Expression of endogenous nuclear bradykinin B2 receptors mediating signaling in immediate early gene activation

Abstract

Bradykinin (BK) represents a pro-inflammatory mediator that partakes in many inflammatory diseases. The mechanism of action of BK is thought to be primarily mediated by specific cell surface membrane B2 receptors (B2Rs). Some evidence has suggested, however, the existence of an intracellular/nuclear B2R population. Whether these receptors are functional and contribute to BK signaling remains to be determined. In this study, by mean of Western blotting, 3D-confocal microscopy, receptor autoradiography and radioligand binding analysis, we showed that plasma membrane and highly purified nuclei from isolated rat hepatocytes contain specific B2R that bind BK. The results depicting B2R nuclear expression in isolated nuclear organelles were reproduced in situ on hepatic sections by immunogold labeling and transmission electron microscopy. Functional tests on single nuclei, by means of confocal microscopy and the calcium-sensitive probe fluo-4AM, showed that BK induces concentration-dependent transitory mobilization of nucleoplasmic calcium; these responses were blocked by B2R antagonist HOE 140, not by the B1R antagonist R954 and, were also found in wild-type C57/Bl6 mice, but not in B2R-KO mice. In isolated nuclei, BK elicited activation/phosphorylation of Akt, acetylation of histone H3 and ensuing pro-inflammatory iNOS gene induction as determined by Western blot and RT-PCR. ChIP assay confirmed binding of acetylated-histone H3 complexes, but not B2R, to promoter region of iNOS gene suggesting that B2R-mediated gene expression is bridged with accessory downstream effectors. This study discloses a previously undescribed mechanism in BK-induced transcriptional events, via intracrine B2R-mediated signaling, occurring in rat autologous hepatic cells.

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.…”

Lack of both bradykinin B1 and B2 receptors enhances nephropathy, neuropathy, and bone mineral loss in Akita diabetic mice

“…The kinins, acting through B2R also increase the expression of inducible NOS (iNOS) (39). Our previous finding that preprandial levels of plasma nitrite/nitrate are lower in BRKO mice than in WT (23), and another report showing that urinary nitrite/nitrate excretion in B2R-null mice is lower than in WT (40) suggest that the KKS is also important in basal NO production.…”

An update on nuclear calcium signalling

“…For example, a C-terminal NLS directs the nuclear import of the bradykinin B2 receptor (Savard et al, 2008). Specific bradykininbinding sites are present in hepatocyte nuclear extracts, and the addition of bradykinin to isolated nuclei was found to evoke a rapid, transient nucleoplasmic Ca 2+ signal.…”