trans-Arachidonic acids induce a heme oxygenase-dependent vasorelaxation of cerebral microvasculature.

Nitrative stress is an important regulator of vascular tone. We have recently described that trans-arachidonic acids (TAA) are major products of NO(2)(.)-mediated isomerization of arachidonic acid in cell membranes and that nitrative stress increases TAA levels leading to neural microvascular degeneration. In the present study, we explored whether TAA exert acute effects on neuromicrovascular tone and investigated potential mechanisms thereof. TAA induced an endothelium-dependent vasorelaxation of rat brain pial microvasculature. This vasorelaxation was independent of nitric oxide, prostanoids, lipoxygenase products, and CYP(450) metabolite trans-hydroxyeicosatetraenoic acids. However, inhibition of heme oxygenase (using zinc protoporphyrin IX) and of dependent soluble guanylate cyclase (sGC; using ODQ) significantly diminished (by approximately 70%) the TAA-induced vasorelaxation. Consistent with these findings, TAA stimulated heme oxygenase (HO)-2-dependent bilirubin (using siRNA HO-2) and cGMP formation, and the HO product carbon monoxide (using CO-releasing CORM-2) reproduced the sGC-dependent cGMP formation and vasorelaxation. Further exploration revealed that TAA-induced vasorelaxation and bilirubin formation (HO activation) were nearly abrogated by large-conductance calcium-dependent potassium channels (BK(Ca)) (using TEA and iberiotoxin). Opening of BK(Ca) with the selective activator NS1619 induced a concentration-dependent vasorelaxation, which was inhibited by HO and sGC inhibitors. Coimmunoprecipitation suggested a molecular complex interaction between BK(Ca) and HO-2 (but not HO-1). Collectively, these findings identify new properties of TAA, specifically cerebral vasorelaxation through interactive activation of BK(Ca) with HO-2 and, in turn, sGC. Our findings provide new insights into the characterization of nitrative stress-derived TAA products, by showing they can act as acute mediators of nitrative stress on neurovascular tone.

G-protein-coupled receptors signalling at the cell nucleus: an emerging paradigm.

G-protein-coupled receptors (GPCRs) comprise a wide family of monomeric heptahelical glycoproteins that recognize a broad array of extracellular mediators including cationic amines, lipids, peptides, proteins, and sensory agents. Thus far, much attention has been given towards the comprehension of intracellular signaling mechanisms activated by cell membrane GPCRs, which convert extracellular hormonal stimuli into acute, non-genomic (e.g., hormone secretion, muscle contraction, and cell metabolism) and delayed, genomic biological responses (e.g., cell division, proliferation, and apoptosis). However, with respect to the latter response, there is compelling evidence for a novel intracrine mode of genomic regulation by GPCRs that implies either the endocytosis and nuclear translocation of peripheral-liganded GPCR and (or) the activation of nuclearly located GPCR by endogenously produced, nonsecreted ligands. A noteworthy example of the last scenario is given by heptahelical receptors that are activated by bioactive lipoids (e.g., PGE(2) and PAF), many of which may be formed from bilayer membranes including those of the nucleus. The experimental evidence for the nuclear localization and signalling of GPCRs will be reviewed. We will also discuss possible molecular mechanisms responsible for the atypical compartmentalization of GPCRs at the cell nucleus, along with their role in gene expression.

Intracrine signaling through lipid mediators and their cognate nuclear G-protein-coupled receptors: a paradigm based on PGE2, PAF, and LPA1 receptors.

Prostaglandins (PGs), platelet-activating factor (PAF), and lysophosphatidic acid (LPA) are ubiquitous lipid mediators that play important roles in inflammation, cardiovascular homeostasis, and immunity and are also known to modulate gene expression of specific pro-inflammatory genes. The mechanism of action of these lipids is thought to be primarily dependent on their specific plasma membrane receptors belonging to the superfamily of G-protein-coupled receptors (GPCR). Increasing evidence suggests the existence of a functional intracellular GPCR population. It has been proposed that immediate effects are mediated via cell surface receptors whereas long-term responses are dependent upon intracellular receptor effects. Indeed, receptors for PAF, LPA, and PGE(2) (specifically EP(1), EP(3), and EP(4)) localize at the cell nucleus of cerebral microvascular endothelial cells of newborn pigs, rat hepatocytes, and cells overexpressing each receptor. Stimulation of isolated nuclei with these lipids reveals biological functions including transcriptional regulation of major genes, namely c-fos, cylooxygenase-2, and endothelial as well as inducible nitric oxide synthase. In the present review, we shall focus on the nuclear localization and signaling of GPCRs recognizing PGE(2), PAF, and LPA phospholipids as ligands. Mechanisms on how nuclear PGE2, PAF, and LPA receptors activate gene transcription and nuclear localization pathways are presented. Intracrine signaling for lipid mediators uncover novel pathways to elicit their effects; accordingly, intracellular GPCRs constitute a distinctive mode of action for gene regulation.

Proangiogenic effects of protease-activated receptor 2 are tumor necrosis factor-alpha and consecutively Tie2 dependent.

OBJECTIVE: Angiogenesis is essential physiologically in growth and pathologically in tumor development, chronic inflammatory disorders, and proliferative retinopathies. Activation of protease-activated receptor 2 (PAR2) leads to a proangiogenic response, but its mechanisms have yet to be specifically described. Here, we investigated the mode of action of PAR2 in retinal angiogenesis. METHODS AND RESULTS: PAR2-activating peptide, SLIGRL, increased retinal angiogenesis associated with an induction of vascular endothelial growth factor and angiopoetin-2 and most notably tie2 in the retina in vivo as well as in cultured neuroretinal endothelial cells. SLIGRL also induced release of the proinflammatory and angiogenic mediator tumor necrosis factor-alpha (TNF-alpha) via the MEK/extracellular signal-regulated kinase (ERK) (MEK/ERK) pathway in these endothelial cells. TNF-alpha, in turn, elicited tie2 expression by activating the MEK/ERK pathway. PAR2-evoked tie2 expression, endothelium proliferation (in vitro), and retinal neovascularization (in vivo) were abrogated by selective TNF-alpha blockers (neutralizing antibody infliximab and soluble TNF-alpha receptor-Fc fusion protein etanercept) as well as the MEK inhibitor PD98059. CONCLUSIONS: The proangiogenic properties of PAR2 are intertwined with its proinflammatory effects, such that in retinal vasculature, they depend on TNF-alpha and subsequent induction of tie2 via the MEK/ERK pathway.