Newly synthesized cAMP is integrated at a membrane protein complex signalosome to ensure receptor response specificity.

Spatiotemporal regulation of cAMP within the cell is required to achieve receptor-specific responses. The mechanism through which the cell selects a specific response to newly synthesized cAMP is not fully understood. In hepatocyte plasma membranes, we identified two functional and independent cAMP-responsive signaling protein macrocomplexes that produce, use, degrade, and regulate their own nondiffusible (sequestered) cAMP pool to achieve their specific responses. Each complex responds to the stimulation of an adenosine G protein-coupled receptor (Ado-GPCR), bound to either A2A or A2B , but not simultaneously to both. Each isoprotein involved in each signaling cascade was identified by measuring changes in cAMP levels after receptor activation, and its participation was confirmed by antibody-mediated inactivation. A2A -Ado-GPCR selective stimulation activates adenylyl cyclase 6 (AC6), which is bound to AKAP79/150, to synthesize cAMP which is used by two other AKAP79/150-tethered proteins: protein kinase A (PKA) and phosphodiesterase 3A (PDE3A). In contrast, A2B -Ado-GPCR stimulation activates D-AKAP2-attached AC5 to generate cAMP, which is channeled to two other D-AKAP2-tethered proteins: guanine-nucleotide exchange factor 2 (Epac2) and PDE3B. In both cases, prior activation of PKA or Epac2 with selective cAMP analogs prevents de novo cAMP synthesis. In addition, we show that cAMP does not diffuse between these protein macrocomplexes or 'signalosomes'. Evidence of coimmunoprecipitation and colocalization of some proteins belonging to each signalosome is presented. Each signalosome constitutes a minimal functional signaling unit with its own machinery to synthesize and regulate a sequestered cAMP pool. Thus, each signalosome is devoted to ensure the transmission of a unique and unequivocal message through the cell.

Oxidative status and fertility of ewes supplemented coffee pulp during estrous ¤ synchronization and early pregnancy / Estado oxidativo y fertilidad en ovejas suplementadas con pulpa de café durante la sincronización estral y gestación temprana / Estado oxidativo e fertilidade nas ovelhas suplementadas com a polpa de café durante sincronização do estro e gestação inicial

Summary Background: it is known that coffee pulp can modify the oxidative status and fertility in dairy cows. Objective: to evaluate the effect of dietary supplementation with coffee pulp on the antioxidant capacity, lipid oxidation and reproductive characteristics of ewes during estrous synchronization and early gestation. Methods: forty Dorset-Suffolk crossbred ewes with 3 or 4 parturitions were allocated to two treatments: T0 (n = 21), ewes supplemented with 450 g of a control feed; and T1 (n = 19), ewes supplemented with 450 g of the feed with 25% coffee pulp. Supplementation began 14 days before estrous synchronization and ended 25 days after breeding. During estrous synchronization, progestogen (CIDR, Controlled Internal Drug Release) was inserted and left in situ for 11 days. Eighteen hours later, estrous detection began with the aid of rams. Blood samples were collected at different times of synchronization and during early pregnancy to determine antioxidant capacity, lipid oxidation and blood progesterone concentration. Pregnancy diagnosis was performed 30 and 60 days after CIDR removal. Results: supplementation with coffee pulp did not affect estrous onset, estrous response or progesterone concentration, but fertility decreased from 100 to 78.95%. The antioxidant capacity measured using the FRAP technique was greater in coffee pulp supplemented ewes only before progestogen insertion. Coffee pulp did not modify lipid oxidation; however, this variable was affected by sampling time, decreasing after progestogen removal to its lowest values at 22 days into pregnancy. Conclusion: although supplementation with coffee pulp at 25% in the concentrate increased antioxidant capacity of ewes before insertion of progestogen, it is not recommended to use this percentage during synchronization or early pregnancy since it can negatively affect gestation rate.

