Mercaptopyruvate acts as endogenous vasodilator independently of 3-mercaptopyruvate sulfurtransferase activity

Accepted Manuscript Mercaptopyruvate acts as endogenous vasodilator independently of 3mercaptopyruvate sulfurtransferase activity Emma Mitidieri, Teresa Tramontano, Danila Gurgone, Valentina Citi, Vincenzo Calderone, Vincenzo Brancaleone, Antonia Katsouda, Noriyuki Nagahara, Andreas Papapetropoulos, Giuseppe Cirino, Roberta d’Emmanuele di Villa Bianca, Raffaella Sorrentino PII:

S1089-8603(17)30260-4

DOI:

10.1016/j.niox.2018.02.003

Reference:

YNIOX 1746

To appear in:

Nitric Oxide

Received Date: 29 September 2017 Revised Date:

30 January 2018

Accepted Date: 12 February 2018

Please cite this article as: E. Mitidieri, T. Tramontano, D. Gurgone, V. Citi, V. Calderone, V. Brancaleone, A. Katsouda, N. Nagahara, A. Papapetropoulos, G. Cirino, R. d’Emmanuele di Villa Bianca, R. Sorrentino, Mercaptopyruvate acts as endogenous vasodilator independently of 3mercaptopyruvate sulfurtransferase activity, Nitric Oxide (2018), doi: 10.1016/j.niox.2018.02.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Mercaptopyruvate

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3-mercaptopyruvate sulfurtransferase knockout mice

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vasorelaxation

ACCEPTED MANUSCRIPT Mercaptopyruvate acts as endogenous vasodilator independently of 3-mercaptopyruvate sulfurtransferase activity

Emma Mitidieria, Teresa Tramontanoa, Danila Gurgonea, Valentina Citib, Vincenzo

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Calderoneb, Vincenzo Brancaleonec, Antonia Katsoudae, Noriyuki Nagaharad, Andreas Papapetropoulose, Giuseppe Cirinoa, Roberta d’Emmanuele di Villa Biancaa,1,#, Raffaella

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Sorrentinoa,1,#

Department of Pharmacy, School of Medicine, University of Naples, Federico II, Naples,

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Italy

Department of Pharmacy, University of Pisa, Pisa, Italy

Department of Science, University of Basilicata, Potenza, Italy

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Isotope Research Center, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo

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Faculty of Pharmacy, University of Athens, Athens, Greece

These authors have equally contributed

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Corresponding authors:

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#

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Raffaella Sorrentino, Department of Pharmacy, School of Medicine, Via D. Montesano, 49, 80131 Naples, Italy. Tel: +39 081 678437, Fax: +39081678107, e-mail address: [email protected].

Roberta d’Emmanuele di Villa Bianca, Department of Pharmacy, School of Medicine, Via D. Montesano, 49, 80131 Naples, Italy. Tel: +39 081 678457, Fax: +39 081 67107 e-mail address: [email protected].

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ACCEPTED MANUSCRIPT Non standard abbreviations: 3-MST: 3-mercaptopyruvate sulfurtransferase CAT: cysteine aspartate aminotransferase CBS:cystathionine-β-synthase

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CSE: cystathionine-γ-lyase DADS: diallyl disulfide DATS: diallyl trisulfide

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eNOS: endothelial nitric oxide syntase

H2S: Hydrogen sulfide

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GYY4137: morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate

L-NIO: N5-(1-Iminoethyl)-L-ornithine dihydrochloride MPT: sodium mercaptopyruvate dehydrate Na2S: sodium sulfide

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NaHS: sodium hydrogen sulfide

ODQ: 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one PE: phenylephrine

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SAC: S-ally cysteine

SF7-AM: Sulfidefluor-7 acetoxymethyl

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sGC: soluble guanylyl cyclase

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ACCEPTED MANUSCRIPT Abstract Hydrogen sulfide (H2S) is produced by the action of cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) or 3-mercaptopyruvate sulfurtransferase (3-MST). 3-MST converts 3-mercaptopyruvate (MPT) to H2S and pyruvate. H2S is recognized as an

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endogenous gaseous mediator with multiple regulatory roles in mammalian cells and organisms. In the present study we demonstrate that MPT, the endogenous substrate of 3MST, acts also as endogenous H2S donor. Colorimetric, amperometric and fluorescence based

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assays demonstrated that MPT releases H2S in vitro in an enzyme-independent manner. A functional study was performed on aortic rings harvested from C57BL/6 (WT) or 3-MST-

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knockout (3-MST-/-) mice with and without endothelium. MPT relaxed mouse aortic rings in endothelium-independent manner and at the same extent in both WT and 3-MST-/- mice. N5(1-Iminoethyl)-L-ornithine dihydrochloride (L-NIO, an inhibitor of endothelial nitric oxide synthase) as well as 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ, a soluble guanylyl

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cyclase inhibitor) did not affect MPT relaxant action. Conversely, hemoglobin (as H2S scavenger), as well as glybenclamide (an ATP-dependent potassium channel blocker) markedly reduced MPT-induced relaxation. The functional data clearly confirmed a non

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enzymatic vascular effect of MPT. In conclusion, MPT acts also as an endogenous H2S donor and not only as 3-MST substrate. MPT could, thus, be further investigated as a means to

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increase H2S in conditions where H2S bioavailability is reduced such as hypertension, coronary artery disease, diabetes or urogenital tract disease.

Keywords: Mercaptopyruvate, hydrogen sulfide, vasorelaxation, aorta, 3-mercaptopyruvate sulfurtransferase knockout mice.

