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

Abstract

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 2ϩ signals and nuclear membrane potential changes. Moreover, pertussis toxin, Ca 2ϩ 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-B inhibitor pyrrolidine dithiocarbamate prevented M&B28767-induced increase in Ca 2ϩ 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-B; the roles for nuclear EP 3 receptors seem different from those on plasma membrane. (Circ Res. 2002;90:682-689.)

Intracrine endothelin signaling evokes IP3-dependent increases in nucleoplasmic Ca2+ in adult cardiac myocytes

“…This increase was prevented by a cellpermeable analog of an ETB-selective antagonist, IRL-2500, but not by adding BQ610 and BQ788 to the extracellular medium, thus confirming a direct role of nuclear ETB in regulating nuclear Ca 2+ signalling in intact ventricular myocytes. Other GPCRs found in nuclear membranes have also been show to increase [Ca 2+ ] n including Ang II [18,91], bradykinin B2 [21], prostaglandin E 2 [22][23][24], lysophosphatidic acid type-1 [25,26], and metabotropic glutamate type-5 [92][93][94][95].…”

“…Similarly, dynorphin B, an agonist of the κ opioid receptor, increases opioid peptide transcription in isolated cardiac nuclei [19]. In nuclei isolated from non-cardiac cells, Ang II [20], bradykinin B2 [21], prostaglandin E 2 [22][23][24], lysophosphatidic acid type-1 [25,26], metabotropic glutamate type-5 [27,28], thromboxane A2 [29,30], and urotensin-II [31] receptor activation also altered gene expression. Hence, endothelin receptors couple to effectors within the nuclear membrane and may be involved in stimulus-transcription coupling.…”

Modulation of Pro-inflammatory Gene Expression by Nuclear Lysophosphatidic Acid Receptor Type-1

“…Prior to stimulation, cells were serum-starved overnight. Isolation of nuclei was achieved by cell fractionation using the hypotonic/Nonidet P-40 lysis method (27). Nuclear envelopes were prepared by incubating nuclear suspensions (8 ϫ 10 6 nuclei/ml) with DNase I (800 units, pancreas type II, Roche Applied Science) and RNase A (32 mg/ml, Promega) for 30 min at 37°C (28).…”

Vaginal Misoprostol vs Vaginal Misoprostol With Estradiol for Labor Induction: A Prospective Double Blind Study

“…Activation of pig peri-nuclear EP receptors by PGE 2 modulates intranuclear calcium transients and transcription of genes such as inducible nitric oxide synthase (NOS) [9] and endothelial NOS [11]. Therefore, PGE 2 might increase NO synthesis and facilitate cervical dilatation during parturition in an intracrine manner via the activation of perinuclear EP 3 receptor in the longitudinal muscle layer.…”