Resumen Antecedentes: se sabe que la pulpa de café puede modificar el estado oxidativo y la fertilidad en vacas lecheras. Objetivo: evaluar los efectos del suministro dietario de pulpa de café y su capacidad antioxidante, oxidación lipídica, y características reproductivas en ovejas durante la sincronización del estro y la gestación temprana. Métodos: cuarenta ovejas cruzadas Suffolk x Dorset de 3 y 4 partos fueron asignadas a dos grupos: T0 (n = 21), suplementación con 450 g de alimento (grupo testigo); y T1 (n = 19), suplementación con 450 g de alimento con 25% de pulpa de café. La suplementación inició 14 días antes de la sincronización del estro y terminó 25 días después del apareamiento. El progestágeno (CIDR, Dispositivo Intravaginal de Liberación Controlada) fue insertado en los animales por 11 días. Dieciocho horas después de su retiro se inició la detección de estros. Se muestreó a diferentes tiempos después de la sincronización y durante la gestación temprana para determinar capacidad antioxidante, oxidación lipídica y concentración de progesterona. El diagnóstico de preñez se realizó 30 y 60 días después de la remoción del CIDR. Resultados: la suplementación con pulpa de café no afectó el inicio del estro, la respuesta al estro ni la concentración de progesterona. Sin embargo, la fertilidad disminuyó de 100 a 78,95%. La capacidad antioxidante, medida mediante la técnica FRAP, fue mayor en ovejas suplementadas con pulpa de café, pero solo antes de la inserción del progestágeno. La pulpa de café no modificó la oxidación lipídica; sin embargo, si fue modificada por el tiempo de muestreo, decreciendo desde la remoción del progestágeno hasta los 22 días de preñez. Conclusión: aunque la suplementación del concentrado con 25% de pulpa de café incrementó la capacidad antioxidante antes de la inserción del progestágeno, no se recomienda ese porcentaje durante la sincronización y gestación temprana, ya que redujo el porcentaje de gestación de las ovejas.

Resumo Antecedentes: a polpa de café pode modificar o estado oxidativo e a fertilidade em vacas leiteiras. Objetivo: avaliar a polpa de café na capacidade antioxidante, oxidação da gordura e nas características reprodutivas das ovelhas durante sincronização do estro e gestação inicial. Métodos: quarenta ovelhas cruzas Suffolk e Dorset de 3 e 4 nascimentos foram agrupadas no T0 (n = 21), suplementação com 450 g de alimento controle e T1 (n = 19), suplementação com 450 g de alimento com 25% de polpa de café. A suplementação iniciou 14 dias antes da sincronização do estro e terminou 25 dias depois do acasalamento. O hormônio progestina (CIDR, dispositivo intravaginal de libertação controlada de fármaco) foi inserido por 11 dias. Dezoito horas depois da retirada iniciouse a detecção do estro. Fizeram-se amostras de diferentes tempos do período e da gestação inicial para determinar a capacidade antioxidante, oxidação dos lipídeos e concentração de progesterona. Realizou-se o diagnóstico de gestação 30 e 60 dias depois de remover o CIDR. Resultados: a suplementação com a polpa de café não afetou o início do estro, a resposta ao estro e a concentração de progesterona, mas a fertilidade decresceu de 100 a 78,95%. A capacidade antioxidante que foi medida pela técnica de FRAP foi maior nas ovelhas suplementadas com a polpa de café somente antes da inserção do progestágeno. A polpa de café não modificou a oxidação dos lipídeos; no entanto, estes foram modificados pelo tempo de amostra, decrescendo depois de remover o progestágeno até 22 dias de gestação. Conclusão: ainda que a polpa de café a 25% de concentração incrementa a capacidade antioxidante antes da inserção do progestágeno, não é recomendado este percentual para as ovelhas durante a sincronização do estro e a gestação inicial, já que decresce a porcentagem de gestação.

NOX2 activated by α1-adrenoceptors modulates hepatic metabolic routes stimulated by ß-adrenoceptors.

