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.

Sickness evokes various neural responses, one of which is activation of the hypothalamo-pituitary-adrenal (HPA) axis. This response can be induced experimentally by injection of bacterial lipopolysaccharide (LPS) or inflammatory cytokines such as IL-1. Although prostaglandins (PGs) long have been implicated in LPSinduced HPA axis activation, the mechanism downstream of PGs remained unsettled. By using mice lacking each of the four PGE receptors (EP1-EP4) and an EP1-selective antagonist, ONO-8713, we showed that both EP1 and EP3 are required for adrenocorticotropic hormone release in response to LPS. Analysis of c-Fos expression as a marker for neuronal activity indicated that both EP1 and EP3 contribute to activation of neurons in the paraventricular nucleus of the hypothalamus (PVN). This analysis also revealed that EP1, but not EP3, is involved in LPS-induced activation of the central nucleus of the amygdala. EP1 immunostaining in the PVN revealed its localization at synapses on corticotropin-releasing hormone-containing neurons. These findings suggest that EP1-and EP3-mediated neuronal pathways converge at corticotropin-releasing hormone-containing neurons in the PVN to induce HPA axis activation upon sickness.