Yellow grease in sheep diets: intake and digestibility / [Óleo residual de fritura em dietas para ovinos: consumo e digestibilidade]

This study aimed to assess the effects of yellow grease supplementation on the intake, digestibility, and nitrogen balance in sheep. Twenty Santa Inês lambs with a mean age of 95 ± 10 d and body weight of 19.29 ± 3.17kg were evaluated in a completely randomized design. The diets were supplemented with oil at concentrations of 0, 20, 40, 60, and 80 gkg-1 of dry matter (DM) of the concentrate. The diets were based on roughage and concentrate (50:50). The experimental period lasted 19 d and included 14 adaptation days and five collection days for the total supplied diet, orts, feces, and urine. Supplementation with yellow grease had no significant effect on the intake of DM, crude protein (CP), neutral detergent fiber (NDF), or non-fiber carbohydrates (NFC). However, the ether extract (EE) intake increased linearly with supplementation of yellow grease. Moreover, no effect was observed for DM, CP, NDF, and NFC digestibility and nitrogen balance. EE digestibility increased linearly with the yellow grease dietary supplementation. Thus, sheep dietary supplementation with yellow grease may be used at a level of up to 80 gkg-1 of DM of concentrate without impairing nutrient intake and digestibility.(AU)

Objetivou-se, com o estudo, avaliar os efeitos do óleo residual de fritura, em dietas para ovinos, sob o consumo, a digestibilidade e o balanço de nitrogênio. Foram utilizados 20 cordeiros Santa Inês, com idade de 95 ± 10 dias e peso corporal de 19,29 ± 3,17kg, em delineamento inteiramente ao acaso. As dietas continham óleo de fritura nas concentrações de 0; 20; 40; 60 e 80gkg-1 da matéria seca (MS) do concentrado. As dietas tinham relação volumoso:concentrado de 50:50. O período experimental foi de 19 dias, incluindo 14 dias em adaptação e cinco dias de coleta do fornecido, das sobras, das fezes e da urina. A suplementação com óleo de fritura não alterou o consumo de MS, proteína bruta (PB), matéria orgânica (MO), fibra em detergente neutro (FDN) e carboidratos não fibrosos (CNF). Entretanto, o consumo de extrato etéreo (EE) aumentou com a inclusão do óleo. Não foi observado efeito na digestibilidade da MS, da PB, da FDN, dos CNF e no balanço de nitrogênio. A digestibilidade do EE aumentou com a inclusão do óleo. Assim, a inclusão de óleo de fritura em dietas para ovinos pode ser utilizada em até 80gkg-1 da MS do concentrado, sem limitar ingestão e digestibilidade dos nutrientes.(AU)

Apparent selectivity of sheep in deferred marandu palisadegrass pastures with variable initial heights / Seletividade aparente de ovinos em pastos de capim-marandu diferidos com alturas iniciais variáveis

The objective of this study was to evaluate the apparent selectivity of sheep in marandu palisadegrass (Urochloa brizantha cv. Marandu) pastures with four heights at the beginning of the deferment period (15, 25, 35 and 45cm). The deferment period was 92 days and started on 03/21/2014. Evaluations occurred in the beginning (first week), middle (45th day) and end (92nd day) of the grazing period, in winter (06/21/2014 to 09/21/2014). Deferred pastures with 15 and 25cm presented lower forage mass (FM), but higher live leaf (LL) percentage in FM than deferred pastures with 35 and 45cm. The live stem percentage in the FM and the apparent selectivity index (ASI) of the LL were superior in the deferred pasture with 45cm. The dead stem (DS) percentage in the grazing simulation (GS) and the ASI of this morphological component were lower in the pasture with 15cm, compared to the deferred pasture with 45cm. The FM and the LL percentages in FM and in the GS sample decreased, while the DS percentages in FM and in GS sample increased with the grazing period. Marandu palisadegrass with 15cm at beginning of the deferment period improves the morphology of the deferred pasture. Selective grazing is difficult during the grazing period.(AU)