The NADPH oxidase (NOX) family of enzymes oxidase catalyzes the transport of electrons from NADPH to molecular oxygen and generates O(2)(•-), which is rapidly converted into H(2)O(2). We aimed to identify in hepatocytes the protein NOX complex responsible for H(2)O(2) synthesis after α(1)-adrenoceptor (α(1)-AR) stimulation, its activation mechanism, and to explore H(2)O(2) as a potential modulator of hepatic metabolic routes, gluconeogenesis, and ureagenesis, stimulated by the ARs. The dormant NOX2 complex present in hepatocyte plasma membrane (HPM) contains gp91(phox), p22(phox), p40(phox), p47(phox), p67(phox) and Rac 1 proteins. In HPM incubated with NADPH and guanosine triphosphate (GTP), α(1)-AR-mediated H(2)O(2) synthesis required all of these proteins except for p40(phox). A functional link between α(1)-AR and NOX was identified as the Gα(13) protein. Alpha(1)-AR stimulation in hepatocytes promotes Rac1-GTP generation, a necessary step for H(2)O(2) synthesis. Negative cross talk between α(1)-/ß-ARs for H(2)O(2) synthesis was observed in HPM. In addition, negative cross talk of α(1)-AR via H(2)O(2) to ß-AR-mediated stimulation was recorded in hepatocyte gluconeogenesis and ureagenesis, probably involving aquaporine activity. Based on previous work we suggest that H(2)O(2), generated after NOX2 activation by α(1)-AR lightening in hepatocytes, reacts with cAMP-dependent protein kinase A (PKA) subunits to form an oxidized PKA, insensitive to cAMP activation that prevented any rise in the rate of gluconeogenesis and ureagenesis.

Adrenaline stimulates H2O2 generation in liver via NADPH oxidase.

It is known that adrenaline promotes hydroxyl radical generation in isolated rat hepatocytes. The aim of this work was to investigate a potential role of NADPH oxidase (Nox) isoforms for an oxidative stress signal in response to adrenaline in hepatocytes. Enriched plasma membranes from isolated rat liver cells were prepared for this purpose. These membranes showed catalytic activity of Nox isoforms, probably Nox 2 based on its complete inhibition with specific antibodies. NADPH was oxidized to convert O(2) into superoxide radical, later transformed into H(2)O(2). This enzymatic activity requires previous activation with either 3 mM Mn(2+) or guanosine 5'-0-(3-thiotriphosphate) (GTPgammaS) plus adrenaline. Experimental conditions for activation and catalytic steps were set up: ATP was not required; S(0.5) for NADPH was 44 microM; S(0.5) for FAD was 8 microM; NADH up to 1 mM was not substrate, and diphenyleneiodonium was inhibitory. Activation with GTPgammaS plus adrenaline was dose- and Ca(2+)-dependent and proceeded through alpha(1)-adrenergic receptors (AR), whereas beta-AR stimulation resulted in inhibition of Nox activity. These results lead us to propose H(2)O(2) as additional transduction signal for adrenaline response in hepatic cells.

In rat hepatocytes, different adenosine receptor subtypes use different secondary messengers to increase the rate of ureagenesis.

In rat hepatocytes, the role of cAMP and Ca(2+) as secondary messengers in the ureagenic response to stimulation of specific adenosine receptor subtypes was explored. Analyzed receptor subtypes were: A(1), A(2A), A(2B) and A(3). Each receptor subtype was stimulated with a specific agonist while blocking all other receptor subtypes with a battery of specific antagonists. For the A(1) and A(3) adenosine receptor subtypes, the secondary messenger was the cytoplasmic Ca(2+) concentration ([Ca(2+)](cyt)). Accordingly, the A(1) or A(3)-mediated increase in [Ca(2+)](cyt) and in ureagenic activity were both inhibited by chelating Ca(2+) with either EGTA or BAPTA-AM. Also, Gd(3+) blocked both the increase in [Ca(2+)](cyt) and ureagenesis, suggesting that a Ca(2+) channel may be involved in the response to both A(1) and A(3). A partial effect was observed with the sarcoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin. The concentration of cyclic AMP ([cAMP]) increased in response to stimulation of either the A(2A) or the A(2B) adenosine receptor subtypes, while it decreased slightly in response to stimulation of either A(1) or A(3). The stimulation of either the A(2A) or A(2B) adenosine receptor subtypes resulted in an increase in [cAMP] and an ureagenic response which were not sensitive to EGTA, BAPTA-AM, Gd(3+) or to thapsigargin. In addition, the adenylyl cyclase inhibitor MDL12,330A blocked the ureagenic response to A(2A) and A(2B), but not the response to either A(1) or A(3). Our results indicate that in the ureagenic liver response to adenosine, the secondary messenger for both, the A(1) and A(3) adenosine receptor subtypes is [Ca(2+)](cyt), while the message from the A(2A) and A(2B) adenosine receptor subtypes is relayed by [cAMP].

