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 (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 cPLA2 translocation to the nuclear envelope and its activation by NE in rabbit VSMCs. 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 10 μM colchicine 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. Coimmunoprecipitation 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 are independent of its translocation to the nuclear envelope in rabbit VSMCs.
Footnotes
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This work was supported by National Heart, Lung, and Blood Institute Grant 19134-29 from the National Institutes of Health and from the Centers for Connective Tissue Diseases and Vascular Biology. F.A.Y. was supported by National Institutes of Health Training Grant 2T32HL007641-16 from National Heart, Lung, and Blood Institute.
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doi:10.1124/jpet.104.081992.
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ABBREVIATIONS: PLA2, phospholipase A2; AA, arachidonic acid; cPLA2, cytosolic phospholipase A2; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase; CaMK-II, calcium/calmodulin-dependent protein kinase-II; NE, norepinephrine; ER, endoplasmic reticulum; MDCK, Madin-Darby canine kidney; ECL, enhanced chemiluminescence; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene; VSMC, vascular smooth muscle cell; CD, cytochalasin D; LA, latrunculin A; KN-93, 2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonly)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; TBST, Tris-buffered saline/Tween 20.
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↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
- Received December 9, 2004.
- Accepted February 8, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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