Signaling pathways involved in the muscarinic acetylcholine receptor activation of arachidonic acid-dependent non-capacitative calcium entry

The oscillatory [Ca2+]i signals typically seen following physiologically relevant stimulation of phospholipase C-linked receptors are associated with a receptor-activated entry of Ca2+, which plays a critical role in driving the oscillations and influencing their frequency. We have recently shown that this receptor-activated entry of Ca2+ does not conform to the widely accepted "capacitative" model and, instead, reflects the activity of a distinct, novel Ca2+ entry pathway regulated by arachidonic acid (Shuttleworth, et al. (1998) J Biol Chem 273, 32636). We now show that the generation of arachidonic acid (AA) under these conditions results from the activity of the type IV phospholipase A2 (PLA2). Based largely on data obtained in systems where large amounts of AA are produced (e.g. in inflammatory responses), type IV PLA2 activation is thought to involve a Ca2+-dependent translocation of the enzyme to the membrane fraction. However, at the low agonist concentrations used in this study, where AA is generated in small amounts as a second messenger, type IV PLA2 activation was independent of increases in [Ca2+]i, and no detectable translocation to the membrane fraction occurs. Despite this result, stimulation of type IV PLA2 activity under these conditions was exclusively found in the plasma membrane fraction, where an increase in phosphorylation of the enzyme was observed. The observed phosphorylation, which elicited a 2 to 4-fold increase in type IV PLA2 activity, was the result of m3 mAChR-mediated activation of Raf-1 and ERK1/2. Contrary to what has been reported by others (Slack, B. E. (2000) Biochem J 348, 381), this activation of the MAPK cascade did not involve transactivation of the EGF receptor. Furthermore, evidence demonstrated that the activation of type IV PLA2 is PTX-insensitive and therefore involves the same family of G-proteins responsible for the parallel, yet independent activation of PLC. We suggest that, at the low physiological agonist concentrations associated with oscillatory [Ca2+] i signals, type IV PLA2 activation involves an ERK1/2-mediated phosphorylation of a discrete pool of the total cellular type IV PLA 2 that is already localized within the membrane fraction at resting [Ca2+]i.