Inosine released after hypoxia activates hepatic glucose liberation through A3 adenosine receptors.

Inosine, an endogenous nucleoside, has recently been shown to exert potent effects on the immune, neural, and cardiovascular systems. This work addresses modulation of intermediary metabolism by inosine through adenosine receptors (ARs) in isolated rat hepatocytes. We conducted an in silico search in the GenBank and complete genomic sequence databases for additional adenosine/inosine receptors and for a feasible physiological role of inosine in homeostasis. Inosine stimulated glycogenolysis (approximately 40%, EC50 4.2 x 10(-9) M), gluconeogenesis (approximately 40%, EC50 7.8 x 10(-9) M), and ureagenesis (approximately 130%, EC50 7.0 x 10(-8) M) compared with basal values; these effects were blunted by the selective A3 AR antagonist 9-chloro-2-(2-furanyl)-5-[(phenylacetyl)amino][1,2,4]-triazolo[1,5-c]quinazoline (MRS 1220) but not by selective A1, A2A, and A2B AR antagonists. In addition, MRS 1220 antagonized inosine-induced transient increase (40%) in cytosolic Ca2+ and enhanced (90%) glycogen phosphorylase activity. Inosine-induced Ca2+ mobilization was desensitized by adenosine; in a reciprocal manner, inosine desensitized adenosine action. Inosine decreased the cAMP pool in hepatocytes when A1, A2A, and A2B AR were blocked by a mixture of selective antagonists. Inosine-promoted metabolic changes were unrelated to cAMP decrease but were Ca2+ dependent because they were absent in hepatocytes incubated in EGTA- or BAPTA-AM-supplemented Ca2+-free medium. After in silico analysis, no additional cognate adenosine/inosine receptors were found in human, mouse, and rat. In both perfused rat liver and isolated hepatocytes, hypoxia/reoxygenation produced an increase in inosine, adenosine, and glucose release; these actions were quantitatively greater in perfused rat liver than in isolated cells. Moreover, all of these effects were impaired by the antagonist MRS 1220. On the basis of results obtained, known higher extracellular inosine levels under ischemic conditions, and inosine's higher sensitivity for stimulating hepatic gluconeogenesis, it is suggested that, after tissular ischemia, inosine contributes to the maintenance of homeostasis by releasing glucose from the liver through stimulation of A3 ARs.

Estrés oxidativo y el uso de antioxidantes en animales domésticos / Oxidative stress and the use of antioxidants in domestic animals

Los radicales libres o especies reactivas al oxigeno (ROS) se producen continuamente en el organismo como consecuencia de los procesos metabólicos normales y fuentes exógenas como el ejercicio intenso, situaciones de estrés, factores ambientales y agentes contaminantes (drogas y pesticidas). Un radical libre es una molécula o átomo que presenta al menos un electrón no apareado. Cuando la producción de ROS es excesiva, el resultado es el estrés oxidativo, término que se relaciona con el daño a las biomoléculas: proteínas, lípidos, carbohidratos y ADN. Asi mismo, se le ha relacionado con enfermedades que afectan a humanos y animales. La presente revisión tiene como objetivo revisar aspectos básicos del estrés oxidativo, así como la utilización de antioxidantes en las diferentes especies de animales domésticos

Prophylactic action of lipoic acid on oxidative stress and growth performance in broilers at risk of developing ascites syndrome.