Objetivou-se avaliar a seletividade aparente de ovinos em pastos de capim-marandu (Urochloa brizantha cv. Marandu) com quatro alturas no início do diferimento (15, 25, 35 e 45cm). O período de diferimento foi de 92 dias e iniciou em 21/03/2014. As avaliações ocorreram no início (primeira semana), meio (45° dia) e fim (92° dia) do período de pastejo, no inverno (21/06/2014 a 21/09/2014). Os pastos diferidos com 15 e 25cm apresentaram menor massa de forragem (MF), mas maior percentual de folha viva (FV) na MF do que os pastos diferidos com 35 e 45cm. O percentual de colmo vivo na MF e o índice de seletividade aparente (ISA) da FV foram superiores no pasto diferido com 45cm. O percentual de colmo morto (CM) na simulação de pastejo (SP) e o ISA desse componente morfológico foram menores no pasto diferido com 15cm, em comparação ao diferido com 45cm. A MF e os percentuais de FV na MF e na amostra de SP se reduziram, enquanto os percentuais de CM na MF e na amostra de SP aumentaram com o período de pastejo. O capim-marandu com 15cm no início do período de diferimento melhora a morfologia do pasto diferido. O pastejo seletivo é dificultado no decorrer do período de pastejo.(AU)

O capim-marandu baixo no início do diferimento melhora a morfologia do pasto e aumenta o desempenho dos ovinos no inverno / The low marandu palisadegrass at the beginning of stockpiling improves pasture morphology and increases sheep performance in winter

Objetivou-se avaliar os efeitos de quatro alturas (15, 25, 35 e 45cm) da Urochloa brizantha cv. Marandu (capim-marandu) no início do diferimento sobre a morfologia do pasto, a seletividade e o desempenho de ovinos no início, meio e fim do período de pastejo, no inverno. Foi utilizado o delineamento inteiramente ao acaso, com três repetições. Os pastos diferidos com 35 e 45cm apresentaram maiores massas de forragem do que aqueles diferidos com 15 e 25cm. O pasto diferido com 15cm apresentou maior porcentagem de folha e menor percentual de colmo morto, em comparação aos demais pastos. A seleção por folha morta aumentou no período de pastejo. Em geral, nos pastos diferidos mais altos, os percentuais de folha viva foram menores e os de colmo morto, maiores nas amostras de pastejo simulado. O desempenho dos ovinos foi superior no pasto diferido com 15cm, intermediário nos pastos diferidos com 25 e 35cm, e inferior no pasto diferido com 45cm. A manutenção do capim-marandu com 15cm no início do diferimento resulta em pasto com melhor morfologia, otimiza a seletividade e aumenta o desempenho dos ovinos no inverno.(AU)

The objective of this study was to evaluate the effects of four heights (15, 25, 35, and 45cm) of Urochloa brizantha cv. Marandu (marandu palisadegrass) at the beginning of the deferment period on the pasture morphology, selectivity, and performance of sheep during the beginning, middle and end of the grazing period in winter. A completely randomized design with three replications was used. Deferred pastures with 35 and 45cm presented larger forage masses than those deferred with 15 and 25cm. Deferred pasture with 15cm presented a higher percentage of live leaf and a lower percentage of dead stem. Dead leaf selection increased during the grazing period. In general, in the higher deferred pastures, the percentages of live leaf were lower and those of dead stalk, higher in the samples of simulated grazing. The sheep performance was higher in the 15cm pasture, intermediate in the 25 and 35cm pastures, and inferior in the 45cm pasture. The maintenance of marandu palisadegrass with 15cm at the beginning of the deferment period results in deferred pasture with better morphology, optimizes the selectivity and the increase the performance of the sheep in the pasture deferred during the winter.(AU)

Study on the practices of silage production and utilization on Brazilian dairy farms.

