Cantalupo, Anna (2013) Role of L-cysteine/hydrogen sulfide (H2S) pathway in cardiovascular system in pathophysiological conditions. [Tesi di dottorato]

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Tipologia del documento:	Tesi di dottorato
Lingua:	English
Titolo:	Role of L-cysteine/hydrogen sulfide (H2S) pathway in cardiovascular system in pathophysiological conditions
Autori:	Autore Email Cantalupo, Anna anna.cantalupo@unina.it
Data:	30 Marzo 2013
Numero di pagine:	283
Istituzione:	Università degli Studi di Napoli Federico II
Dipartimento:	Farmacia
Scuola di dottorato:	Scienze farmaceutiche
Dottorato:	Scienza del farmaco
Ciclo di dottorato:	25
Coordinatore del Corso di dottorato:	nome email D'Auria, Maria Valeria madauria@unina.it
Tutor:	nome email Cirino, Giuseppe cirino@unina.it
Data:	30 Marzo 2013
Numero di pagine:	283
Parole chiave:	Hydrogen sulfide, cGMP, PKG-I, hypertension, ACE-inhibitors, Nogo-B, myogenic tone
Settori scientifico-disciplinari del MIUR:	Area 05 - Scienze biologiche > BIO/14 - Farmacologia
Depositato il:	09 Apr 2013 11:55
Ultima modifica:	23 Lug 2014 08:52
URI:	http://www.fedoa.unina.it/id/eprint/9286
DOI:	10.6092/UNINA/FEDOA/9286

Abstract

Hydrogen sulphide (H2S) is a novel gaseous mediator enzimatically produced by cystathionine-beta-synthase (CBS) and cystathionine-gamma-lyase (CSE). It is involved in physiological homeostatic processes and several pathological conditions; however, more importantly, H2S has a prominent role in cardiovascular system, where CSE deletion results in hypertension. H2S-induced vasodilation is hypothesized to occur through ATP-sensitive potassium channels (KATP); however, we recently demonstrated that it also increases cGMP levels in tissues. Herein, we studied the involvement of cGMP-dependent protein kinase-I in H2S-induced vasorelaxation. The effect of H2S on vessel tone was studied in phenylephrine-contracted aortic rings with or without endothelium. cGMP levels were determined in cultured cells or isolated vessel by enzyme immunoassay. Pretreatment of aortic rings with sildenafil attenuated NaHS-induced relaxation, confirming previous findings that H2S is a phosphodiesterase inhibitor. In addition, vascular tissue levels of cGMP in cystathionine gamma lyase knockouts were lower than those in wild-type control mice. Treatment of aortic rings with NaHS, a fast releasing H2S donor, enhanced phosphorylation of vasodilator-stimulated phosphoprotein in a time-dependent manner, suggesting that cGMP-dependent protein kinase (PKG) is activated after exposure to H2S. Incubation of aortic rings with a PKG-I inhibitor (DT-2) attenuated NaHS-stimulated relaxation. Interestingly, vasodilatory responses to a slowly releasing H2S donor (GYY 4137) were unaffected by DT-2, suggesting that this donor dilates mouse aorta through PKG-independent pathways. Dilatory responses to NaHS and L-cysteine (a substrate for H2S production) were reduced in vessels of PKG-I knockout mice (PKG-I2/2). Moreover, glibenclamide inhibited NaHS-induced vasorelaxation in vessels from wild-type animals, but not PKG-I2/2, suggesting that there is a cross-talk between KATP and PKG. Our results confirm the role of cGMP in the vascular responses to NaHS and demonstrate that genetic deletion of PKG-I attenuates NaHS and L-cysteine-stimulated vasodilation. ACE inhibitors are widely used in controlling blood pressure in hypertensive patients and they represent first line treatment in different cardiovascular diseases, since they also show additional beneficial effects unrelated to ACE inhibition. In particular, therapeutic use of sulfhydrylated inhibitor S-zofenopril has raised different hypotheses regarding the role played by its thiol group in the beneficial clinical effects it exerts over other ACEi. Here, we investigated whether H2S pathway contributes to the beneficial vascular effect described. SHR and WKY rats were treated with either zofenopril or enalapril in vivo. Aorta and carotid were harvested and vascular reactivity in vitro to Ach and L-cysteine assessed. CBS, CSE expression as well as H2S levels were evaluated in both vascular tissues. The vascular response to Ach in both carotid artery and aorta was impaired in SHR (~30%, P<0.001). Zofenopril, but not enalapril, treatment rescued the vascular response to Ach, while the vasodilatory response to L-cysteine was enhanced. CSE protein expression in vessels, and tissue and plasma H2S levels were restored to WKY levels in SHR rats receiving zofenopril. In contrast, CBS expression was unchanged. Zofenoprilat, the active metabolite of zofenopril, released in vitro detectable amount of H2S as well as it relaxed vessels in vitro in a concentration-dependent manner (P<0.001). Adminstration in vivo of the R-zofenoprilat diasteroisomer that does not inhibit ACE, did not modify blood pressure, but still displayed a beneficial vascular profile. Our findings establish that S-zofenopril modulates vascular function by rescuing the H2S pathway in a model of spontaneous hypertension, suggesting a unique mechanism unrelated to ACE inhibition and based on H2S release, which maybe explain the beneficial effects of sulfhydrylated ACE inhibitors reported in literature. The reticulon (Rtn) family of proteins are localized primarily to the endoplasmic reticulum (ER) of most cells. The Rtn-4 family, (aka Nogo) consists of 3 splice variants of a common gene called Rtn-4A, Rtn-4B, and Rtn-4C. Recently, we identified the Rtn-4B (Nogo-B) protein in endothelial and smooth muscle cells of the vessel wall, and showed that Nogo-B is a regulator of cell migration in vitro and vascular remodeling and angiogenesis in vivo. However, it is still unknown the biological function of Nogo-B in the peripheral system. Thus, we aimed to investigate the role of Nogo-B in the vasculature by using Nogo-A/B-/- mice. We found that Nogo-A/B-/- mice were markedly hypotensive compared to WT mice and moreover, mesenteric resistance vessels undergo vascular remodeling as both the media wall thickness and wall thickness:radius ratio were significantly reduced in Nogo-A/B-/- mice. Finally, by using pressure myograph system we found that both cholinergic and adrenergic response were not affected in Nogo-A/B-/- mice, conversely, the myogenic tone was markedly impaired in those mice. In conclusion, the present study reports a previously unidentified role of Nogo-B plays in the regulation of blood pressure in physiological conditions. Further studies are needed to understand the mechanism defining the physiological and biological function of Nogo-B.

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