Lysophosphatidic acid (LPA) is a bioactive molecule involved in inflammation, immunity, wound healing, and neoplasia. Its pleiotropic actions arise presumably by interaction with their cell surface G protein-coupled receptors. Herein, the presence of the specific nuclear lysophosphatidic acid receptor-1 (LPA 1 R) was revealed in unstimulated porcine cerebral microvascular endothelial cells (pCMVECs), LPA 1 R stably transfected HTC4 rat hepatoma cells, and rat liver tissue using complementary approaches, including radioligand binding experiments, electron-and cryomicroscopy, cell fractionation, and immunoblotting with three distinct antibodies. Coimmunoprecipitation studies in enriched plasmalemmal fractions of unstimulated pCMVEC showed that LPA 1 Rs are dually sequestrated in caveolin-1 and clathrin subcompartments, whereas in nuclear fractions LPA 1 R appeared primarily in caveolae. Immunofluorescent assays using a cell-free isolated nuclear system confirmed LPA 1 R and caveolin-1 co-localization. In pCMVEC, LPA-stimulated increases in cyclooxygenase-2 and inducible nitricoxide synthase RNA and protein expression were insensitive to caveolea-disrupting agents but sensitive to LPAgenerating phospholipase A 2 enzyme and tyrosine kinase inhibitors. Moreover, LPA-induced increases in Ca 2؉ transients and/or iNOS expression in highly purified rat liver nuclei were prevented by pertussis toxin, phosphoinositide 3-kinase/Akt inhibitor wortmannin and Ca 2؉ chelator and channel blockers EGTA and SK&F96365, respectively. This study describes for the first time the nucleus as a potential organelle for LPA intracrine signaling in the regulation of pro-inflammatory gene expression.In the mammalian system, LPA 1 signaling cascades regulate important cellular processes, including gene expression, cell proliferation and growth, cell survival and death, and cell motility and secretion (1-3). These plethora of activities are characteristic features of inflammation that occur in various physiological as well as pathological states (e.g. ontogenic change, wound healing, cancer, etc.) (1-3). In humans, physiological responses induced by LPA arise from specific interactions with at least three genetically identified receptors designated LPA 1 , LPA 2 , and LPA 3 (formerly referred to as EDG 2 , EDG 4 , and EDG 7 receptors, respectively), which belong to the heptahelical transmembrane-spanning G protein-coupled receptor (GPCR) superfamily (4). These receptors show a broad, virtually distinct distribution and may couple in a cell-dependent manner to numerous heterotrimeric G proteins. In this context, LPA 1 and LPA 2 receptors have been shown to interact with G i/o , G q/11/14 , and G 12/13 proteins, whereas the LPA 3 receptor combines with G i/o and G q/11/14 proteins (5). Although many responses induced by extracellular LPA can result from its interaction with plasma membrane GPCRs, there is circumstantial evidence for an intracrine mode of action of LPA. For instance, putative biogenesis (e.g. secretory and cytosolic calcium-depen...

Abstract-We reported upregulation of endothelial nitric oxide synthase (eNOS) by PGE 2 in tissues and presence of perinuclear PGE 2 receptors (EP). We presently studied mechanisms by which PGE 2 induces eNOS expression in cerebral microvessel endothelial cells (ECs). 16,16-Dimethyl PGE 2 and selective EP 3 receptor agonist M&B28767 increased eNOS expression in ECs and the NO-dependent vasorelaxant responses induced by substance P on cerebral microvessels. These effects could be prevented by prostaglandin transporter blocker bromcresol green and actinomycin D. EP 3 immunoreactivity was confirmed on plasma and perinuclear membrane of ECs. M&B28767 increased eNOS RNA expression in EC nuclei, and this effect was augmented by overexpression of EP 3 receptors. M&B28767 also induced increased phosphorylation of Erk-1/2 and Akt, as well as changes in membrane potential revealed by the potentiometric fluorescent dye RH421, which were prevented by iberiotoxin; perinuclear K Ca channels were detected, and their functionality corroborated by NS1619-induced Ca 2ϩ signals and nuclear membrane potential changes. Moreover, pertussis toxin, Ca 2ϩ chelator, and channel blockers EGTA, BAPTA, and SK&F96365, as well as K Ca channel blocker iberiotoxin, protein-kinase inhibitors wortmannin and PD 98059, and NF-B inhibitor pyrrolidine dithiocarbamate prevented M&B28767-induced increase in Ca 2ϩ transients and/or eNOS expression in EC nuclei. We describe for the first time that PGE 2 through its access into cell by prostaglandin transporters induces eNOS expression by activating perinuclear EP 3 receptors coupled to pertussis toxin-sensitive G proteins, a process that depends on nuclear envelope K Ca channels, protein kinases, and NF-B; the roles for nuclear EP 3 receptors seem different from those on plasma membrane. (Circ Res. 2002;90:682-689.)

