Bani, Daniele

Bigazzi, M.

Failli, Paola

Mazzetti, L.

Nistri, Silvia

Quattrone, S.

Sacchi, Tatiana

English

Florence Research

Relaxin up-regulates inducible nitric oxide synthaseexpression and nitric oxide generation in ratcoronary endothelial cells1PAOLA FAILLI, SILVIA NISTRI,* SILVIA QUATTRONE,* LUCA MAZZETTI,MARIO BIGAZZI,† TATIANA BANI SACCHI,* AND DANIELE BANI*,2Department of Preclinical and Clinical Pharmacology, *Department of Anatomy, Histology andForensic Medicine, Section of Histology, University of Florence, and †Prosperius Institute,Florence, ItalySPECIFIC AIMSNitric oxide (NO) -mediated vasodilatation appears tobe a primary effect of relaxin (RLX), a peptide hor-mone of the insulin/IGF family produced mainly by theovaries and best known for its actions on the femalereproductive system that has recently been shown tomarkedly dilatate heart blood vessels. We used ratcoronary endothelial (RCE) cells in primary culture asa model to address the hypothesis that RLX can have adirect action on the coronary endothelium by inducingchanges in its ability to generate NO and, if so, to clarifythe possible mechanisms of action and influence oncell function.PRINCIPAL FINDINGS1. RLX increases the expression of inducible NOsynthase (NOS II) protein and mRNA in RCE cellsbut has no effect on the constitutive NO synthase(NOS III)Examination of RCE cell cultures immunostained withantibodies specific for NOS II and NOS III isoformsrevealed that the control cells were almost negative forNOS II and showed a clear-cut cytoplasmic immunore-activity for NOS III. A 24 h incubation with RLX (60ng/ml, prepared from a highly purified porcine RLXpreparation, 2500 to 3000 U/mg, provided by O. D.Sherwood) caused a marked increase in the immuno-reactivity for NOS II, leaving NOS III immunoreactivityunchanged. Western blot analysis confirmed that con-trol RCE cells did not express NOS II but did expressdetectable amounts of NOS III. A 24 h incubation withRLX (60 ng/ml) caused the expression of NOS II andhad a negligible effect on NOS III. Addition of dexa-methasone (8 mol/l), a well-known inhibitor of theNOS transcription factor NF-B, together with RLXcompletely abolished the RLX-induced expression ofNOS II but had no significant effect on NOS III.Replacement of the authentic hormone with an equiv-alent concentration of RLX inactivated with 1,2-ciclo-hexanedione, which binds to the arginines of thereceptor binding domain of RLX and prevents theligand-receptor interaction, failed to increase NOS IIexpression, thus confirming the specificity of the RLXeffect. Evaluation of NOS mRNA by RT-PCR (Fig. 1)showed that, under basal conditions, RCE cells ex-pressed both NOS II and NOS III mRNAs. A 24 hincubation with RLX at the noted concentration causeda clear-cut, significant elevation of the amount of NOSII mRNA and had no effect on NOS III mRNA. Asexpected, addition of dexamethasone (8 mol/l) to-gether with RLX caused a marked, significant reduc-tion of NOS II mRNA, which dropped to a level similarto that of the control cultures, and a negligible reduc-tion of NOS III mRNA.2. RLX increases NO release and intracellular NOgeneration by RCE cellsDetermination of nitrite, the stable end product of NOmetabolism, in RCE cell supernatants with the Griessreaction (expressed as nmol of nitrite/ml of culturemedium) showed that, in a 24 h incubation in mediumwith no RLX added, RCE cells produced detectableamounts of NO (8.80.1). Addition of RLX (60 ng/ml) to the RCE cell cultures caused a significantincrease in nitrite in the medium (17.91.2; P0.001vs. control). This effect of RLX was abrogated by theaddition of the NOS inhibitor l-NMMA (10 mol/l)during the last 20 min of RLX incubation (9.70.4;P0.001 vs. RLX alone), as well as by a 24 h incubationwith dexamethasone (8 mol/l) together with RLX(3.80.1; P0.001 vs. RLX alone). In one experiment,inactivated RLX given in the place of authentic RLXwas ineffective in increasing nitrite amount (8.9). AnRLX-induced increase in endogenous generation of1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01–0569fje; to cite this article,use FASEB J. (December 14, 2001) 10.1096/fj.01–0569fje2 Correspondence: Dipartimento di Anatomia, Istologiae Medicina Legale. Sezione di Istologia, Universita` diFirenze, V.le G. Pieraccini, 6, I-50139 Florence, Italy.E-mail: