Analisi dei dati delle colture cellular

Negli esperimenti riguardanti la valutazione dell’iperpolarizzazione, la fluorescenza è stata misurata ad una lunghezza d’onda di eccitazione di 450 nm ed una lunghezza d’onda di emissione di 520 nm mediante il lettore multipiastra EnSpire (Perkin Elmer®). Inoltre, ad ogni dato registrato sia per le cellule trattate con i nuovi composti di sintesi testati sia per le cellule trattate con la sostanza di riferimento oppure esposte al solo veicolo è stato sottratto il valore dei corrispettivi “bianchi”, ovvero la fluorescenza registrata nei pozzetti contenenti i medesimi trattamenti ma privi di cellule. Nell’esperimento con la sonda DiBac(4)3, i valori acquisiti sono stati espressi come variazione della fluorescenza, che viene calcolata mediante la seguente formula:

ΔF = (Ft – F0) / F0

dove F0 è la fluorescenza basale prima dell’aggiunta dell’agente iperpolarizzante, mentre Ft rappresenta la fluorescenza al tempo t dopo l’aggiunta del composto iperpolarizzante. I grafici così ottenuti sono stati poi rielaborati al fine di calcolare l’area sotto la curva (AUC) e, infine, i dati di tutti i composti in esame sono stati espressi come AUC percentuale rispetto al valore di AUC della sostanza iperpolarizzante di riferimento NS1619 alla concentrazione di 10 µM. L’elaborazione e l’analisi dei dati sono state effettuate mediante l’impiego del software Graph Pad Prism 6.0. L’analisi statistica è stata condotta mediante test statistico One way ANOVA seguito dal post test Bonferroni, in cui un livello di p<0.05 è stato considerato come limite di significatività statistica (*p<0.05; **p<0.01; ***p<0.001).

44

4. RISULTATI E DISCUSSIONE

Il solfuro di idrogeno è stato ampiamente descritto in letteratura come un agente in grado di evocare, a livello vascolare, un effetto iperpolarizzante. È stato dimostrato che tale attività è determinata dall’interazione dell’idrogeno solforato con varie tipologie di target, quali canali del potassio, in particolare i canali KATP e KV7, ed enzimi come la fosfodiesterasi (PDE). Pertanto, l’esigenza di trovare composti che siano in grado di indurre effetti vascolari positivi, come l’effetto vasorilasciante, ha portato al disegno di molecole di nuova sintesi, che fossero H2S-donors.

In questo lavoro di tesi sono state prese in esame tre molecole di nuova sintesi, precedentemente identificate come H2S-donors, per testarne l’attività iperpolarizzante di membrana che, relativamente alla membrana della muscolatura liscia vasale, si configura come il primo step per l’evento vasodilatante. Il primo composto testato, BW 502, ha mostrato, alle due concentrazioni somministrate, ovvero 30 µM e 100 µM, un’azione iperpolarizzante che, seppur modesta rispetto alla molecola di riferimento NS1619 10 µM, si è configurata come concentrazione-dipendente (Grafico 1).

45 Infatti, considerata l’iperpolarizzazione data da NS1619 come 100%, la concentrazione 30 µM ha esibito un effetto iperpolarizzante, espresso come area sotto la curva (AUC), del 14.7 ± 3.4%, mentre la concentrazione 100 µM ha mostrato un’azione iperpolarizzante del 30.1 ± 1.4%, raggiungendo il limite di significatività statistica rispetto all’effetto iperpolarizzante del veicolo (Grafico 2).

Grafico 1: Il grafico mostra la variazione di fluorescenza rilevata nel tempo correlata all’effetto

iperpolarizzante di membrana sulle cellule di muscolatura liscia vascolare umana (HASMC) in seguito al trattamento con Veicolo allo 0.5% di DMSO, NS1619 10 µM, BW 502 30 µM e BW

46

Il secondo composto testato BW 102 ha mostrato, invece, un profilo diverso. La sua azione iperpolarizzante, infatti, non ha evidenziato le caratteristiche di concentrazione- dipendenza (Grafico 3). Grafico 2: Area sotto la curva (AUC) del grafico relativo all’effetto iperpolarizzante di membrana sulle cellule HASMC in seguito al trattamento con veicolo allo 0.5% di DMSO, NS1619 10 µM, BW 502 30 µM e BW 502 100 µM. L’effetto iperpolarizzante di membrana di NS1619 è posto come 100%. Le barre verticali mostrano l’errore standard. Gli asterischi (*) sopra le colonne indicano un valore significativamente differente rispetto al veicolo con ***p < 0.001