Dairy farmers across Brazil were invited to participate in a study on silage production and utilization practices. Two hundred sixty farmers filled out a questionnaire, which was made available on a website. The questionnaire consisted of 14 questions, including information about the characteristics of the herd (n=3), the crop(s) used in the ensiling process, the use of additives, the harvest (n=3), the type of silo (n=1), aspects related to sealing (n=2), and management practices applied during feed-out (n=3). Farmers were also asked a final question about the main barriers they faced when producing and using silage. The main dairy-producing regions of Brazil had a strong influence on the number of participants. The profiles of farmers were heterogeneous and divided into 5 groups, which was considered a positive attribute of the study, allowing better analysis and assessment of current circumstances. Corn was the most widely grown crop for silage. Sorghum, tropical grasses, and sugarcane were the other species most cited. Additives were used by a small number of farmers (27.7%). Approximately 40% of farmers still depended on loaned equipment or outsourced services. The pull-type forage harvester was the main piece of equipment used on dairy farms (90.4%). Only 54.6% of respondents answered that they sharpen their harvester knives daily. Horizontal silos (bunker and stack) were the structures most commonly used to store silage. Most farmers sealed silos with double-sided plastic film (black-on-white) and with soil. However, almost one-fifth of all farmers still use black plastic. Manual removal of silage from the silos was practiced at most farms (i.e., the lack of equipment was also reflected in the stage of silage utilization). Disposal of spoiled silage before inclusion in the livestock feed was not a common practice on the farms. The main barriers encountered on the farms were lack of equipment, lack of manpower, and climatic variations. The results of this research may guide researchers, industries, extension workers, and governments to seek efficiency in milk production on farms using silage in the diet of livestock throughout the year or during part of the year in Brazil.

Mitochondrial- and endoplasmic reticulum-associated oxidative stress in Alzheimer's disease: from pathogenesis to biomarkers.

Alzheimer's disease (AD) is the most common cause of dementia in the elderly, affecting several million of people worldwide. Pathological changes in the AD brain include the presence of amyloid plaques, neurofibrillary tangles, loss of neurons and synapses, and oxidative damage. These changes strongly associate with mitochondrial dysfunction and stress of the endoplasmic reticulum (ER). Mitochondrial dysfunction is intimately linked to the production of reactive oxygen species (ROS) and mitochondrial-driven apoptosis, which appear to be aggravated in the brain of AD patients. Concomitantly, mitochondria are closely associated with ER, and the deleterious crosstalk between both organelles has been shown to be involved in neuronal degeneration in AD. Stimuli that enhance expression of normal and/or folding-defective proteins activate an adaptive unfolded protein response (UPR) that, if unresolved, can cause apoptotic cell death. ER stress also induces the generation of ROS that, together with mitochondrial ROS and decreased activity of several antioxidant defenses, promotes chronic oxidative stress. In this paper we discuss the critical role of mitochondrial and ER dysfunction in oxidative injury in AD cellular and animal models, as well as in biological fluids from AD patients. Progress in developing peripheral and cerebrospinal fluid biomarkers related to oxidative stress will also be summarized.

Amyloid beta peptide 1-42 disturbs intracellular calcium homeostasis through activation of GluN2B-containing N-methyl-d-aspartate receptors in cortical cultures.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that leads to debilitating cognitive deficits. Recent evidence demonstrates that glutamate receptors are dysregulated by amyloid beta peptide (Aß) oligomers, resulting in disruption of glutamatergic synaptic transmission which parallels early cognitive deficits. Although it is well accepted that neuronal death in AD is related to disturbed intracellular Ca(2+) (Ca(2+)(i)) homeostasis, little is known about the contribution of NMDARs containing GluN2A or GluN2B subunits on Aß-induced Ca(2+)(i) rise and neuronal dysfunction. Thus, the main goal of this work was to evaluate the role of NMDAR subunits in dysregulation of Ca(2+)(i) homeostasis induced by Aß 1-42 preparation containing both oligomers (in higher percentage) and monomers in rat cerebral cortical neurons. The involvement of NMDARs was evaluated by pharmacological inhibition with MK-801 or the selective GluN2A and GLUN2B subunit antagonists NVP-AAM077 and ifenprodil, respectively. We show that Aß, like NMDA, increase Ca(2+)(i) levels mainly through activation of NMDARs containing GluN2B subunits. Conversely, GluN2A-NMDARs antagonism potentiates Ca(2+)(i) rise induced by a high concentration of Aß (1µM), suggesting that GluN2A and GluN2B subunits have opposite roles in regulating Ca(2+)(i) homeostasis. Moreover, Aß modulate NMDA-induced responses and vice versa. Indeed, pre-exposure to Aß (1µM) decrease NMDA-evoked Ca(2+)(I) rise and pre-exposure to NMDA decrease Aß response. Interestingly, simultaneous addition of Aß and NMDA potentiate Ca(2+)(I) levels, this effect being regulated by GluN2A and GluN2B subunits in opposite manners. This study contributes to the understanding of the molecular basis of early AD pathogenesis, by exploring the role of GluN2A and GluN2B subunits in the mechanism of Aß toxicity in AD.