daniele.bani@unifi.it252 0892-6638/02/0016-0252 © FASEBNO ([NO]i) by RCE cells is also shown by imageanalysis studies with the NO-sensitive fluorescent probeDAF-2/DA. A 24 h incubation with RLX at the notedconcentration increased [NO]i in DAF-2/DA-loadedRCE cell cultures challenged with the vasoconstrictoragonist angiotensin II (AT II). This effect of RLX wasprevented by the NOS inhibitor l-NAME (10 mol/l)added 20 min before challenging the cells with AT II.3. RLX depresses the agonist-induced intracellularCa2 ([Ca2]i) transient in RCE cellsIn basal conditions, [Ca2]i in RCE cells evaluated byloading the cells with the Ca2-sensitive fluorophoreFura 2-AM was similar in control and RLX-treated cells(1107 vs. 1267 nmol/l, respectively). Stimulation ofcontrol RCE cells with the vasoconstrictor agonists ATII or thrombin caused a prompt increase in [Ca2]i,attaining a peak 60 s after addition of the agonists andwith a decay time of 21.6  1.7 s (Fig. 2A). A 24 hincubation of RCE cells with RLX dramatically de-creased both peak and decay time of the agonist-induced [Ca2]i rise (Fig. 2A). A 20 min preincubationwith either the nonspecific NOS inhibitor l-NAME(10 mol/l) or the selective NOS II inhibitor 1400W(1 mol/l) modified the agonist-induced [Ca2]i tran-sient in control and RLX-treated RCE cells in differentways (Fig. 2B). Although in the control cells, onlyFigure 1. Analysis of NOS mRNA expression by RT-PCR.A) Representative gel electrophoresis of amplification prod-ucts for NOS II, NOS III, and the housekeeping control gene-actin from RCE cell cultures. Lanes, A: untreated controls;B: cultures treated with RLX (60 ng/ml for 24 h); C: culturestreated with RLX and dexamethasone (8 mol/l for 24 h).B) Densitometric analysis of the bands from 3 separateexperiments expressed as % changes of the values of thecontrols (one-way ANOVA). Gray columns: NOS II. a) P 0.05 vs. control; b) P  0.05 vs. RLX alone. Open column:NOS III. RLX vs. control: not significant.Figure 2. Evaluation of [Ca2]i. A) Representative tracings of[Ca2]i-associated fluorescence in Fura 2-AM-loaded RCEcells from control and RLX-treated cultures on challengewith angiotensin II (AT II, 1 mol/l). RLX (60 ng/ml for24 h) markedly reduces both the peak and decay time of the[Ca2]i transient as compared with the control. AT II (1mol/l) was added at time 0 (arrow) and maintainedthroughout the experiment as indicated. B) Percent [Ca2]iincrease over the basal (left chart) and [Ca2]i transientdecay time (right chart) in 3 separate RCE cell cultureexperiments. Compared with controls, RLX (60 ng/ml for24 h) caused a significant decrease of both the parametersassayed. Coincubation with the nonspecific NOS inhibitorl-NMMA (10 mol/l for 20 min) or the selective NOS IIinhibitor 1400W (1 mol/l for 20 min) blunted the effect ofRLX. Significance of differences (one-way ANOVA): RLX vs.control: P  0.001 (increase), P  0.01 (decay time); RLX vs.RLX l-NAME: P 0.01 (both); RLX vs. RLX 1400W: P0.05 (increase), P  0.01 (decay time); RLX  l-NAME vs.RLX  1400W: not significant (both).253RELAXIN STIMULATES NO PATHWAY IN ENDOTHELIAL CELLSl-NAME caused a moderate increase in the decay timeof [Ca2]i transient, in the RLX-treated cells both NOSinhibitors were able to significantly raise both peak anddecay time. 1400W was only slightly less effective thanl-NAME in blunting the effect of RLX. These findingssuggest a predominant role of NOS II in mediating theaction of RLX on [Ca2]i in RCE cells.CONCLUSIONS AND SIGNIFICANCEThere is general agreement that under physiologicalconditions, the vasodilatatory action of NO is primarilyan endothelium-dependent process. In fact, endothe-lial cells contain the constitutive Ca2/calmodulin-dependent NOS III isoform that continuously producessmall amounts of NO, which cause relaxation of thevascular wall by an autocrine/paracrine mechanismdevoted to a moment-to-moment regulation of thevascular tone. Endothelial cells can also express induc-ible NOS II, which synthesizes higher amounts of NOthan NOS III and can be up-regulated by differentstimuli, especially inflammatory cytokines and media-tors. The current study provides evidence that thevasorelaxant hormone RLX has a direct effect onendothelial cells from rat coronary vessels. This effectconsists mainly of the up-regulation of NOS II mRNAand protein, apparently