47 Rispetto alla iperpolarizzazione manifestata da NS1619, presa anche in questo caso come 100% di riferimento, la concentrazione 30 µM del composto BW 102 ha dato un effetto iperpolarizzante, espresso come area sotto la curva (AUC), del 40.1 ± 6.6%; la concentrazione 100 µM, sebbene il valore dell’area sotto la curva risulti leggermente inferiore (33.6 ± 7.1%), può essere considerata del tutto sovrapponibile al valore registrato con la concentrazione 30 µM, in quanto dal punto di vista statistico non esiste alcuna differenza tra i due valori percentuali ottenuti (Grafico 4). Grafico 3: Il grafico mostra la variazione di fluorescenza rilevata nel tempo correlata all’effetto iperpolarizzante di membrana sulle cellule di muscolatura liscia vascolare umana (HASMC) in seguito al trattamento con Veicolo allo 0.5% di DMSO, NS1619 10 µM, BW 102 30 µM e BW 102 100 µM. Le barre verticali stanno ad indicare l’errore standard.

48 Tra tutti i composti testati la molecola denominata BW-HP 301 si è dimostrata quella più promettente (Grafico 5). Grafico 4: Area sotto la curva (AUC) del grafico relativo all’effetto iperpolarizzante di membrana sulle cellule HASMC in seguito al trattamento con veicolo allo 0.5% di DMSO, NS1619 10 µM, BW 102 30 µM e BW 102 100 µM. L’effetto iperpolarizzante di membrana di NS1619 è posto come 100%. Le barre verticali mostrano l’errore standard. Gli asterischi (*) sopra le colonne indicano un valore significativamente differente rispetto al veicolo con ***p < 0.001

49 Infatti, dall’analisi dei valori ottenuti mediante il calcolo dell’area sotto la curva (AUC) le due concentrazioni testate, ovvero 30 µM e 100 µM, hanno esibito un profilo concentrazione-dipendente: la concentrazione di 30 µM ha evocato un effetto iperpolarizzante pari al 51.8 ± 4.5% rispetto a NS1619 e la concentrazione più alta testata, ovvero la 100 µM, ha manifestato un valore dell’effetto iperpolarizzante del tutto sovrapponibile (108.4 ± 4.9%) a quello evocato dalla molecola di riferimento NS1619 (Grafico 6).

Grafico 5: Il grafico mostra la variazione di fluorescenza rilevata nel tempo correlata all’effetto

iperpolarizzante di membrana sulle cellule di muscolatura liscia vascolare umana (HASMC) in seguito al trattamento con Veicolo allo 0.5% di DMSO, NS1619 10 µM, BW-HP 301 30 µM e

50

In conclusione, lo screening effettuato relativamente all’attività iperpolarizzante dei composti BW 502, BW 102 e BW-HP 301 ha evidenziato profili differenti dei tre H2S- donors; in particolare BW-HP 301 si è dimostrato essere il composto più promettente tra i tre.

Quindi, pur non essendo possibile eseguire una valutazione dei rapporti struttura-attività, poiché, per motivi di confidenzialità, non possono essere divulgate le strutture delle molecole testate, senza ombra di dubbio il composto BW-HP 301 si configura come quello meritevole di approfondimenti futuri da svolgere sul vaso isolato. Grafico 6: Area sotto la curva (AUC) del grafico relativo all’effetto iperpolarizzante di membrana sulle cellule HASMC in seguito al trattamento con veicolo allo 0.5% di DMSO, NS1619 10 µM, BW-HP 301 30 µM e BW-HP 301 100 µM. L’effetto iperpolarizzante di membrana di NS1619 è posto come 100%. Le barre verticali mostrano l’errore standard. Gli asterischi (*) sopra le colonne indicano un valore significativamente differente rispetto al veicolo con ***p < 0.001

51

5. BIBLIOGRAFIA

Abe K, Kimura H. The possible role of hydrogen sulfide as an endogenous neuromodulator. J Neurosci. 1996; 16(3): 1066-1071.

Altaany Z, Moccia F, Munaron L, Mancardi D, Wang R. Hydrogen sulfide and endothelial dysfunction: relationship with nitric oxide. Curr Med Chem. 2014; 21(32): 3646-3661.

