The effects of prostaglandin E 2 are thought to be mediated via G protein-coupled plasma membrane receptors, termed EP. However recent data implied that prostanoids may also act intracellularly. We investigated if the ubiquitous EP 3 and the EP 4 receptors are localized in nuclear membranes. Radioligand binding studies on isolated nuclear membrane fractions of neonatal porcine brain and adult rat liver revealed the presence of EP 3 and EP 4 . A perinuclear localization of EP 3␣ and EP 4 receptors was visualized by indirect immunocytofluorescence and confocal microscopy in porcine cerebral microvascular endothelial cells and in transfected HEK 293 cells that stably overexpress these receptors. Immunoelectron microscopy clearly revealed EP 3␣ and EP 4 receptors localization in the nuclear envelope of endothelial cells; this is the first demonstration of the nuclear localization of these receptors. Data also reveal that nuclear EP receptors are functional as they affect transcription of genes such as inducible nitric-oxide synthase and intranuclear calcium transients; this appears to involve pertussis toxin-sensitive G proteins. These results define a possible molecular mechanism of action of nuclear EP 3 receptors.Prostaglandin E 2 (PGE 2 ) 1 is one of the most abundant prostanoids in the brain (1) and plays an important role in many cerebral functions, particularly in the newborn (2). PGE 2 also influences mitogenesis (3), promotes growth and metastasis of tumors (4), and stimulates gene transcription (5). To date, the biological actions of PGE 2 have been attributed to result from its interaction with plasma membrane G protein-coupled receptors termed EP, which include EP 1 , EP 2 , EP 3 , and EP 4 subtypes (6). Recent studies have shown that the nuclear membrane contains high levels of cyclooxygenase-1 and -2 and of PGE 2 (7). Possible intracellular sites of action for prostanoids are also suggested by other data. For example, a transporter that mediates the influx of prostanoid has been identified (8). Cytosolic phospholipase A 2 undergoes a calcium-dependent translocation to the nuclear envelope (9), and cyclooxygenase-2 has been shown to translocate to the nucleus in response to certain growth factors (10). It is thus possible that prostanoids may exert some of their effects via intracellular EP receptors, to have a direct nuclear action as recently proposed by Goetzl et al. (11), and Morita et al. (12).It has generally been assumed that the signal transduction cascades are initiated at the plasma membrane and not the nuclear membranes. However, recent studies have disclosed that the nuclear envelope plays a major role in signal transduction cascades (13,14). In fact, a novel nuclear lipid metabolism that is a part of unique nuclear signaling cascade termed NEST (nuclear envelope signal transduction) has been hypothesized (15). Both heterotrimeric and low molecular weight G proteins (15, 16), phospholipase C (13), phospholipase D (15), and adenylate cyclase (17) have shown to be localized at the nucleus. Th...

Prostaglandin E 2 receptors (EP) were detected by radioligand binding in nuclear fractions isolated from porcine brain and myometrium. Intracellular localization by immunocytofluorescence revealed perinuclear localization of EPs in porcine cerebral microvascular endothelial cells. Nuclear association of EP 1 was also found in fibroblast Swiss 3T3 cells stably overexpressing EP 1 and in human embryonic kidney 293 (Epstein–Barr virus-encoded nuclear antigen) cells expressing EP 1 fused to green fluorescent protein. High-resolution immunostaining of EP 1 revealed their presence in the nuclear envelope of isolated (cultured) endothelial cells and in situ in brain (cortex) endothelial cells and neurons. Stimulation of these nuclear receptors modulate nuclear calcium and gene transcription.

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.)