Elsevier

Biochemical Pharmacology

Volume 65, Issue 3, 1 February 2003, Pages 397-405
Biochemical Pharmacology

Endothelium-dependent and -independent relaxation and VASP serines 157/239 phosphorylation by cyclic nucleotide-elevating vasodilators in rat aorta

https://doi.org/10.1016/S0006-2952(02)01523-XGet rights and content

Abstract

Endothelium-dependent vasodilation is thought to be mediated primarily by the NO/cGMP signaling pathway whereas cAMP-elevating vasodilators are considered to act independent of the endothelial cell layer. However, recent functional data suggest that cAMP-elevating vasodilators such as β-receptor agonists, adenosine or forskolin may also be endothelium-dependent. Here we used functional and biochemical assays to analyze endothelium-dependent, cGMP- and cAMP-mediated signaling in rat aorta. Acetylcholine and sodium nitroprusside (SNP) induced a concentration-dependent relaxation of phenylephrine-precontracted aorta. This response was reflected by the phosphorylation of the vasodilator-stimulated phosphoprotein (VASP), a validated substrate of cGMP- and cAMP-dependent protein kinases (cGK, cAK), on Ser157 and Ser239. As expected, the effects of acetylcholine were endothelium-dependent. However, relaxation induced by the β-receptor agonist isoproterenol was also almost completely impaired after endothelial denudation. At the biochemical level, acetylcholine- and isoproterenol-evoked cGK and cAK activation, respectively, as measured by VASP Ser239 and Ser157 phosphorylation, was strongly diminished. Furthermore, the effects of isoproterenol were repressed by eNOS inhibition when endothelium was present. We also observed that the relaxing and biochemical effects of forskolin were at least partially endothelium-dependent. We conclude that cAMP-elevating vasodilators, i.e. isoproterenol and to a lesser extent also forskolin, induce vasodilation and concomitant cyclic nucleotide protein kinase activation in the vessel wall in an endothelium-dependent way.

Introduction

Vascular tone can be regulated by a variety of vasoconstricting and vasodilating agents. Two major vasodilator pathways utilize the cyclic nucleotides cAMP and cGMP as messenger molecules. The cAMP pathway is initiated by receptor-mediated stimulation of adenylyl cyclase and leads to the activation of cAMP-dependent protein kinase (cAK). cGMP, which is synthesized in the smooth muscle cell layer following the activation of soluble guanylyl cyclase by endothelium-derived nitric oxide (NO), regulates ion-channels, phosphodiesterases and cGMP-dependent protein kinases (cGK) [1]. Type I cGK has been shown to mediate the vasodilatory action of NO in mouse aorta [2], at least at low concentrations of NO [3]. A validated substrate of cGK I as well as cAK is the vasodilator-stimulated phosphoprotein (VASP), a protein localized at actin filaments, focal adhesions and dynamic membrane regions [4]. Other described substrates of cGK I in smooth muscle are the cytoskeletal proteins RhoA [5] and the myosin binding subunit of myosin phosphatase [6]. cGK I also regulates the release of calcium from intracellular stores, e.g. via the IP3-receptor-interacting protein IRAG [7]. Usually the NO/cGMP pathway is considered to be endothelium-dependent, whereas cAMP-elevating pathways are not. Recently however, a number of reports have challenged this concept suggesting that cAMP-elevating agents such as β-adrenoreceptor agonists and adenosine have an endothelium-dependent component to the vasorelaxant response [8], [9], [10], [11], [12]. One possible mechanism involved could be the activation of eNOS and endothelial NO formation by cAMP [12], [13]. A promising new tool for the analysis of cAMP- and cGMP-dependent pathways is the phosphorylation state of the cAK and cGK I substrate VASP. VASP phosphorylation has been shown to be a marker of the activity of the NO/cGMP pathway in a number of studies of animal models of endothelial function and dysfunction [14], [15], [16], [17], [18] as well as in humans [19]. These studies were performed using an antibody against the phosphorylation site Ser239 of VASP. This antibody was generated by us initially to study cGK I function in platelets [20]. VASP contains two additional cGK phosphorylation sites, namely Ser157 and Thr278, although the latter is phosphorylated with low efficiency [21]. In general the VASP Ser157 and Ser239 sites are phosphorylated by both cAK as well as cGK I, although studies with human platelets and endothelial cells suggested a preference of cGK I for the Ser239 site whereas cAK phosphorylates Ser157 faster [20], [22].

In the present study we determined the extent of endothelium-dependence of cAMP-mediated vasodilation of rat aorta in comparison with the cGMP pathway. In order to gain more information on the mechanisms involved we compared direct activation of adenylyl cyclase using forskolin with indirect receptor-mediated activation of the cAMP pathway using isoproterenol. In addition to vasodilation as common endpoint of the signaling pathways the kinase activity of cAK and cGK was measured directly using VASP Ser157 and Ser239 phosphorylation.

Section snippets

Vascular reactivity studies

Male Wistar rats (200–250 g) were purchased from Charles River and anaesthetized with 60 mg/kg pentobarbital administered intraperitoneally (i.p.). The chest was opened and the descending thoracic aorta was dissected, cleaned of connective tissue and cut into 3 mm rings which were mounted in an organ bath (FMI) for isometric force measurements. In some rings endothelium was removed by gentle rubbing of the intimal surface with a stainless steel forceps and only rings with contractile responses to

Measurement of VASP phosphorylation in rat aorta

We studied the activity of cAMP- and cGMP-regulated protein kinases by measuring the phosphorylation state of their substrate VASP. VASP content and phosphorylation state were studied in Western blots using antibodies against native VASP (Fig. 1) and phosphospecific antibodies against the phosphorylation sites Ser239 and Ser157. The phospho-Ser157 antibody is a newly developed antibody which was tested in this study for the first time on vascular tissue samples. In rat aorta this antibody

Discussion

The most important finding of the present study is the pronounced endothelium-dependence of cAMP-elevating vasodilators. Especially isoproterenol-induced vasodilation and cyclic nucleotide regulated kinase activation were almost completely blocked after endothelial denudation. In this respect isoproterenol effects were very similar to the classic endothelium-dependent vasodilator acetylcholine. Furthermore the effect of the direct adenylyl cyclase activator forskolin was at least partially

Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (SFB355).

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