Amagase H. Clarifying the real bioactive constituents of garlic. J Nutr. 2006; 136: 716S–725S. Banerjee SK, Maulik SK. Effect of garlic on cardiovascular disorders: A review. Nutr J. 2002; 1: 4. Barresi E, Nesi G, Citi V, Piragine E, Piano I, Taliani S, Da Settimo F, Rapposelli S, Testai L, Breschi MC, Gargini C, Calderone V, Martelli A. Iminothioethers as Hydrogen Sulfide Donors: From the Gasotransmitter Release to the Vascular Effects. J Med Chem. 2017; 60: 7512-7523. Beltowski J. Hydrogen sulfide in pharmacology and medicine- An update. Pharmacol Rep. 2015; 67: 647-658. Benavides GA, Squadrito GL, Mills RW, Patel HD, Isbell TS, Patel RP, Darley- Usmar VM, Doeller JE, Kraus DW. Hydrogen sulfide mediates the vasoactivity of garlic. Proc Natl Acad Sci USA. 2007; 104: 17977–17982. Brunton PJ, Sausbier M, Wiezorrk G, Sausbier U, Kraus HG, Russel JA, Ruth P, Shipston MJ. Hypothalamic-pituitary-adrenal axis hyporesponsiveness to restraint stress in mice deficient for large-conductance calcium- and voltage- activated potassium (BK) channels. Endocrinology. 2007; 148: 5496–5506.

Bryan J, Vila-Carriles WH, Zhao J, Babenko AP, Aguilar-Bryan L. Toward linking structure with function in ATP-sensitive K1 channels. Diabetes. 2004; 53: S104– S112.

52 Bucci M, Cirino G. Hydrogen sulphide in heart and systemic circulation. Inflamm Allergy drug Targets. 2011; 10: 103-108. Bucci M, Papapetropoulos A, Vellecco V, Zhou Z, Pyriochou A, Roussos F, Brancaleone V, Cirino G. Hydrogen sulfide is an endogenous inhibitor of phosphodiesterase activity. Arterioscler Thromb Vasc Biol. 2010; 30: 1998-2004. Calderone V, Martelli A, Testai L, Citi V, Breschi MC. Using hydrogen sulfide to design and develop drugs. Expert Opin Drug Discov. 2016; 11(2): 163-175. Calderone V. Large-conductance, Ca2+_{-activation K} 1 channels: Function, pharmacology and drugs. Curr Med Chem. 2002; 9: 1385–1395.

Caliendo G, Cirino G, Santagada V, Wallace JL. Synthesis and biological effects of hydrogen sulfide: development of H2S-releasing drugs as pharmaceutical. J Med Chem. 2010; 53: 6275-6286. Calvert JW, Jha S, Gundewar S, Elrod JW, Ramachandran A, Pattillo CB, Kevil CG, Lefer DJ. Hydrogen sulfide mediates cardioprotection through Nrf2 signaling. Circ Res. 2009, 105: 365-374. Cao X, Ding L, Xie Z-Z, Yang Y, Whiteman M, Moore PK, Bian J-S. A review of hydrogen sulfide synthesis, metabolism and measurement: is modulation of hydrogen sulfide a novel therapeutic for cancer?. Antioxid Redx Signal. 2019; 31(1): 1-38.

Carballal S, Trujillo M, Cuevasanta E, Bartesaghi S, Moller M N, Folkes LK, Garcia- Bereguian M A, Gutierrez-Merino C, Wardmann P, Denicola A, Radi R, Alvarez B. Reactivity of hydrogen sulfide with peroxynitrite and other oxidants of biological interest. Free Radical Biol Med. 2011; 50: 196.

Cavallini D, Modovi B, De Marco C, Sciosciasantoro A. Inhibitory effect of mercaptoethanol and hypotaurine on the desulfhydration of cysteine by cystathionase. Arch Biochem Biophys. 1962; 96: 456-457.

53 Chadha PS, Zunke F, Zhu HL, Davis AJ, Jepps TA, Olesen SP, Cole WC, Moffatt JD, Greenwood IA. Reduced KCNQ4-encoded voltage-dependent potassium channel activity underlies impaired β-adrenoceptor-mediated relaxation of renal arteries in hypertension. Hypertension. 2012; 59: 877-884.

