Nitric Oxide Signaling via Nuclearized Endothelial Nitric-oxide Synthase Modulates Expression of the Immediate Early Genes iNOS and mPGES-1

Abstract

Stimulation of freshly isolated rat hepatocytes with lysophosphatidic acid (LPA) resulted in LPA 1 receptor-mediated and nitricoxide-dependent up-regulation of the immediate early genes iNOS (inducible nitric-oxide synthase (NOS)) and mPGES-1 (microsomal prostaglandin E synthase-1). Because LPA is a ligand for both cell surface and intracellular receptor sites and a potent endothelial NOS (eNOS) activator, we hypothesized that NO derived from activated nuclearized eNOS might participate in gene regulation. Herein we show, by confocal microscopy performed on porcine cerebral endothelial cells expressing native LPA 1 -receptor and eNOS and on HTC4 rat hepatoma cells co-transfected with recombinant human LPA 1 -receptor and fused eNOS-GFP cDNA, a dynamic eNOS translocation from peripheral to nuclear regions upon stimulation with LPA. Nuclear localization of eNOS and its downstream effector, soluble guanylate cyclase, were demonstrated in situ in rat liver specimens by immunogold labeling using specific antibodies. Stimulation of this nuclear fraction with LPA and the NO donor sodium nitroprusside resulted, respectively, in increased production of nitrite (and eNOS phosphorylation) and cGMP; these separate responses were also correspondingly blocked by NOS inhibitor L-NAME and soluble guanylate cyclase inhibitor ODQ. In addition, sodium nitroprusside evoked a sequential increase in nuclear Ca 2؉ transients, activation of p42 MAPK, NF-B binding to DNA consensus sequence, and dependent iNOS RNA. This study describes a hitherto unrecognized molecular mechanism by which nuclear eNOS through ensuing NO modulates nuclear calcium homeostasis involved in gene transcription-associated events. Moreover, our findings strongly support the concept of the nucleus as an autonomous signaling compartment.Nitric oxide (NO) is a short-lived uncharged free radical involved in numerous complex biological processes such as blood pressure regulation, vascular inflammation, cell-mediated cytotoxicity, and survival (1, 2). One of the most biologically relevant actions of NO is its binding to the heme moiety in the heterodimeric enzyme soluble guanylate cyclase (sGC), 3 which leads to the production of the intracellular second messenger molecule cGMP and activation of cGMP-dependent protein kinases G (PKG). However, NO can also interact with and modify the bioactivity of a number of protein macromolecules through a series of reversible and nonreversible chemical reactions (e.g. S-, N-, and hemenitrosylation, tyrosine and tryptophan nitration) providing multifaceted regulatory mechanisms for cellular functions (3,4). NO is synthesized from L-arginine by a tripartite family of nitric-oxide synthase (NOS) isozymes composed of NOS-1 (nNOS), NOS-2 (iNOS), and NOS-3 (eNOS) originally found in brain, macrophages, and endothelium, respectively, and later discovered, for each NOS, in many other cell types (5).NOS isozymes, especially eNOS, are highly regulated by a number of mechanisms. Emerging findings show that eNOS bioactivity and mobilization to...

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.…”

Long-term continuous sildenafil treatment ameliorates corporal veno-occlusive dysfunction (CVOD) induced by cavernosal nerve resection in rats

“…[11][12][13] It is likely that this occurs through the maintenance of high levels of cGMP, since this compound reduces collagen synthesis and the activation of the pro-fibrotic TGFb1 pathway and protects SMC from apoptosis, while stimulating the spontaneous induction of inducible nitric oxide synthase (iNOS, also known as NOS2). [14][15][16][17][18][19][20][21][22][23] The expression of iNOS in certain non-immunological tissues is assumed to be a defense mechanism against fibrosis. [24][25][26][27][28][29] The nitric oxide produced by iNOS, besides inhibiting collagen synthesis and the TGFb1 pathway, also quenches reactive oxygen species, and in some cases, the differentiation of fibroblasts to myofibroblasts, the cells that produce collagen in many fibrotic conditions.…”

Use of the most recent reagent (CuF) for stimulation of NO synthesis by the medicinal leech salivary cell secretion in the cultures of human endothelium cells (HUVEC) and in rat cardiomiocytes

“…Эти сигналы не появляются в присутствии ингибитора синтеза NO (L-NAME), а также в клетках, где не наблюдается интенсивного окрашивания цитоплазмы. Мы полагаем, что образующееся в цитоплазме HUVEC производное NO-FL диффундирует в ядро и сорбируется на структурах ядрышка, однако нельзя полностью исключить и синтез NO в ядре [30,31].…”