Received for publication December 9, 2004.
Revised February 8, 2005.
Accepted for publication February 8, 2005.

Intact Actin Filaments are Required for Cytosolic Phospholipase A₂ Translocation but not for its Activation by Norepinephrine in Vascular Smooth Muscle Cells

Abstract

Cytosolic phospholipase A₂ (cPLA₂) is activated and translocated to the nuclear envelope by various vasoactive agents including norepinephrine (NE), and releases arachidonic acid (AA) from tissue phospholipids. We previously demonstrated that NE-induced cPLA₂ translocation to the nuclear envelope is mediated via its phosphorylation by calcium/calmodulin dependent kinase-II in rabbit vascular smooth muscle cells (VSMC). Cytoskeletal structures actin and microtubule filaments have been implicated in the trafficking of proteins to various cellular sites. This study was conducted to investigate the contribution of actin and microtubule filaments to cPLA₂ translocation to the nuclear envelope and its activation by NE in rabbit VSMC. NE (10 µM) caused cPLA₂ translocation to the nuclear envelope as determined by immunofluorescence. Cytochalasin D (CD, 0.5 µM) and latrunculin A (LA, 0.5 µM) that disrupted actin filaments, blocked cPLA₂ translocation elicited by NE. On the other hand, disruption of microtubule filaments by colchicine (10 µM) did not block NE-induced cPLA₂ translocation to the nuclear envelope. CD and LA did not inhibit NE-induced increase in cytosolic calcium and cPLA₂ activity, determined from the hydrolysis of L-1-¹⁴C-arachidonyl phosphatidylcholine and release of AA. Co-immunoprecipitation studies showed an association of actin with cPLA₂, which was not altered by CD or LA. Far-Western analysis showed that cPLA₂ interacts directly with actin. Our data suggest that NE-induced cPLA₂ translocation to the nuclear envelope requires an intact actin but not microtubule filaments and that cPLA₂ phosphorylation and activation and AA release is independent of its translocation to the nuclear envelope in rabbit VSMC.

Key words: arachidonic acid, cytochalasin D, cytosolic pospholipase A₂ translocation, latrunculin A, norepinephrine, vascular smooth muscle cells

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