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Phospholipase a2 activity in rat embryonic brain and in primary cultures of cortical neurons
The developmental changes in the activity of 85 kDa calcium-dependent cytosolic PLA2 in pre-term embryonic rat brain and in primary cultures of rat cortical neurons has been assessed with the aid of a radio-enzymatic method. We have further investigated the modulation of the enzymatic activity in primary cortical neurons, namely the inhibition of PLA2 with MAFP and the activaton with mellitin.
Brains of Wistar rat embryos were obtained at gestational days E18 through E21 and dissected in order to isolate hippocampal, cortical, and total brain tissue. Neural samples were either homogenised in Tris-Sucrose buffer (pH7.4), or dissociated into single cell suspensions and cultured for 2 to 9 days in Neurobasal medium (starting from E18/E19 embryos only) prior to radio-enzymatic determination of PLA2 activity. Cultured neurons were treated with different doses of MAFP (ranging from 5µM to 250µM) or mellitin (1 to 20µg/mL) for 1, 5, 10, 15, 30, and 60 minutes.
PLA2 activity was highest in the brains of 18 day embryos (E18) and gradually decreased toward birth. No significant differences were found regarding PLA2 activity in different topographies, namely hippocampus, cortex, and total brain. In primary cultures of cortical neurons, PLA2 activity was higher than the respective embryonic tissue activity, straight from early development. In vitro PLA2 activity peaked on the 4th day in culture, and decreased after six days of incubation, when the cultures became mature. MAFP inhibited PLA2 activity in a dose-dependent fashion (maximum inhibition at concentrations of 100µM and 250µM). Such effect was observed after 1 minute of incubation, and peaked at 15 minutes). Mellitin stimulated PLA2 activity in a dose-dependent effect.
This study reinforces previous evidence that PLA2 plays an important role in the early development of the nervous system, and further suggests that it may be implicated in neurodevelopmental processes and the in vitro survival of neurons. The in vitro modulation of neuronal PLA2 is a useful model for the study of PLA2 metabolism and biochemistry, which can be further addressed to the investigation of the biology of neurodegenerative and neuropsychiatric disorders.