Regulation of eNOS expression in brain endothelial cells by perinuclear EP3 receptors

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 PGE2 induces eNOS expression in cerebral microvessel endothelial cells (ECs). 16,16-Dimethyl PGE(2) and selective EP3 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. EP3 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 EP3 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 Ca2+ signals and nuclear membrane potential changes. Moreover, pertussis toxin, Ca2+ 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-kappaB inhibitor pyrrolidine dithiocarbamate prevented M&B28767-induced increase in Ca2+ 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 EP3 receptors coupled to pertussis toxin-sensitive G proteins, a process that depends on nuclear envelope K-Ca channels, protein kinases, and NF-kappaB; the roles for nuclear EP3 receptors seem different from those on plasma membrane.