IGF-1 protects against diabetic features in an in vivo model of Huntington's disease.

Huntington's disease (HD) is the most prevalent polyglutamine expansion disorder. HD is caused by an expansion of CAG triplet in the huntingtin (HTT) gene, associated with striatal and cortical neuronal loss. Central and peripheral metabolic abnormalities and altered insulin-like growth factor-1 (IGF-1) levels have been described in HD. Thus, we hypothesized that restoration of IGF-1-mediated signaling pathways could rescue R6/2 mice from metabolic stress and behavioral changes induced by polyglutamine expansion. We analyzed the in vivo effect of continuous peripheral IGF-1 administration on diabetic parameters, body weight and motor behavior in the hemizygous R6/2 mouse model of HD. We used 9 week-old and age-matched wild-type mice, subjected to continuously infused recombinant IGF-I or vehicle, for 14 days. IGF-1 treatment prevented the age-related decrease in body weight in R6/2 mice. Although blood glucose levels were higher in R6/2 mice, they did not reach a diabetic state. Even though, IGF-1 ameliorated poor glycemic control in HD mice. This seemed to be associated with a decrease in blood insulin levels in R6/2 mice, which was increased following IGF-1 infusion. Similarly, blood IGF-1 levels decreased during aging in both wild-type and R6/2 mice, being significantly improved upon its continuous infusion. Although no significant differences were found in motor function in R6/2-treated mice, IGF-1 treatment highly improved paw clasping scores. In summary, these results suggest that IGF-1 has a protective role against HD-associated impaired glucose tolerance, by enhancing blood insulin levels.

Multiple defects in energy metabolism in Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia in old age. Cognitive impairment in AD may be partially due to overall hypometabolism. Indeed, AD is characterized by an early region-specific decline in glucose utilization and by mitochondrial dysfunction, which have deleterious consequences for neurons through increased production of reactive oxygen species (ROS), ATP depletion and activation of cell death processes. In this article, we provide an overview of the alterations on energetic metabolism occurring in AD. First, we resume the evidences that link the 'metabolic syndrome' with increased risk for developing AD and revisit the major changes occurring on both extra-mitochondrial and mitochondrial metabolic pathways, as revealed by imaging studies and biochemical analysis of brain and peripheral samples obtained from AD patients. We also cover the recent findings on cellular and animal models that highlight mitochondrial dysfunction as a fundamental mechanism in AD pathogenesis. Recent evidence posits that mitochondrial abnormalities in this neurodegenerative disorder are associated with changes in mitochondrial dynamics and can be induced by amyloid-beta (Aß) that progressively accumulates within this organelle, acting as a direct toxin. Furthermore, Aß induces activation of glutamate N-methyl-D-aspartate receptors (NMDARs) and/or excessive release of calcium from endoplasmic reticulum (ER) that may underlie mitochondrial calcium dyshomeostasis thereby disturbing organelle functioning and, ultimately, damaging neurons. Throughout the review, we further discuss several therapeutic strategies aimed to restore neuronal metabolic function in cellular and animal models of AD, some of which have reached the stage of clinical trials.

Toxicity of beta-amyloid in HEK293 cells expressing NR1/NR2A or NR1/NR2B N-methyl-D-aspartate receptor subunits.

