An optimized spectrophotometric assay reveals increased activity of enzymes involved in 2-arachidonoyl glycerol turnover in the cerebral cortex of a rat model of Alzheimer's disease
| Autor: | Matilde S. Rodrigues, Cláudia Ferreira, Lisiane Souza, Ana Ledo, João Laranjinha, Anna Pliássova, Attila Köfalvi, Cândida Dias, Rodrigo A. Cunha |
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| Rok vydání: | 2020 |
| Předmět: |
Glycerol
Diacylglycerol lipase 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Receptor Cannabinoid CB1 Alzheimer Disease Animals JZL184 030304 developmental biology Cerebral Cortex 0303 health sciences biology Chemistry General Neuroscience Serine hydrolase Endocannabinoid system Monoacylglycerol Lipases Rhc80267 Rats Monoacylglycerol lipase Biochemistry biology.protein Methoxy arachidonyl fluorophosphonate 030217 neurology & neurosurgery Ex vivo Endocannabinoids |
| Zdroj: | The European journal of neuroscienceREFERENCES. 55(4) |
| ISSN: | 1460-9568 |
| Popis: | The endocannabinoid system is implicated in a plethora of neuropsychiatric disorders. However, it is technically challenging to assess the turnover of 2-arachidonoyl glycerol (2-AG), the principal endocannabinoid molecule in the brain. Two recent studies showed that diacylglycerol lipase α (DAGLα), an enzyme chiefly responsible for the cerebral production of 2-AG, also accepts the surrogate chromogenic substrate 4-nitrophenyl butyrate (4-NPB). Here, we aimed to optimize this spectrophotometric assay for ex vivo brain tissue, in particular, rat cerebrocortical homogenates, to measure the activity of the major enzymes responsible for the production and degradation of 2-AG. The initial velocity of 4-NPB hydrolysis was dependent on protein, substrate, and Ca2+ concentrations, and was sensitive to the non-selective serine hydrolase inhibitor, methoxy arachidonyl fluorophosphonate, the DAGLα inhibitors, OMDM188, tetrahydrolipstatin, and RHC80267, as well as the monoacylglycerol lipase (MAGL) inhibitor, JZL184, respectively. Next, we tested the usefulness of this assay in ex vivo brain tissue of rat models of human health conditions known to affect cerebrocortical 2-AG production, i.e. pathological stress and sporadic Alzheimer's disease (AD). In rats submitted to chronic restraint stress, cortical CB1 R density was significantly decreased, as assessed with radioligand binding. Nevertheless, 4-NPB hydrolysis remained at control levels. However, in rats 4 weeks after intracerebroventricular injection with streptozotocin - an established model of sporadic AD -, both CB1 R levels and 4-NPB hydrolysis and its DAGL- and MAGL-dependent fractions were significantly increased. Altogether, we optimized a simple complementary ex vivo technique for the quantification of DAGL and MAGL activity in brain samples. |
| Databáze: | OpenAIRE |
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