Regulation of eNOS Expression in Brain Endothelial Cells by Perinuclear EP 3 Receptors

Abstract

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 PGE 2 induces eNOS expression in cerebral microvessel endothelial cells (ECs). 16,16-Dimethyl PGE 2 and selective EP 3 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. EP 3 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 EP 3 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 Ca 2+ signals and nuclear membrane potential changes. Moreover, pertussis toxin, Ca 2+ 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-κB inhibitor pyrrolidine dithiocarbamate prevented M&B28767-induced increase in Ca 2+ 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 EP 3 receptors coupled to pertussis toxin-sensitive G proteins, a process that depends on nuclear envelope K Ca channels, protein kinases, and NF-κB; the roles for nuclear EP 3 receptors seem different from those on plasma membrane.