4] - Phosphatidylethanol Formation as Index of Phospholipase D Activity in Rat Brain Cortex Slices

Abstract

Publisher Summary This chapter describes assay method and the mechanisms involved in the activation of phospholipase D (PLD) in rat brain cortex. PLD activity has been identified in a wide variety of mammalian cells, where its stimulation by receptor agonists, phorbol esters, and Ca 2+ ionophores has been extensively documented. Most of the receptor agonists associated with the activation of PLD also stimulate phosphoinositide-specific phospholipase C (PLC). The generation of phosphatidylethanol (PEt), a product with a high degree of metabolic stability, has been taken as an unequivocal indicator of PLD activity, because PEt results exclusively from PLD-catalyzed transphosphatidylation and is formed neither by base exchange enzymatic activities nor by de novo synthesis. A simple assay for PLD activity in rat brain cortical slices was used, taking advantage of its unique transphosphatidylation activity. Slices provide a functionally intact system where PLD activity can be investigated under more physiological conditions. Thus, the production of [ 32 P]PEt in cortical slices labeled with 32 P i , was monitored as a measure of PLD activity. Therefore, by monitoring PEt formation as an index of PLD activity in rat brain cortical slices, it was reported about the involvement of α 1 -adrenoceptors in receptor-mediated activation of PLD. PLD activity can be regulated by both PKC-dependent and PKC-independent mechanisms. Furthermore, this assay provides a useful and highly reproducible method for the study of PLD activity.