos/metabolismo , Fibrose Cística/tratamento farmacológico , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Diminazena/farmacologia , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Masculino , Mucina-5AC/genética , Mucina-5AC/metabolismo , Mucina-5B/genética , Mucina-5B/metabolismo , Depuração Mucociliar/efeitos dos fármacos , Muco/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Suínos , Traqueia/efeitos dos fármacos , Traqueia/metabolismo , Traqueia/patologia
17.
Sensors (Basel) ; 20(6)2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32168979

RESUMO

Bicarbonate plays a central role in human physiology from cellular respiration to pH homeostasis. However, so far, the measurement of bicarbonate concentration changes in living cells has only been possible by measuring intracellular pH changes. In this article, we report the development of a genetically encoded pH-independent fluorescence-based single-use sensory cellular test system for monitoring intracellular bicarbonate concentration changes in living cells. We describe the usefulness of the developed biosensor in characterizing the bicarbonate transport activities of anionophores-small molecules capable of facilitating the membrane permeation of this anion. We also demonstrate the ability of the bicarbonate sensory cellular test system to measure intracellular bicarbonate concentration changes in response to activation and specific inhibition of wild-type human CFTR protein when co-expressed with the bicarbonate sensing and reporting units in living cells. A valuable benefit of the bicarbonate sensory cellular test system could be the screening of novel anionophore library compounds for bicarbonate transport activity with efficiencies close to the natural anion channel CFTR, which is not functional in the respiratory epithelia of cystic fibrosis patients.


Assuntos
Bicarbonatos/análise , Técnicas Biossensoriais , Adenilil Ciclases/metabolismo , Animais , Bicarbonatos/metabolismo , Transporte Biológico/fisiologia , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Células CHO , Cricetinae , Cricetulus , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Espaço Intracelular/química , Espaço Intracelular/metabolismo
18.
Sci Rep ; 10(1): 5507, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32218474

RESUMO

Cerebral metabolism, which can be monitored by magnetic resonance spectroscopy (MRS), changes rapidly after brain ischaemic injury. Hyperpolarisation techniques boost 13C MRS sensitivity by several orders of magnitude, thereby enabling in vivo monitoring of biochemical transformations of hyperpolarised (HP) 13C-labelled precursors with a time resolution of seconds. The exogenous administration of the metabolite L-lactate was shown to decrease lesion size and ameliorate neurological outcome in preclinical studies in rodent stroke models, as well as influencing brain metabolism in clinical pilot studies of acute brain injury patients. The aim of this study was to demonstrate the feasibility of measuring HP [1-13C] L-lactate metabolism in real-time in the mouse brain after ischaemic stroke when administered after reperfusion at a therapeutic dose. We showed a rapid, time-after-reperfusion-dependent conversion of [1-13C] L-lactate to [1-13C] pyruvate and [13C] bicarbonate that brings new insights into the neuroprotection mechanism of L-lactate. Moreover, this study paves the way for the use of HP [1-13C] L-lactate as a sensitive molecular-imaging biosensor in ischaemic stroke patients after endovascular clot removal.


Assuntos
Isquemia Encefálica/metabolismo , Ácido Láctico/metabolismo , Fármacos Neuroprotetores/metabolismo , Acidente Vascular Cerebral/metabolismo , Animais , Bicarbonatos/metabolismo , Técnicas Biossensoriais/métodos , Isquemia Encefálica/diagnóstico por imagem , Isquemia Encefálica/terapia , Isótopos de Carbono , Sistemas Computacionais , Modelos Animais de Doenças , Estudos de Viabilidade , Humanos , Imuno-Histoquímica , Infarto da Artéria Cerebral Média/diagnóstico por imagem , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/terapia , Ácido Láctico/administração & dosagem , Espectroscopia de Ressonância Magnética/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Imagem Molecular/métodos , Transportadores de Ácidos Monocarboxílicos/metabolismo , Fármacos Neuroprotetores/administração & dosagem , Ácido Pirúvico/metabolismo , Acidente Vascular Cerebral/diagnóstico por imagem , Acidente Vascular Cerebral/terapia
19.
Ann Biomed Eng ; 48(5): 1562-1572, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32072384

RESUMO

Extracorporeal CO2 removal is a highly promising support therapy for patients with hypercapnic respiratory failure but whose clinical implementation and patient benefit is hampered by high cost and highly specialized expertise required for safe use. Current approaches target removal of the gaseous CO2 dissolved in blood which limits their ease of clinical use as high blood flow rates are required to achieve physiologically significant CO2 clearance. Here, a novel hybrid approach in which a zero-bicarbonate dialysis is used to target removal of bicarbonate ion coupled to a gas exchange device to clear dissolved CO2, achieves highly efficiently total CO2 capture while maintaining systemic acid-base balance. In a porcine model of acute hypercapnic respiratory failure, a CO2-reduction of 61.4 ± 14.4 mL/min was achieved at a blood flow rate of 248 mL/min using pediatric-scale priming volumes. The dialyzer accounted for 81% of total CO2 capture with an efficiency of 33% with a minimal pH change across the entire circuit. This study demonstrates the feasibility of a novel hybrid CO2 capture approach capable of achieving physiologically significant CO2 removal at ultralow blood flow rates with low priming volumes while leveraging widely available dialysis platforms to enable clinical adoption.


Assuntos
Circulação Extracorpórea , Hipercapnia/terapia , Animais , Bicarbonatos/metabolismo , Circulação Sanguínea , Dióxido de Carbono/metabolismo , Hipercapnia/metabolismo , Masculino , Suínos
20.
J Sep Sci ; 43(11): 2125-2132, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32073721

RESUMO

Methanol-chloroform based protein precipitation is an essential step in many liquid chromatography-tandem mass spectrometry-based cellular proteomics applications. However, re-solubilization of the total protein precipitate is difficult using regular in-solution digestion protocol. Sodium deoxycholate is reported as an efficient surfactant for re-solubilization of membrane fractions. In this study, we demonstrated an application combining methanol-chloroform based protein precipitations and deoxycholic acid assisted re-solubilization of pellets to evaluate the improvement of protein identifications in mass spectrometry-based bottom-up proteomics. We evaluated the modified method using an equal amount of Raw 264.7 mouse macrophage cell lysate. Detailed in-solution trypsin digestion studies were presented on methanol-chloroform precipitated samples with or without deoxycholic acid treatments and compared with popular sample digestion methods. A mass spectrometric analysis confirmed an 82% increase in protein identification in deoxycholic acid-treated samples compared to other established methods. Furthermore, liquid chromatography-tandem mass spectrometry analysis of an equal amount of proteins from methanol-chloroform precipitated, and methanol-chloroform/deoxycholic acid-treated macrophage cell lysate showed a 14% increase and 27% unique protein identifications. We believe this improved digestion method could be a complementary or alternative method for mammalian cell sample preparations where sodium dodecyl sulfate based lysis buffer is frequently used.


Assuntos
Clorofórmio/metabolismo , Metanol/metabolismo , Proteômica , Tripsina/análise , Tripsina/metabolismo , Animais , Bicarbonatos/química , Bicarbonatos/metabolismo , Clorofórmio/química , Cromatografia Líquida , Metanol/química , Camundongos , Células RAW 264.7 , Soluções , Espectrometria de Massas em Tandem
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