Chang L, Geng B, Yu F, Zhao J, Jian H, Du J, Tang C. Hydrogen sulfide inhibits myocardial injury induced by homocysteine in rats. Amino acids. 2008; 34: 573– 585.

Chatterji T, Gates KS. Reaction of thiols with 7-methylbenzopentathiepin. Bioorg Med Chem Lett. 2003; 13: 1349–1352.

Chen SL, Yang CT, Yang ZL, Guo RX, Meng JL, Cui Y, Lan AP, Chen PX, Feng JQ. Hydrogen sulphide protects H9c2 cells against chemical hypoxia-induced cell injuries. Clin Exp Pharmacol Physiol. 2010; 37(3): 316-321.

Cheng Y, Ndisang JF, Tang G, Cao K, Wang R. Hydrogen sulfide-induced relaxation of resistance mesenteric artery beds of rats. Am J Physiol Heart Circ Physiol. 2004; 287: 2316–2323.

Cheng HM, Koutsidis G, Lodge JK, Ashor A, Siervo M, Lara J. Tomato and lycopene supplementation and cardiovascular risk factors: a systematic review and meta- analysis. Atherosclerosis. 2017; 257: 100-108.

Citi V, Martelli A, Gorica E, Brogi S, Testai L, Calderone V. Role of hydrogen sulfide in endothelial dysfunction: Pathophysiology and therapeutic approaches. Journal of Advanced Research. https://doi.org/10.1016/j.jare.2020.05.015

Citi V, Martelli A, Testai L, Marino A, Breschi MC, Calderone V. Hydrogen sulfide releasing capacity of natural isothiocyanates: is it a reliable explanation for the multiple biological effects of Brassicaceae?. Planta Med. 2014; 80: 610-613.

Del Camino D, Kanevsky M, Yellen G. Status of the intracellular gate in the activated- not-open state of shaker K+ channels. J Gen Physiol. 2005; 126: 419–428.

54 Del Camino D, Yellen G. Tight steric closure at the intracellular activation gate of a voltage-gated K+ channel. Neuron. 2001; 32: 649–656.

Dinkova-Kostova AT, Kostov RV. Glucosinolates and isothiocyanates in health and disease. Trends Mol Med. 2012; 18: 337–347.

Doeller JE, Isbell TS, Benavides G, Koenitzer J, Patel H, Patel RP, Lancaster JR. Polarographic measurement of Hydrogen Sulfide production and consumption by mammalian tissue. Anal Biochem. 2005; 341: 40-51.

Dorman DC, Moulin FJ, McManus BE, Mahle KC, James RA, Struve MF. Cytochrome oxidase inhibition induced by acute hydrogen sulfide inhalation: Correlation with tissue sulfide concentration in the rat brain, liver, lung, and nasal epithelium. Toxicol Sci. 2002; 65: 18– 25.

Doyle DA, Morais Cabral J, Pfuetzner RA, Kuo A, Gulbis JM, Cohen SL, Chait BT, MacKinnon R. The structure of the potassium channel: molecular basis of K+

conduction and selectivity. Science. 1998; 280: 69–77.

Eichhorn B, Dobrev D. Vascular large conductance calcium-activated potassium channels: Functional role and therapeutic potential. Naunyn Schmiedebergs Arch Pharmacol. 2007; 376: 145–155.

Eto K, Kimura H. The production of hydrogen sulfide is regulated by testosterone and S-adenosyl-L-methionine in mouse brain. J Neurochem. 2002; 83: 80–86.

Eto K, Ogasawara M, Umemura K, Nagai Y, Kimura H. Hydrogen sulfide is produced in response to neuronal excitation. J Neurosci. 2002; 22: 3386-3391.

Filipovic MR, Miljkovic J, Allgauer A, Chaurio R, Shubina T, Herrmann M, Ivanovic- Burmazovic I. Biochemical insight into physiological effects of H S: reaction with peroxynitrite and formation of a new nitric oxide donor, sulfinyl nitrite. Biochem J. 2012A; 441: 609.

55 Filipovic MR, Miljkovic JL, Nauser T, Royzen M, Klos K, Shubina T, Koppenol WH, Lippard SJ and Ivanovic-Burmazovic I. Chemical characterization of the smallest S- nitrosothiol, HSNO; cellular cross-talk of H2S and S-nitrosothiols. J Am Chem Soc. 2012B; 134: 12016.