Neurotoxicity induced by beta-amyloid peptide (Abeta) involves glutamate toxicity, resulting from overactivation of N-methyl-D-aspartate (NMDA) receptors and elevation of intracellular calcium. However, the heterogeneity of the NMDA receptors, frequently composed of NR1 and NR2A-D subunits, has been less studied. Thus, we determined the contribution of NMDA receptor subtypes on Abeta(1-40) toxicity in HEK293 cells transiently expressing NR1/NR2A or NR1/NR2B subunits. Analysis of lactate dehydrogenase (LDH) release and trypan blue exclusion revealed an increase in Abeta(1-40) toxicity upon NR1/NR2A expression, compared to NR1/NR2B, indicating loss of plasma membrane integrity. Furthermore, Abeta(1-40) decreased intracellular ATP in cells expressing NR1/NR2A. MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate), a noncompetitive NMDA receptor antagonist, partially prevented the decrease in cell viability and the energy impairment. These differences were not accounted for by the activation of caspases 2, 3, 8 and 9 or calpains or by DNA fragmentation, excluding the hypothesis of apoptosis. Functional NR1/NR2A and NR1/NR2B receptor subtypes were further evidenced by single-cell calcium imaging. Stimulation of NR1/NR2A receptors with NMDA/glycine revealed an increase in intracellular calcium in cells pre-exposed to Abeta(1-40). Opposite effects were observed upon activation of NR1/NR2B receptors. These results suggest that NR1/NR2A-composed NMDA receptors mediate necrotic cell death in HEK293 cells exposed to Abeta(1-40) through changes in calcium homeostasis.

Protective effect of zinc on amyloid-beta 25-35 and 1-40 mediated toxicity.

Amyloid beta-peptide (Abeta) is widely held to be associated with Alzheimer's disease, the insoluble aggregates of the peptide being the major constituents of senile plaques. In this study, we evaluated the effect of Zn(2+) (5, 50 and 200 microM) on Abeta induced toxicity using the human teratocarcinome (NT2) cell line. Our results proved that 50 and 200 microM Zn(2+) protected NT2 cells from Abeta 25-35 toxicity. Zinc was also shown to be effective by preventing the loss of mitochondrial membrane potential (DeltaPsi(m)) induced by Abeta 25-35, not allowing cytochrome c release from mitochondria, and subsequently, caspase 3 activation. However, when the cells were treated with Abeta 1-40, only Zn(2+) 5 microM had a protective effect. We have further observed that 5 microM Zn(2+) prevented Abeta 1-40 aggregation into a beta-sheet structure. Considering the results presented, we argue that Zn(2+) has a concentration-dependent protective effect.

Drugs of abuse induce apoptotic features in PC12 cells.

Drugs of abuse induce the release of dopamine in the central nervous system, particularly in the mesolimbic-mesocortical pathway. As dopamine may act as a neurotoxin, in this study, we analyzed the effects of the drugs of abuse, cocaine, heroin, and amphetamine, on the neurodegeneration of PC12 cells, a dopaminergic cell line, by evaluating the activity of caspase-3 and mitochondrial cytochrome c release. All the drugs were shown to induce caspase-3 activation, similarly to staurosporine, a classical inducer of apoptotic cell death. Furthermore, like staurosporine, the drugs of abuse induced a decrease in mitochondrial cytochrome c content, suggesting the involvement of the mitochondrial apoptotic pathway.

Toxic effects of opioid and stimulant drugs on undifferentiated PC12 cells.

Cell death and reactive oxygen species production have been suggested to be involved in neurodegeneration induced by the drugs of abuse. In this study we analyze the toxicity of the following drugs of abuse: heroin, morphine, d-amphetamine, and cocaine in undifferentiated PC12 cells, used as dopaminergic neuronal models. Our data show that opioid drugs (heroin and morphine) are more toxic than stimulant drugs (d-amphetamine and cocaine). Toxic effects induced by heroin are associated with a decrease in intracellular dopamine, an increase in DOPAC levels, and the formation of ROS, whereas toxic effects induced by amphetamine are associated with a decrease in intracellular dopamine and in ATP/ADP levels. In contrast with cocaine, both amphetamine and heroin induced features of apoptosis. The data suggest that the death of cultured PC12 cells induced by the drugs of abuse is correlated with a decrease in intracellular dopamine levels, which can be associated with an increased dopamine turnover and oxidative cell injury.

The mechanism of mitochondrial membrane potential retention following release of cytochrome c in apoptotic GT1-7 neural cells.