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Background-Intravitreal neovascular diseases, as in ischemic retinopathies, are a major cause of blindness. Because inflammatory mechanisms influence vitreal neovascularization and cyclooxygenase (COX)-2 promotes tumor angiogenesis, we investigated the role of COX-2 in ischemic proliferative retinopathy. Methods and Results-We describe here that COX-2 is induced in retinal astrocytes in human diabetic retinopathy, in the murine and rat model of ischemic proliferative retinopathy in vivo, and in hypoxic astrocytes in vitro. Specific COX-2 but not COX-1 inhibitors prevented intravitreal neovascularization, whereas prostaglandin E 2 , mainly via its prostaglandin E receptor 3 (EP 3 ), exacerbated neovascularization. COX-2 inhibition induced an upregulation of thrombospondin-1 and its CD36 receptor, consistent with the observed antiangiogenic effects of COX-2 inhibition; EP 3 stimulation reversed effects of COX-2 inhibitors on thrombospondin-1 and CD36. Conclusion-These findings point to an important role for COX-2 in ischemic proliferative retinopathy, as in diabetes.

Maternal protein deprivation, which leads to hypertension in the offspring, is associated with diminished NO-dependent relaxation of major organ (cerebral) microvasculature, which seems to be largely attributed to decreased sGC expression and cGMP levels. The study provides an additional explanation for abnormal vasorelaxation in nutrient-deprived subjects in utero.

Prostanoids exert significant effects on circulatory beds. They play a role in the response of the vasculature to adjustments in perfusion pressure and oxygen and carbon dioxide tension, and they mediate the actions of numerous factors. The role of prostanoids in governing circulation of the perinate is suggested to surpass that in the adult. Prostanoids are abundantly generated in the perinate. They have been implicated in autoregulation of blood flow as studied in brain and eyes. Prostaglandins are also dominant regulators of ductus arteriosus tone. The effects of these autacoids are mediated through specific G protein-coupled receptors. In addition to the pharmacological characterization of the prostanoid receptors, important advances in understanding the biology of these receptors have been made in the last decade. Their cloning and the development of animals with disrupted genes of these receptors have been very informative. The involvement of prostanoid receptors in the developing subject, especially on brain and ocular vasculature and on ductus arteriosus, has also begun to be investigated; the expression of these receptors changes with development. Some but not all of the ontogenic changes in these receptors are attributed to homologous regulation. Interestingly, in the process of elucidating their effects, functional perinuclear prostaglandin E2 receptors have been uncovered. This article reviews prostanoid receptors and addresses implications on the developing subject with attention to vascular physiology.

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IL-1 is a major proinflammatory cytokine which interacts with the IL-1 receptor I (IL-1RI) complex, composed of IL-1RI and IL-1R accessory protein subunits. Currently available strategies to counter pathological IL-1 signaling rely on a recombinant IL-1 receptor antagonist, which directly competes with IL-1 for its binding site. Presently, there are no small antagonists of the IL-1RI complex. Given this void, we derived 15 peptides from loops of IL-1R accessory protein, which are putative interactive sites with the IL-1RI subunit. In this study, we substantiate the merits of one of these peptides, rytvela (we termed “101.10”), as an inhibitor of IL-1R and describe its properties consistent with those of an allosteric negative modulator. 101.10 (IC50 ≈ 1 nM) blocked human thymocyte proliferation in vitro, and demonstrated robust in vivo effects in models of hyperthermia and inflammatory bowel disease as well as topically in contact dermatitis, superior to corticosteroids and IL-1ra; 101.10 did not bind to IL-1RI deficient cells and was ineffective in vivo in IL-1RI knockout mice. Importantly, characterization of 101.10, revealed noncompetitive antagonist actions and functional selectivity by blocking certain IL-1R pathways while not affecting others. Findings describe the discovery of a potent and specific small (peptide) antagonist of IL-1RI, with properties in line with an allosteric negative modulator.