Furne J, Springfield J, Koenig T, DeMaster E, Levitt MD. Oxidation of hydrogen sulfide and methanethiol to thiosulfate by rat tissues: A specialized function of the colonic mucosa. Biochem Pharmacol. 2001; 62: 255–259.

Geng B, Yang J, Qi Y, Zhao J, Pang Y, Du J, Tang C. H2S generated by heart in rat and its

effects on cardiac function. Biochem Biphys Res Commun. 2004; 313(2): 362-368. Germain E, Chevalier J, Siess MH, Teyssier C. Hepatic metabolism of diallyl disulphide in rat and man. Xenobiotica. 2003; 33: 1185–1199.

Goubern M, Andriamihaja M, Nubel T, Blachier F, Bouillaud F. Sulfide, the first inorganic substrate for human cells. Faseb J. 2007; 21: 1699– 1706 .

Grambow E, Mueller-Graf F, Deliagina E, Frank M, Kuhula A, Vollmar B. Effect of the hydrogen sulfide donor GYY4137 on platelet activation and microvascular thrombus formation in mice. Platelets. 2014; 25: 166–174. Greenwood IA, Ohya S. New tricks for old dogs: KCNQ expression and role in smooth muscle. Br J Pharmacol. 2009 Apr; 156(8): 1196-1203. Hildebrandt TM, Grieshaber MK. Three enzymatic activities catalyze the oxidation of sulfide to thiosulfate in mammalian and invertebrate mitochondria. Febs J. 2008; 275: 3352–3361. Holmgren M., Shin KS, Yellen G. The activation gate of a voltage-gated K+ _{channel can} be trapped in the open state by an intersubunit metal bridge. Neuron. 1998; 21: 617– 621.

56 Hoorigan FT, Aldrich RW. Coupling between voltage sensor activation, Ca2+ binding and channel opening in large conductance (BK) potassium channels. J Gen Physiol. 2002; 120: 267–305.

Hosoki R, Matsuki N, Kimura H. The possibile role of hydrogen sulfide as an endogenous smooth muscle relaxant in sinergy with nitric oxide. Biochem Biophys Res Commun. 1997; 237: 527–531. Hughes MN, Centelles MN, Moore KP. Making and working with hydrogen sulfide: The chemistry and generation of hydrogen sulfidein vitro and its measurement in vivo: A review. Free Radic Biol Med. 2009; 47(10): 1346-1353. Iciek M, Bilska A, Ksiazek L, Srebro Z, Wlodek L. Allyl disulfide as donor and cyanide as acceptor of sulfane sulfur in the mouse tissues. Pharmacol Rep. 2005; 57: 212–218.

Ishii I, Akahoshi N, Yu XN, Kobayashi Y, Namekata K, Komaki G, Kimura H. Murine cystathionine gamma-lyase: complete cDNA and genomic sequences, promoter activity, tissue distribution and developmental expression. Biochem J. 2004; 381:113– 123.

Jackson SJ, Singletary KW, Venema RC. Sulforaphane suppresses angiogenesis and disrupts endothelial mitotic progression and microtubule polymerization. Vascul Pharmacol. 2007; 46: 77–84.

Jackson-Weaver O, Osmond JM, Riddle MA, Naik JS, Gonzalez Bosc LV, Walker BR, Kanagy NL. Hydrogen sulfide dilates rat mesenteric arteries by activating endothelial large-conductance Ca2+-activated K+ channels and smooth muscle Ca2+ sparks. Am J Physiol Heart Circ Physiol. 2013; 304: H1446–H1454.

Jesberger M, Davis TP, Barner L. Applications of Lawesson’s reagent in organic and organometallic syntheses. Synthesis. 2003; 13: 1929–1958.

Jiang B, Tang G, Cao K, Wu L, Wang R. Molecular mechanism for H2S-induced

57 Jones CM, Lawrence A, Wardman P, Burkitt MJ. Free Electron paramagnetic resonance spin trapping investigation into the kinetics of glutathione oxidation by the superoxide radical: re-evaluation of the rate constant. Radical Biol Med. 2002; 32: 982. Khanamiri S, Soltysinska E, Jepps TA, Bentzen BH, Chadha PS, Schmitt N, Greenwood IA, Olesen SP. Contribution of Kv7 channels to basal coronary flow and active response to ischemia. Hypertension. 2013; 62: 1090-1097. Kimura H. Hydrogen sulfide: its production, release and functions. Amino Acids. 2011; 41: 113-121.