mediated by activation of adexamethasone-sensitive transcription factor. This fac-tor could be NF-B, because it has been shown to bindto the NOS II gene promoter and to be involved in theinduction of NOS II caused by inflammatory cytokinesand lipopolysaccharides. NF-B was also found to me-diate RLX-induced NOS II expression by arterialsmooth muscle cells in vitro.In RCE cells, the up-regulation of NOS II ostensiblyleads to increased NO generation, which is concurrentwith a diminished responsiveness of the cells to vaso-constrictors as judged by the clear-cut decrease in theagonist-induced [Ca2]i transient observed in the RLX-treated cells vs. the untreated controls. These effects ofRLX on coronary endothelial cells may account for theoverall vasodilatatory properties of this hormone inheart blood vessels, as reported in our previous studieswhere RLX was found to increase coronary flow byacting through the NO biosynthetic pathway.At variance with estrogens or insulin, which up-regulate the constitutive, endothelial-type NOS III inhuman and bovine endothelial cells in vitro, RLX doesnot seem to influence NOS III isoform in RCE cells. Infact, NOS III expression in terms of detectable proteinand mRNA is not significantly modified by a 24 hincubation with RLX at the concentration tested. Thisfinding suggests that NOS III is not substantially in-volved in the response of RCE cells to RLX. This view isalso indirectly supported by the results of experimentson the depression by RLX of agonist-induced [Ca2]itransient. In these experiments, the reduction of theeffect of RLX afforded by the selective NOS II inhibitor1400W was nearly the same as that afforded by thenonspecific NOS inhibitor l-NAME, suggesting thatNOS II contributes the major amount of biologicallyactive NO in response to RLX. Evidence shows thatNOS II may function as a ‘constitutive’ enzyme in somecell types under physiological conditions. Once synthe-sized on the action of different inducers, NOS II isactive for hours to days and generates large amounts ofNO, which can have beneficial effects such as anti-atherogenic and antiapoptotic. At variance with inflam-matory cytokines, which lead to massive NOS II induc-tion and consequent generation of harmful NO levels,RLX could up-regulate NOS II in RCE cells within aphysiological range. This property of RLX may there-fore underlie a variety of cardiovascular protectivefunctions of endothelial-derived NO beyond vasodilata-tion. These include the reduced expression of chemo-kines and adhesion molecules as well as the inhibitionof smooth muscle cell proliferation in the coronaryvascular wall. RLX may afford significant protectionagainst cardiovascular ischemic disease, in agreementwith the results of our previous studies in rat andguinea pig hearts subjected to ischemia and reperfu-sion. In the above studies, RLX was also found tosignificantly decrease neutrophil extravasation into thedamaged myocardium, an effect that can be ascribed toreduced neutrophil adhesion to the coronary endothe-lium possibly mediated by increased generation of NOby the endothelial cells.In conclusion, the effects of RLX on coronary endo-thelial cells reported here could account for at leastpart of the cardiovascular protective action of thishormone we demonstrated in animal models. To ourknowledge, RLX emerges as the only hormone capableto up-regulate NOS II in endothelial cells. Togetherwith estrogens, which can modulate endothelial NOgeneration acting via the constitutive NOS III isoform,RLX can be regarded as a physiological regulator ofheart microcirculation and hence of myocardial bloodperfusion. Further support for this view comes fromprevious findings that the heart possesses specific re-ceptors for RLX. On these grounds, the presence ofcirculating RLX in women during fertile life may helpexplain the low incidence of coronary heart diseasecompared with age-matched men and postmenopausalwomen.254 Vol. 16 February 2002 FAILLI AL.The FASEB Journal

Relaxin up regulates inducile nitric oxide synthase expression and nitric oxide generation in rat coronary endothelial

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Relaxin up regulates inducile nitric oxide synthase expression and nitric oxide generation in rat coronary endothelial

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