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Thrombin effect increasing swelling-induced glutamate efflux was examined in cultured cortical astrocytes, cerebellar granule neurons (CGN), hippocampal and cortical neurons. Hypotonic glutamate efflux (monitored by d-[H]aspartate) from cortical astrocytes was increased by thrombin (5 U/mL) to reach 16 % of the cell pool in 5 min. Thrombin had lower effects in CGN, and marginal effects in hippocampal and cortical neurons. These differences were related to the magnitude of thrombin-evoked cytosolic calcium rise. The protease-activated receptor 1 is expressed in astrocytes and neurons. In astrocytes exposed to chemical ischemia (sodium iodoacetate plus sodium azide) d-[H]aspartate release showed a first phase (20-40 min) of initial low efflux which progressively increases; and a second phase (40-60 min) of larger efflux coincident with cell swelling. Efflux at the first phase was 52 % inhibited by the glutamate transporter blocker DL- threo-β-benzyloxyaspartic-acid (TBOA) and 11 % by the volume-sensitive pathway blocker phloretin. At the second phase, efflux was reduced 52 and 38 % respectively, by these blockers. In CGN d-[H]aspartate efflux increased sharply and then decreased. This efflux was 32 % reduced by calcium omission, 46 % by TBOA and 32 % by 4-[(2-butyl-6,7dichloro-2-cyclopentyl-2,3-dihydro-1oxo-1 H-inden-5-yl)oxy] butanoic-acid. Thrombin enhanced this release by 32 %. Ischemic treatment increased astrocyte mortality from 4 % in controls to 39 and 61 % in ischemia and ischemia plus thrombin, respectively. Cell death was prevented by phloretin. CGN viability was unaffected by the treatment. These results suggest that coincidence of swelling and thrombin during ischemia elevates extracellular glutamate prominently from astrocyte efflux, which may endanger neurons in vivo. [ABSTRACT FROM AUTHOR]

Increased levels of circulating fatty acids, such as palmitic acid (PA), are associated with the development of obesity, insulin resistance, type-2 diabetes and metabolic syndrome. Furthermore, these diseases are linked to an increased risk of cancer, cardiovascular diseases, mild cognitive impairment and even Alzheimer's disease (AD). However, the precise actions of elevated PA levels on neurons and their association with neuronal metabolic disruption that leads to the expression of pathological markers of AD, such as the overproduction and accumulation of the amyloid-β peptide, represent an area of intense investigation. A possible molecular mechanism involved in the effects of PA may be through dysfunction of the NAD + sensor enzyme, SIRT1. Therefore, the aim of the present study was to analyze the relationship between the effects of PA metabolism on the function of SIRT1 and the upregulation of BACE1 in cultured hippocampal neurons. PA reduced the total amount of NAD + in neurons that caused an increase in p65 K310 acetylation due to inhibition of SIRT1 activity and low protein content. Furthermore, BACE1 protein and its activity were increased, and BACE1 was relocated in neurites after PA exposure.