The relationship is investigated between mitochondrial membrane potential (Delta Psi(M)), respiration and cytochrome c (cyt c) release in single neural bcl-2 transfected cells (GT1-7bcl-2) or GT1-7puro cells during apoptosis induced by staurosporine (STS). Bcl-2 inhibited the mitochondrial release of cyt c and apoptosis. Three different cell responses to STS were identified in GT1-7puro cells: (i) neither Delta Psi(M) nor cyt c were significantly affected; (ii) a decrease in Delta Psi(M) was accompanied by a complete release of cyt c; or (iii) cyt c release occurred independently of a loss of Delta Psi(M). The endogenous inner membrane proton leak of the in situ mitochondria, monitored by respiration in the presence of oligomycin, was increased by STS by 92% in puro cells, but by only 23% in bcl-2 cells. STS decreased respiratory capacity, in the presence of protonophore, by 31% in puro cells and by 20% in bcl-2 cells. In the absence of STS, oligomycin hyperpolarized mitochondria within both puro and bcl-2-transfected cells, indicating that the organelles were net generators of ATP. However after 15 h exposure to STS oligomycin rapidly collapsed residual mitochondrial polarization in the puro cells, indicating that Delta Psi(M) had been maintained by ATP synthase reversal. bcl-2 cells in contrast, maintained Delta Psi(M) until protonophore was added. These results indicate that the maintenance of Delta Psi(M) following release of cyt c may be a consequence of ATP synthase reversal and cytoplasmic ATP hydrolysis in STS-treated GT1-7 cells.

Antioxidant effect of flavonoids after ascorbate/Fe(2+)-induced oxidative stress in cultured retinal cells.

In this study, we investigated the structure-activity relationship of four flavonoids, i.e. eriodictyol, luteolin, quercetin, and taxifolin, in cultured retinal cells after ascorbate/Fe(2+)-induced oxidative stress. The relative order of antioxidant efficacy, determined by the thiobarbituric acid method, was the following: eriodictyol > quercetin > luteolin > taxifolin. Upon preincubation, the flavonoids were also effective in reducing the extent of lipid peroxidation. Oxidative stress, determined by the changes in fluorescence of 2',7'-dichlorodihydrofluorescein, was also decreased in the presence of the flavonoids, showing the following order of antioxidant efficacy: eriodictyol > taxifolin approximately quercetin > luteolin. Ascorbate/Fe(2+)-induced oxidative stress or incubation in the presence of the flavonoids did not significantly affect the viability of retinal cells. We also evaluated the degree of membrane partition of the flavonoids. In this system, the results strongly suggest that the higher antioxidant activity of the flavonoids is not correlated with the presence of a double bond at C(2)-C(3) and/or a hydroxyl group at C(3) on the C ring, but rather may depend on the capacity to inhibit the production of reactive oxygen species to interact hydrophobically with membranes. Eriodictyol was shown to be the most efficient antioxidant in protecting against oxidative stress induced by ascorbate/Fe(2+) in the retinal cells.

Glutamate regulates the viability of retinal cells in culture.

In this study, we show that glutamate regulates the viability of cultured retinal cells upon transient glucose deprivation. At low concentrations (10-100 microM) glutamate decreased MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction to about 50% of control and decreased intracellular ATP levels (about 4-fold) after transient glucose removal. Under these conditions, the decrease in MTT reduction was associated with the activation of NMDA (N-methyl-D-aspartate) receptors. Upon exposure to high (10 mM) glutamate and transient glucose deprivation, the intracellular levels of glutamate increased. High glutamate significantly counteracted the decrease in MTT reduction and ATP production observed in the presence of low glutamate concentrations. AOAA (aminooxyacetic acid), a non-specific inhibitor of mitochondrial transaminases, enhanced the intracellular glutamate levels, but did not largely affect glutamate-mediated changes in MTT reduction or ATP production. Furthermore, the intracellular levels of pyruvate were not significantly altered, suggesting that changes in ATP production were not due to an increase in glycolysis. Thus, the recovery from glucose deprivation seems to be facilitated in retinal neuronal cells that had been exposed to high glutamate, in comparison with low glutamate, suggesting a role for high glutamate and glucose in maintaining retinal cell function following conditions of glucose scarcity.

Mitochondria control ampa/kainate receptor-induced cytoplasmic calcium deregulation in rat cerebellar granule cells.

