Phosphorylation of Blood Vessel Vasodilator-Stimulated Phosphoprotein at Serine 239 as a Functional Biochemical Marker of Endothelial Nitric Oxide/Cyclic GMP Signaling C ´ ESAR IBARRA-ALVARADO, JAN GALLE, VOLKER O. MELICHAR, ALEXANDER MAMEGHANI, and HARALD H. H. W. SCHMIDT Rudolf-Buchheim-Institute for Pharmacology, Justus-Liebig-University, Giessen, Germany (C.I.A., H.H.H.W.S.); Departments of Pharmacology and Toxicology (C.I.A., V.O.M.) and Medicine/Nephrology (J.G., A.M.), Julius-Maximilians-University, Wu ¨ rzburg, Germany; and Faculty of Chemistry, University of Quere ´ taro, Centro Universitario, Quere ´ taro, Me ´ xico (C.I.A.) Received May 29, 2001; accepted October 30, 2001 This paper is available online at http://molpharm.aspetjournals.org ABSTRACT The endothelium-derived relaxing factors nitric oxide (NO) and prostacyclin (PGI 2 ) are important antithrombotic, relaxant, and antiproliferative agents of the blood vessel wall that exert their intracellular effects primarily via cGMP- and cAMP-dependent protein kinases (cGK, cAK). However, no biochemical marker for their activity in the intact blood vessel is available except for transient increases in the concentration of cGMP and cAMP. Using Western blot analysis and specific antibodies, we show here that phosphorylation of the vasodilator-stimulated phos- phoprotein (VASP) at Ser239 (P Ser239 -VASP) in rabbit aorta was detectable only in segments with an intact endothelium, al- though at least one third of VASP is contained in the remaining vascular wall. In endothelium-denuded aorta, VASP phosphor- ylation was increased by the NO donor sodium nitroprusside (SNP). Levels of P Ser239 -VASP, in the presence of endothelium and either SNP or 8-bromo-cAMP, were maximal. VASP phos- phorylation elicited by 8-bromo-cAMP was inhibited signifi- cantly by the cGK inhibitor Rp-8-Br-PET-cGMPS. Stimulated P Ser239 -VASP formation was fully reversible, reaching basal levels after 10 min of repeated washouts. Consistent with the important role that the NO/cGMP pathway plays in the forma- tion of P Ser239 -VASP in rabbit aorta, inhibition of NO synthase by N  -nitro-L-arginine methyl ester (L-NAME; 1 mM) or of sol- uble guanylyl cyclase by 1H-[1,2,4]oxadiazolo[3,4-a]quinoxalin- 1-one (ODQ; 50 M) almost completely abolished P Ser239 - VASP formation in endothelium intact blood vessels. These data suggest that vascular P Ser239 -VASP is primarily regulated by the NO/cGMP pathway and may thus serve as a biochemical marker for the activity state of this essential pathway in endo- thelial function. Vascular endothelium releases a variety of relaxing factors including prostacyclin (PGI 2 ) (Bunting et al., 1976) and nitric oxide (NO) (Palmer et al., 1987). Both are involved in the acute and long-term regulation of vascular tone (Furchgott and Vanhoutte, 1989; Somlyo and Somlyo, 1994). An abnor- mal increase in smooth muscle tone, due to endothelial dys- function and alterations in the production or action of these factors, has been implicated in the pathogenesis of several cardiovascular disease states, including arterial hyperten- sion, coronary heart disease, and atherosclerosis (Somlyo and Somlyo, 1994; Kojda and Harrison, 1999). Relaxation of vascular smooth muscle can result from ei- ther cyclooxygenase-catalyzed PGI 2 formation, receptor-me- diated activation of adenylyl cyclase (AC), and increased cAMP levels or via Ca 2+ -triggered activation of NO synthase in endothelial cells leading to activation of soluble guanylyl cyclase (sGC) and increased cGMP levels (Rasmussen et al., 1990; Murad, 1994). Vasodilators that elevate either cGMP or cAMP inhibit the contraction of vascular smooth muscle cells and the aggregation of platelets. NO- and cGMP-in- duced smooth muscle relaxation and platelet inhibition are primarily mediated by cGMP-dependent protein kinase (cGK) as evidenced by the absence of this response in cGK-I knock-out mice (Pfeifer et al., 1999; Hofmann et al., 2000). This study was supported by the Deutsche Forschungsgemeinschaft (SFB547/C7) and the Interdisciplinary Center for Clinical Research, IZKF, Wu ¨ rzburg (D7, C4). C.I.A. is a fellow of the Deutsche Akademische Austaus- chdienst (grant A/97/00418). This study is part of a thesis by C.I.A. submitted to the Faculty of Chemistry, Julius-Maximilians-University, Wu ¨ rzburg, Ger- many. ABBREVIATIONS: NO, nitric oxide; PGI 2 , prostacyclin; AC, adenylyl cyclase; sGC, soluble guanylyl cyclase; cGK, cGMP-dependent protein kinase; VASP, vasodilator-stimulated phosphoprotein; cAK, cAMP-dependent protein kinase; Ena, enabled; Evl, Ena-VASP-like protein; PE, phenylephrine; ODQ, 1H-[1,2,4]oxadiazolo[3,4-a]quinoxalin-1-one; DMSO, dimethyl sulfoxide; KHB, Krebs-Henseleit buffer; 8-Br-cAMP, 8-bro- mo-cAMP; Rp-8-Br-PET-cGMPS, -Phenyl-1,N 2 -etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp-isomer; P Ser239 -VASP, VASP phosphorylated at serine 239; L-NAME, N  -nitro-L-arginine methyl ester; SNP, sodium nitroprusside; PAGE, polyacrylamide gel electro- phoresis; TBS, Tris-buffered saline; NOS, nitric-oxide synthase. 0026-895X/02/6102-312–319$3.00 MOLECULAR PHARMACOLOGY Vol. 61, No. 2 Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics 1127/959249 Mol Pharmacol 61:312–319, 2002 Printed in U.S.A. 312