The objective of this study was to assess the effects of dietary supplementation with lipoic acid (LA) on broilers maintained at 2235 m above sea level with high risk to develop ascites syndrome (AS). A total of 2040 chicks were fed under commercial conditions with water and specific diets ad libitum during 7 weeks in two consecutive experiments. Mortality and indicators of performance and oxidative stress were compared weekly in broilers fed a basal diet plus 0, 10, 20, or 40 parts/10(6) LA. The effects of LA at 40 parts/10(6) were also studied during the initial 3 weeks or the last 4 weeks of the production cycle. Diets supplemented with 40 parts/10(6) of LA during 7 weeks significantly improved feed conversion, decreased general mortality and mortality attributable to AS, and lowered thiobarbituric acid reactive substances and hydroxyl radicals in liver, and increased total glutathione pool. Smaller doses or shorter periods of exposure to LA were partially effective. In conclusion, LA under our experimental conditions has a prophylactic action in broilers with high risk to develop AS due to oxygen availability limitation.

Effects of dietary supplementation with vitamin C or vitamin E on cardiac lipid peroxidation and growth performance in broilers at risk of developing ascites syndrome.

OBJECTIVE: To assess effects of high dietary amounts of vitamin C or vitamin E and oxidative stress on the heart and growth performance of broilers maintained at an altitude of 2,200 m above sea level. ANIMALS: 360 chicks (1-day-old broilers). PROCEDURE: Birds were randomly assigned to 3 groups (120 chicks/group). Each group of birds was fed a specific diet (control group, basal diet containing 12 mg of vitamin E (DL-alpha-tocopherol acetate)/kg of feed without additional ascorbic acid; vitamin E group, basal diet supplemented with 75 mg of vitamin E/kg of feed; and vitamin C group, basal diet supplemented with 400 mg of ascorbic acid/kg of feed) throughout the entire 7 weeks of the study. Feed consumption and body weight of chicks were recorded on a weekly basis. Nine randomly selected birds from each group were euthanatized each week. Remaining birds were euthanatized at the end of the study. Samples of cardiac tissues were obtained to measure thiobarbituric acid reactive substances (TBARS), an indicator of oxidative stress. RESULTS: Vitamin E-supplemented diets resulted in better growth performance, lower rates of feed conversion, and lower TBARS content. Vitamin C-supplemented diets resulted in lower feed consumption and lower rates of feed conversion. When used separately, neither of the vitamins had any effect on mortality attributable to ascites syndrome. CONCLUSION AND CLINICAL RELEVANCE: It is recommended that diets supplemented with vitamin C, vitamin E, or both be fed to broilers maintained at an altitude of 2,200 m above sea level to improve growth performance.

Regulation of glycogen metabolism in hepatocytes through adenosine receptors. Role of Ca2+ and cAMP.

The objective of this work is to identify the adenosine receptor subtype and the triggered events involved in the regulation of hepatic glycogen metabolism. Glycogenolysis, gluconeogenesis, cAMP, and cytosolic Ca2+ ([Ca2+](cyt)) were measured in isolated hepatocytes challenged with adenosine A1, A2A, and A3 receptor-selective agonists. Stimulation of adenosine receptor subtypes with selective agonists in Ca2+ media produced a dose-dependent increase in [Ca2+]cyt with A1>A2=A3, cAMP with A2A, glycogenolysis with A1>A2A>A3, and gluconeogenesis with A2A>A1>A3, in addition, a decrease in cAMP was observed with A1=A3. Comparatively, in Ca2+-free media or with a cell membrane-permeant Ca2+ chelator, activation of these adenosine receptors with the same selective agonists produced a smaller and transient rise in [Ca2+]cyt with A1=A3>A2, no rise in glycogenolysis and gluconeogenesis with A3>A1, but a full rise with A2A. Thus, in isolated rat hepatocytes activation of the adenosine A1 receptor triggered Ca2+-mediated glycogenolysis, activation of the adenosine A2A receptor stimulated cAMP-mediated gluconeogenesis, and activation of the adenosine A3 receptor increased [Ca2+]cyt and decreased cAMP with minor changes in glycogen metabolism.

