Localization of functional prostaglandin E2 receptors in the nuclear envelope

Prostaglandin E2 is thought to act via G protein-coupled plasma membrane EP receptors. For the first time, studies reported in this thesis provide several lines of evidence suggesting the presence of functional nuclear membrane EP receptors in a variety of cells and tissues, in both newborn and adult animals. Radioligand binding studies revealed that the kinetics of PGE 2 binding to plasma membrane and nuclear membrane fractions isolated from pig newborn brain, adult uterus and adult rat liver were similar; these tissues are known to express high levels of PGE2 receptors. However in each tissue, the relative distribution of EP receptors in plasma membrane and nuclear membrane differed. A perinuclear localization of EP1, EP3alpha and EP4 receptors was visualized by indirect immunocytofluorescence in cultured porcine newborn cerebral microvascular endothelial cells, in transfected human embryonic kidney (HEK) 293 cells over-expressing these receptors, in fibroblast Swiss 3T3 cells over-expressing EP1 receptor and in HEK 293 cells expressing EP1 receptor fused to green fluorescent protein. High resolution immunostaining of EP1, EP3alpha and EP4 receptors revealed their presence in the nuclear envelope of cultured porcine endothelial cells and in situ, in adult rat brain (cortex) endothelial cells and neurons. These nuclear membrane EP receptors were found to increase the transcription of two target genes (iNOS, c-fos) that are known to be stimulated by PGE2. Nuclear calcium transients were also modulated by stimulation of nuclear EP receptors and this appears to involve pertussis toxin-sensitive G proteins. The discovery of nuclear EP receptors opens new avenues for the intracellular actions of prostanoids.**Please refer to dissertation for diagrams