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...

It has been postulated that intracellular binding sites for platelet-activating factor (PAF) contribute to proinflammatory responses to PAF. Isolated nuclei from porcine cerebral microvascular endothelial cells (PCECs) produced PAF-molecular species in response to H2O2. Using FACS analysis, we demonstrated the expression of PAF receptors on cell and nuclear surfaces of PCECs. Confocal microscopy studies performed on PCECs, Chinese hamster ovary cells stably overexpressing PAF receptors, and isolated nuclei from PCECs also showed a robust nuclear distribution of PAF receptors. Presence of PAF receptors at the cell nucleus was further revealed in brain endothelial cells by radioligand binding experiments, immunoblotting, and in situ in brain by immunoelectron microscopy. Stimulation of nuclei with methylcarbamate-PAF evoked a decrease in cAMP production and a pertussis toxin-sensitive rise in nuclear calcium, unlike observations in plasma membrane, which exhibited a pertussis toxin-insensitive elevation in inositol phosphates. Moreover, on isolated nuclei methylcarbamate-PAF evoked the expression of proinflammatory genes inducible nitric oxide synthase and cyclooxygenase-2 (COX-2) and was associated with augmented extracellular signal-regulated kinase 1/2 phosphorylation and NF-κB binding to the DNA consensus sequence. COX-2 expression was prevented by mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 and NF-κB inhibitors. This study describes for the first time the nucleus as a putative organelle capable of generating PAF and expresses its receptor, which upon stimulation induces the expression of the proinflammatory gene COX-2.

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.

Background and Purpose-Oxidant stress, especially in the premature, plays a major role in the pathogenesis of hypoxic-ischemic encephalopathies mostly manifested in the periventricular region. We studied the vasomotor mode of actions of the peroxidation product 15-F 2t -isoprostane (15-F 2t -IsoP) (8-iso-prostaglandin F 2␣ ) on periventricular region during development. Methods-Effects of 15-F 2t -IsoP on periventricular microvessels of fetal, newborn, and juvenile pigs were studied by video imaging and digital analysis techniques. Thromboxane formation and intracellular Ca 2ϩ were measured by radioimmunoassay and by using the fluorescent indicator fura 2-AM. Results-15-F 2t -IsoP-mediated constriction of periventricular microvessels decreased as a function of age such that in the fetus it was Ϸ2.5-fold greater than in juvenile pigs. 15-F 2t -IsoP evoked more thromboxane formation in the fetus than in the newborn, which was greater than that in the juvenile periventricular region; this was associated with immunoreactive thromboxane A 2 (TXA 2 ) synthase expression in the fetus that was greater than that in newborn pigs, which was greater than that in juvenile pigs. 15-F 2t -IsoP-induced vasoconstriction was markedly inhibited by TXA 2 synthase and receptor blockers (CGS12970 and L670596). Vasoconstrictor effects of the TXA 2 mimetic U46619 on fetal, neonatal, and juvenile periventricular microvessels did not differ. 15-F 2t -IsoP increased TXA 2 synthesis by activating Ca 2ϩ influx through non-voltage-gated channels in endothelial cells (SK&F96365 sensitive) and N-type voltage-gated channels (-conotoxin sensitive) in astrocytes; smooth muscle cells were not responsive to 15-F 2t -IsoP but generated Ca 2ϩ transients to U46619 via L-type voltage-sensitive channels. Conclusions-15-F 2t -IsoP causes periventricular brain region vasoconstriction in the fetus that is greater than that in the newborn, which in turn is greater than that in the juvenile due to greater TXA 2 formation generated through distinct stimulatory pathways, including from endothelial and astroglial cells. The resulting hemodynamic compromise may contribute to the increased vulnerability of the periventricular brain areas to oxidant stress-induced injury in immature subjects. (Stroke. 2000;31:516-525.)