Estado oxidativo hepático y comportamiento productivo en pollos de engorda, alimentados con dos fuentes de selenio y niveles altos de vitaminas E y C / Hepatic oxidative status and performance in broiler chiks fed with two sources of selenium and high levels of viatamins E and C

Se evaluó la adición de dosis elevadas de vitaminas E y C, y dos fuentes de selenio (selenometionina y selenito de sodio) en la dieta, sobre el estado oxidativo hepático y los principales indicadores productivos comerciales en pollos de engorda, explorando una posible relación entre ambos fenómenos. Se utilizaron 720 pollos mixtos de un día de edad, alojados en una caseta de ambiente natural, con equipo estándar. Se utilizó un diseño completamente aleatorizado, con 3 tratamientos: 1. Dieta convencional, 2. dieta convencional con 75 Ul de vitamina E/kg de alimento y 400 ppm de vitamina C suplementarias, 3. dieta semejante a la anterior, pero con selenometionina como fuente suplementaria de selenio a dosis convencional. No se encontró diferencia significativa (P>0.05) en ganancia de peso, conversión alimentaria, consumo de alimento, mortalidad general o mortalidad por síndrome ascítico, entre los diferentes tratamientos. Los niveles de glutatión total hepático (GT) no se modificaron significativamente (P>0.05) entre tratamientos, pero sí en función del tiempo. La lipoperoxidación hepática (LP) disminuyó significativamente en las aves de los dos últimos tratamientos, respecto de las del primer tratamiento; paralelamente se encontró una relación inversa entre los niveles de GT y de LP. Finalmente no se pudo establecer una relación entre el estado oxidativo hepático y el comportamiento productivo de las aves.

Cambios químicos en el fermentado con sorgo / Chemical changes in bovine colostrum fermented with sorghum

Se fermentó calostro bovino por 8 ó 21 días a temperatura ambiente (18-20§C) agregando sorgo molido (7.5 por ciento o sin agregar sorgo (testigo)). Tanto en el calostro testigo como con sorgo, antes y después de fermentarlos, se determino pH, humedad, proteína cruda, proteína digestible, amoníaco, ácido láctico y energía bruta. No se observaron diferencias (P>0.01) en el porcentaje de proteína cruda en el calostro testigo (7.12, 5.76, 5.70) y con sorgo (6.66, 5.71, 5.98) a los 0,8 y 21 días de fermentación respectivamente. El calostro con sorgo presentó una menor proporción (P<0.01) de proteína digestible (89.0, 81.0, 86.0 por ciento) que el testigo (90.0, 93.0, 93.0 por ciento), sin embargo, la producción de amoníaco fue menor (P<0.01) en el calostro con sorgo (0.23, 0.097, 1.20 por ciento) que en el testigo (0.25, 1.31, 1.37 por ciento) El contenido de ácido láctigo aumento (P<0.01) en el calostro con sorgo después de 21 días de fermentación (1.24 g/100 ml), en relación al testigo (0.82 g/100 ml). Los valores de energía bruta fueron mayores (P<0.01) a los 8 y 21 días de fermentación en el calostro con sorgo (1.16, 0.97 Kcal/g) en relación con el testigo (0.91, 0.84 Kcal/g). Al agregar sorgo al calostro se redujo la degradación de proteína cruda y disminuyó la producción de amoníaco, también aumentó el contenido de energía bruta al fermentarlo por 8 y 21 días y el ácido láctico después de 21 días de fermentación

Influencia de la fuente de energía sobre el efecto ureogénico de nucleósidos y hormonas en hepatocitos aislados de rata / Energy source over nucleoside and hormone ureogenic effect in isolated rat hepatocytes

La adenosina y la inosina son nucleósidos formados durante la degradación del ATP, los cuales juegan un papel importante en la regulación del metabolismo hepático. En células aisladas de hígado de rata e incubadas bajo diferentes condiciones experimentales, ambos nucleósidos presentan un efecto ureogénico, el cual presenta las siguientes características; a) Se manifiesta cuando los hepatocitos son incubados con glutamina, alanina o carbonato de amonio; b) este proceso metabólico regulado por adenosina e inosina es dependiente del sustrato oxidable; c) un efecto inhibitorio de la ureogénesis es observado cuando las células son incubadas en ausencia de calcio; d) este efecto es antagonizado por glucagon, pero no por epinefrina. Los efectos metabolicos de la denosina e inosina aquí descritos, forman parte de un sistema muy fino de control metabólico, cuyo mecanismo de regulación genera una respuesta a corto plazo, lo que determinará la funciónalidad del órgano. Por lo tanto, se considera que los datos presentados en este trabajo son de gran importancia fisiológica