King SB. Potential biological chemistry of Hydrogen Sulfide (H2S) with the Nitrogen

Oxides. Free Radic Biol Med. 2013 Feb; 55: 1-7.

Kondo K, Bhushan S, King AL, Prabhu SD, Hamid T, Koenig S, Murohara T, Predmore BL, Gojon G Sr, Gojon G Jr, Wang R, Karusula N, Nicholson CK, Calvert JW, Lefer DJ. H2S

protects against pressure overload-induced heart failure via upregulation of endothelial nitric oxide synthase. Circulation. 2013; 127: 1116-1127.

Kuo SM, Lea TC, Stipanuk MH. Developmental pattern, tissue distribution, and subcellular distribution of cysteine: Alpha-ketoglutarate aminotransferase and 3- mercaptopyruvate sulfurtransferase activities in the rat. Biol Neonate. 1983; 43: 23– 32.

Kurzban GP, Chu L, Ebersole JL, Holt SC. Sulfhemoglobin formation in human erythrocytes by cystalysin, an L-cysteine desulfhydrase from Treponema denticola. Oral Microbiol Immunol. 1999; 14: 153– 164.

Lecher HZ, Greenwood RA, Whitehouse KC, Chao TH. The Phosphonation of Aromatic Compounds with Phosphorus Pentasulfide. J Am Chem Soc. 1956; 78: 5018.

Li L, Hsu A, Moore PK. Actions and interactions of nitric oxide, carbon monoxide and hydrogen sulphide in the cardiovascular system and in inflammation-a tale of three gases. Pharmacol Ther. 2009; 123: 386–400.

58 Li L, Moore PK. Putative biological roles of hydrogen sulfide in health and disease: A breath of not so fresh air? Trends Pharmacol Sci. 2008; 29: 84–90.

Li L, Whiteman M, Guan YY, Neo KL, Cheng Y, Lee SW, Zhao Y, Baskae R, Tan CH, Moore PK. Characterization of a novel, water-soluble hydrogen sulfide-releasing molecule (GYY4137): New insights into the biology of hydrogen sulfide. Circulation. 2008; 117: 2351–2360. Li YF, Xiao CS, Hui RT. Calcium sulfide (CaS), a donor of hydrogen sulfide (H2S): A new antihypertensive drug? Med Hypoth. 2009; 73: 445-447. Liu Z, Han Y, Li L, Lu H, Men G, Li X, Shirhan M, Peh MT, Xie L, Zhou S, Wang X, Chen Q, Dai W, Tan CH, Pan S, Moore PK, Ji Y. The hydrogen sulfide donor, GYY4137, exhibits anti-atherosclerotic activity in high fat fed apolipoprotein E(-/-) mice. Br J Pharmacol. 2013; 169: 1795–1809.

Lowicka E and Beltowski J. Hydrogen sulfide (H2S) – the third gas of interest for

pharmacologists. Pharmacological Reports. 2007; 59: 4-24. Mani BK, Byron KL. Vascular KCNQ channels in humans: the sub- threshold brake that regulates vascular tone? Br J Pharmacol. 2011; 162: 38–41. Martelli A, Citi V, Testai L, Brogi S, Calderone V. Organic isothiocyanates as hydrogen sulfide donors. Antioxidants & Redox Signaling. 2020; 32: 2. Martelli A, Testai L, Breschi MC, Blandizzi C, Virdis A, Taddei S, Calderone V. Hydrogen sulphide: novel opportunity for drug discovered. Medicinal Reserce Review. 2010; 32(6): 1093-1130.

Martelli A, Testai L, Breschi MC, Lawson K, McKay NG, Miceli F, Taglialatela M, Calderone V. Vasorelaxation by hydrogen sulphide involves activation of Kv7 potassium channels. Pharmacol Res. 2013A; 70(1): 27-34.

Martelli A, Testai L, Citi V, Marino A, Bellagambi FG, Ghimenti S, Breschi MC, Calderone V. Pharmacological characterization of the vascular effects of aryl

59 isothiocyanates: is hydrogen sulfide the real player? Vascul Pharmacol. 2014; 60: 32- 41. Martelli A, Testai L, Citi V, Marino A, Pugliesi I, Barresi E, Nesi G, Rapposelli S, Taliani S, Da Settimo F, Breschi MC, Calderone V. Arylthioamides as H2S Donors: l-cisteine-

activated releasing properties and vascular effects in vitro and in vivo. ACS Med Chem Lett. 2013 Aug; 4(10): 904-908.