Although mitochondria mediate the delayed failure of cytoplasmic Ca(2+) homeostasis [delayed Ca(2+) deregulation (DCD)] in rat cerebellar granule cells resulting from chronic activation of NMDA receptors, their role in AMPA/KA-induced DCD remains to be established. The mitochondrial ATP synthase inhibitor oligomycin protected cells against KA- but not NMDA-evoked DCD. In contrast to NMDA-evoked DCD, no additional protection was afforded by the further addition of rotenone. The effects of KA on cytoplasmic Ca(2+) homeostasis, including the protection afforded by oligomycin, could be reproduced by veratridine. KA exposure induced a partial mitochondrial depolarization that was enhanced by oligomycin, indicating ATP synthase reversal. The nonglycolytic substrates pyruvate and lactate were unable to maintain Ca(2+) homeostasis in the presence of KA. In contrast to NMDA, KA exposure did not cause mitochondrial Ca(2+) loading. The data indicate that Na(+) entry via noninactivating AMPA/KA receptors or voltage-activated Na(+) channels compromises mitochondrial function sufficiently to cause ATP synthase reversal. Oligomycin may protect by preventing the consequent mitochondrial drain of cytoplasmic ATP.

Developmental profile of excitatory GABA(A) responses in cultured rat cerebellar granule cells.

GABA induced a transient increase in cytosolic free Ca2+ in cerebellar granule cells, which decreased from 3 to 8 days in vitro (DIV). Cytosolic Ca2+ changes induced by glutamate/glycine were comparable at 3 and 7 DIV. The GABA response was ascribed to GABA(A)-receptor mediated depolarization activating L-type Ca2+ channels since the response was inhibited by bicuculline or nifedipine. GABA-mediated Ca2+ rise at 4 DIV was potentiated by pentobarbital or by the neurosteroid 5beta-pregnan-3alpha-ol-20-one, or by decreasing the extracellular Cl- concentration. Neurons cultured for > 7 DIV showed no rise in intracellular Ca2+ in response to GABA regardless of the Cl- gradient. GABA(A) receptor-mediated cytosolic Ca2+ rise suggests an important role for the excitatory activity of GABA in developing cerebellar granule neurons.

Mitochondrial membrane potential and glutamate excitotoxicity in cultured cerebellar granule cells.

The relationship between changes in mitochondrial membrane potential (Deltapsi(m)) and the failure of cytoplasmic Ca(2+) homeostasis, delayed Ca(2+)deregulation (DCD), is investigated for cultured rat cerebellar granule cells exposed to glutamate. To interpret the single-cell fluorescence response of cells loaded with tetramethylrhodamine methyl ester (TMRM(+)) or rhodamine-123, we devised and validated a mathematical simulation with well characterized effectors of Deltapsi(m) and plasma membrane potential (Deltapsi(P)). Glutamate usually caused an immediate decrease in Deltapsi(m) of <10 mV, attributable to Ca(2+) accumulation rather than enhanced ATP demand, and these cells continued to generate ATP by oxidative phosphorylation until DCD. Cells for which the mitochondria showed a larger initial depolarization deregulated more rapidly. The mitochondria in a subpopulation of glutamate-exposed cells that failed to extrude Ca(2+) that was released from the matrix after protonophore addition were bioenergetically competent. The onset of DCD during continuous glutamate exposure in the presence or absence of oligomycin was associated with a slowly developing mitochondrial depolarization, but cause and effect could not be established readily. In contrast, the slowly developing mitochondrial depolarization after transient NMDA receptor activation occurs before cytoplasmic free Ca(2+) ([Ca(2+)](c)) has risen to the set point at which mitochondria retain Ca(2+). In the presence of oligomycin no increase in [Ca(2+)](c) occurs during this depolarization. We conclude that transient Ca(2+) loading of mitochondria as a consequence of NMDA receptor activation initiates oxidative damage to both plasma membrane Ca(2+) extrusion pathways and the inhibition of mitochondrial respiration. Depending on experimental conditions, one of these factors becomes rate-limiting and precipitates DCD.

Adenosine A2A receptors regulate the extracellular accumulation of excitatory amino acids upon metabolic dysfunction in chick cultured retinal cells.

