Intact actin filaments are required for cytosolic phospholipase A2 translocation but not for its activation by norepinephrine in vascular smooth muscle cells.
Cytosolic phospholipase A(2) (cPLA(2)) 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 cPLA(2) translocation to the nuclear envelope is mediated via its phosphorylation by calcium/calmodulin-dependent kinase-II in rabbit vascular smooth muscle cells (VSMCs). 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 cPLA(2) translocation to the nuclear envelope and its activation by NE in rabbit VSMCs. NE (10 microM) caused cPLA(2) translocation to the nuclear envelope as determined by immunofluorescence. Cytochalasin D (CD; 0.5 microM) and latrunculin A (LA; 0.5 microM) that disrupted actin filaments, blocked cPLA(2) translocation elicited by NE. On the other hand, disruption of microtubule filaments by 10 microM colchicine did not block NE-induced cPLA(2) translocation to the nuclear envelope. CD and LA did not inhibit NE-induced increase in cytosolic calcium and cPLA(2) activity, determined from the hydrolysis of l-1-[(14)C]arachidonyl phosphatidylcholine and release of AA. Coimmunoprecipitation studies showed an association of actin with cPLA(2), which was not altered by CD or LA. Far-Western analysis showed that cPLA(2) interacts directly with actin. Our data suggest that NE-induced cPLA(2) translocation to the nuclear envelope requires an intact actin but not microtubule filaments and that cPLA(2) phosphorylation and activation and AA release are independent of its translocation to the nuclear envelope in rabbit VSMCs.
Fatima
Soghra
S
Department of Pharmacology, College of Medicine, University of Tennessee Health Science Center, Memphis, 38163, USA.
Yaghini
Fariborz A
FA
Pavicevic
Zoran
Z
Kalyankrishna
Shailaja
S
Jafari
Nauzanene
N
Luong
Elizabeth
E
Estes
Anne
A
Malik
Kafait U
KU
D000199:Actins / Q000502:physiology*; D021581:Active Transport, Cell Nucleus; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology*; D004285:Dogs; D004789:Enzyme Activation / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology*; D032389:Myocytes, Smooth Muscle / Q000201:enzymology*; D009638:Norepinephrine / Q000494:pharmacology*; D009685:Nuclear Envelope / Q000201:enzymology; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010766:Phosphorylation; D021381:Protein Transport; D011817:Rabbits
D000199:Actins; D016718:Arachidonic Acid; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium; D009638:Norepinephrine