Assay of Arachidonic Acid Release Coupled to α1- and α2-Adrenergic Receptors
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AA is the precursor for prostaglandins, eicosanoids, and the endogenous cannabinoid ligands, such as anandamide ( 1 ). The major pathways that lead to the liberation of free AA from phospholipids are shown in Fig. 1 . Phospholipase A 2 (PLA 2 ) provides a direct pathway for AA release and is the most common source of cellular AA liberation. PLA 2 is the primary source of free AA in most cell systems, including neurons. Physiological regulation of PLA 2 occurs primarily through the action of calcium, which binds to several PLA 2 isoforms and is required for activity ( 2 , 3 ). The low-molecular-weight secretory PLA 2 isoforms require millimolar calcium, whereas the cytosolic classes of PLA 2 need only micromolar concentrations. Calcium is required for translocation to membranes, where the cytosolic form of the enzyme can have access to its phospholipid substrate. Other isoforms are completely independent of calcium, but these variants may be primarily involved in membrane remodeling rather than signal transduction ( 4 ). The calcium-independent forms of PLA 2 play an important role in the rapid incorporation of free AA into phospholipids, which is taken advantage of in the AA release assay described here.
Fig. 1. Possible pathways for generation of AA from phospholipids. Hypothetical model of the biochemical pathways contributing to the liberation of AA. AA = arachidonic acid, PLA 2 =phospholipase A 2 , PLC=phospholipase C, PLD=phospholipase D, PA=phosphatidic acid, DAG=diacylglyceride, MAG=monoacylglyceride, EtOH=ethanol, PEt=phosphatidylethanol.