Martelli A, Testai L, Marino A, Breschi MC, Da Settimo F, Calderone V. Hydrogen sulphide: biopharmacological roles in the cardiovascular system and pharmaceutical perspectives. Curr Med Chem. 2012; 19: 3325-3336.

Martelli A, Piragine E, Citi V, Testai L, Pagnotta E, Ugolini L, Lazzeri L, Di Cesare Mannelli L, Manzo OL, Bucci M, Ghelardini C, Breschi MC, Calderone V. Erucin exhibits vasorelaxing effects and antihypertensive activity by H2S-releasing properties. Br J

Pharmacol. 2020 Feb; 177(4): 824-835.

Medeiros JV, Bezerra VH, Gomes AS, Barbosa AL, Lima-Junior RC, Soares PM, Brito GA, Ribeiro RA, Cunha FQ, Souza MH. Hydrogen sulfide prevents ethanol- induced gastric damage in mice: Role of ATP-sensitive potassium channels and capsaicin-sensitive primary afferent neurons. J Pharmacol Exp Ther. 2009; 330: 764–770.

Melchini A, Traka MH. Biological profile of erucin: A new promising anticancer agent from cruciferous vegetables. Toxins (Brasel). 2010 Apr; 2(4): 593-612.

Miki T, Seino S. Roles of KATP channels as metabolic sensors in acute metabolic changes. J Mol Cell Cardiol. 2005; 38: 917–925.

Mitsuhashi H, Yamashita S, Ikeuchi H, Kuroiwa T, Kaneko Y, Hiromura K, Ueki K, Nojima Y. Oxidative stress-dependent conversion of hydrogen sulfide to sulfite by activated neutrophils. Shock. 2005; 24: 529–534.

Munchberg U, Anwar A, Mecklenburg S, Jacob C. Polysulfides as biologically active ingredients of garlic. Org Biomol Chem. 2007; 5: 1505–1518.

60 Nicholls P, Kim JK. Sulphide as an inhibitor and electron donor for the cytochrome c oxidase system. Can J Biochem. 1982; 60(6):613-623. Nichols CG. KATP channels as molecular sensors of cellular metabolism. Nature. 2006; 440: 470–476. O'Rourke B. Myocardial K(ATP) channels in preconditioning. Circ Res. 2000; 87: 845- 855. Ogasawara Y, Ishii K, Togawa T, Tanabe S. Determination of bound sulfur in serum by gas dialysis/high-performance liquid chromatography. Anal Biochem. 1993; 215: 73– 81.

Ogasawara Y, Isoda S, Tanabe S. Tissue and subcellular distribution of bound and acid-labile sulfur, and the enzymic capacity for sulfide production in the rat. Biol Pharm Bull. 1994; 17: 1535–1542.

Pan J, Carroll KS. Persulfide reactivity in the detection of protein s- sulfhydration. ACS Chem Biol. 2013; 8: 1110.

Paulsen CE, Carroll KS. Cysteine-mediated redox signaling: chemistry, biology, and tools for discovery. Chem Rev. 2013; 113: 4633.

Picton R, Eggo MC, Merrill GA, Langman MJ, Singh S. Mucosal protection against sulphide: importance of the enzyme rhodanese. Gut. 2002; 50: 201–205.

Polhemus DJ, Li Z, Pattillo CB, Gojon GSr, Gojon GJr, Giordano T, Krum H. A novel hydrogen sulfide prodrug, SG1002, promotes hydrogen sulfide and nitric oxide bioavailability in heart failure patients. Cardiovasc Ther. 2015; 33: 216–226.

Porter P, Grishaver MS, Jones OW. Characterization of human cystathionine beta- synthase: Evidence for the identity of human L-serine dehydratase and cystathionine beta-synthase. Biochim Biophys Acta. 1974; 364: 128-139.

Powell CR, Dillon KM, Matso JB. A Review of Hydrogen Sulfide (H2S) Donors: Chemistry

61 Pun PB, Lu J, Kan EM, Moochhala S. Gases in the mitochondria. Mitochondrion. 2010; 10: 83–93. Puranik M, Weeks CL, Lahaye D, Kabil O, Taoka S, Nielsen SB, Groves JT. Dynamics of