The role of endogenous extracellular adenosine as a tonic modulator of the extracellular accumulation of excitatory amino acids (glutamate and aspartate) caused by metabolic inhibition was investigated in cultured retinal cells. The selective adenosine A2A receptor antagonist, 4-[2-[7-amino-2-(2-furyl)(1,2,4)-triazin-5-ylamino]-ethyl]ph enol (ZM241385) (50 nM), increased the release of glutamate (three- to four-fold) and of aspartate (nearly two-fold) upon iodoacetic acid-induced glycolysis inhibition, in the presence or in the absence of Ca2+. Blockade of tonic activation of A2A receptors by ZM241385 also increased (nearly two-fold) the ischemia-induced release of glutamate and aspartate. Furthermore, another selective A2A receptor antagonist, 5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5- c] pyrimidine (SCH58261), also increased the release of aspartate and glutamate by about two-fold in cells submitted to glycolysis inhibition. In contrast, the selective adenosine A1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (100 nM), did not significantly modify the extracellular accumulation of either glutamate or aspartate caused by inducers of chemical ischemia or glycolytic inhibitors. Inhibition of glycolysis also increased (about three-fold) the extracellular accumulation of GABA, which was virtually unchanged by ZM241385. Furthermore, the GABAA receptor antagonist, bicuculline (10 microM), only increased (nearly two-fold) the iodoacetic acid-induced Ca(2+)-dependent release of glutamate, whereas the GABAB receptor antagonist, 3-aminopropyl(diethoxymethyl) phosphinic acid, CGP35348 (100 microM), was devoid of effects on the extracellular accumulation of glutamate and aspartate. These results show that endogenous extracellular adenosine, which rises under conditions of inhibited glycolysis, tonically inhibits the extracellular accumulation of excitatory amino acid through the activation of A2A, but not A1, adenosine receptors, and this effect is independent of GABAA and GABAB functions in the cultured retinal cells.

Adenosine modulation of D-[3H]aspartate release in cultured retina cells exposed to oxidative stress.

In this study we evaluated the role of adenosine receptor activation on the K+-evoked D-[3H]aspartate release in cultured chick retina cells exposed to oxidant conditions. Oxidative stress, induced by ascorbate (3.5 mM)/Fe2+ (100 microM), increased by about fourfold the release of D-[3H]aspartate, evoked by KCl 35 mM in the presence and in the absence of Ca2+. The agonist of A1 adenosine receptors, N6-cyclopentyladenosine (CPA; 10 nM), inhibited the K+-evoked D-[3H]aspartate release in control in oxidized cells. The antagonist of A1 adenosine receptor, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 50 nM), potentiated the release of D-[3H]aspartate in oxidized cells, and reverted the effect observed in the presence of CPA 10 nM. However, in oxidized cells, when DPCPX was tested together with CPA 100 nM the total release of D-[3H]aspartate increased from 5.1 +/- 0.4% to 11.4 +/- 1.0%, this increase being reverted by 3,7-dimethyl-1-propargylxanthine (DMPX; 100 nM), an antagonist of A2A adenosine receptors. In cells of both experimental conditions, the K+-evoked release of D-[3H]aspartate was potentiated by the selective agonist of A2A adenosine receptors, 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosin e (CGS 21680; 10 nM), whereas the antagonist of these receptors, DMPX (100 nM), inhibited the release of D-[3H]aspartate in oxidized cells, but not in control cells. Adenosine deaminase (ADA; 1 U/ml), which is able to remove adenosine from the synaptic space, reduced the K+-evoked D-[3H]aspartate release, from 5.1 +/- 0.4% to 3.1 +/- 0.3% in oxidized cells, and had no significant effect in control cells. The extracellular accumulation of endogenous adenosine, upon K+-depolarization, was higher in oxidized cells than in control cells, and was reduced by the inhibitors of adenosine transporter (NBTI) and of ecto-5'-nucleotidase (AOPCP). This suggests that adenosine accumulation resulted from the outflow of adenosine mediated by the transporter, and from extracellular degradation of adenine nucleotide. Our data show that both inhibitory A1 and excitatory A2A adenosine receptors are present in cultured retina cells, and that the K+-evoked D-[3H]aspartate release is modulated by the balance between inhibitory and excitatory responses. Under oxidative stress conditions, the extracellular accumulation of endogenous adenosine seems to reach levels enough to potentiate the release of D-[3H]aspartate by the tonic activation of A2A adenosine receptors.