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ACCEPTED MANUSCRIPT 1. Introduction Hydrogen sulfide (H2S) is a colorless, flammable and water-soluble gas [1,2]. Although it was known for its toxic properties, it is now recognized as an endogenous gaseous transmitter with a regulatory role in many physiological and pathological processes [3-6]. In mammalian cells,

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endogenous H2S is produced from desulfhydration of cysteine catalyzed by two pyridoxal-5phosphate-dependent enzymes, cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) [7,8]. The third pathway for H2S generation requires two enzymes, cysteine aspartate

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aminotransferase (CAT) and 3-mercaptopyruvate sulfurtransferase (3-MST). CAT catalyzes the transamination reaction between L-cysteine and α-ketoglutarate to produce 3-

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mercaptopyruvate (MPT) and L-glutamate, whereas 3-MST converts MPT to pyruvate and H2S [8].

The vascular effects of H2S have been recently reviewed [9]. Most of the papers demonstrate that the primary action of H2S in vasculature is vasodilatory, but it is important to point out

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that there is heterogeneity in the vascular responses to H2S depending on the species and experimental models used. Moreover, the effects of H2S are concentration and vascular beddependent. In particular, in isolated vessels such as rat gastric artery, aorta and mesenteric

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vascular bed or human internal mammary artery as well as rabbit arteries, a biphasic effect of H2S has been demonstrated i.e. a contracting effect at lower concentration and a vasodilating

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effect at higher concentration [10-15]. In general, CSE is recognized as the major H2S producing enzyme in cardiovascular system. More recently, the existence of the 3-MST/H2S pathway in vasculature was demonstrated. Both 3-MST and CAT are localized in vascular endothelium [16] and smooth muscle cells [1]. The 3-MST/H2S pathway has been shown to have an important role in the regulation of vasculature function, causing relaxation, and in angiogenesis [17]. During the course of their studies, Coletta and co-workers noted a nonenzymatic release of H2S by MPT in bEnd3 endothelial cells. This effect was evident in a

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ACCEPTED MANUSCRIPT 100-300 micromolar range while enzymatically produced H2S was shown to occur at the 13mM range [17]. Current knowledge suggests that restoration of H2S levels could have therapeutic value in cardiovascular diseases [5,18]. Several studies reported a protective role for H2S donors in

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different disease models [19]. According to their chemical structure and source, H2S donors are divided into different classes including inorganic salts, phosphorodithioate derivates, thioaminoacids, aminothiols, and natural organosulfur compounds from Alliaceae or

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Brassicaceae [2, 20]. The most widely used inorganic compounds to generate H2S are sodium hydrogen sulfide (NaHS) and sodium sulfide (Na2S). These compounds rapidly dissociate in

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solution leading to immediate H2S formation. The uncontrolled and rapid release of H2S makes accurate control of its concentration difficult to achieve. The organic donors can be divided, according to their source of origin, into naturally occurring and synthetic. Sulfurcontaining compounds from Allium species such as diallyl disulfide (DADS), diallyltrisulfide

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(DATS) and S-allyl cysteine (SAC) act as H2S donors [21]. Similarly, the H2S-release from natural isothiocyanates accounts for several biological effects of plants belonging to the Brassicaceae botanical family [22]. In addition, S-propargyl cysteine (ZYZ-802) and S-

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propyl cysteine, two synthetic cysteine analogues, have been proposed for the treatment of ischemic heart disease via modulation of the H2S pathway [23]. Similarly, to previously

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reported H2S donors, morpholin-4-ium 4-methoxyphenyl (morpholino) phosphinodithioate (GYY4137) is a water-soluble molecule, which releases H2S upon hydrolysis. More recently, the H2S release from synthetic isothiocyanates has also been reported to account for the vasorelaxing and the cardio-protective activities of these compounds [24,25]. The H2S releasing capability of each donor category is quite different; such variable profiles may lead to significant differences in the biological and pharmacological effects of each donor. Indeed, contrary to inorganic salts that release H2S in a narrow time frame in large

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ACCEPTED MANUSCRIPT amounts, organic donors release H2S more slowly. The rate of H2S release among organic donors shows substantial differences. Another major difference for donors is that they release H2S either spontaneously or they require triggers such as ROS, low pH or cysteine. In addition, different physicochemical features (i.e. stability in aqueous solution) and

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pharmacokinetics (i.e. bioavailability and distribution) deeply influence the pharmacological behavior and the therapeutic indication of each H2S-releasing compound [20]. Thus, the search for further classes of H2S-releasing compounds is still an attractive area in drug

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discovery.

Here, we have addressed the contribution of the 3-MST/H2S pathway in mouse aorta and we

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with direct vasodilatory effects.

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have demonstrated that MPT, an endogenous occurring substrate, can act also as a H2S donor

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ACCEPTED MANUSCRIPT 2. Materials and methods 2.1. H2S measurement 2.1.1. H2S release in cell-free assay: colorimetric assay H2S determination in solution was performed by using the methylene blue based assay

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[26,27]. Briefly, MPT (code 90374; Sigma-Aldrich, Milan, Italy) was dissolved in a potassium phosphate buffer (PPB), 100 mM pH 7.4. A concentration-response curve (3 µM10 mM) was performed in eppendorf tubes in a total volume of 500 µl. Zinc acetate (ZnAc,

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1%, 250 µl, Sigma-Aldrich, Milan, Italy) was added to trap evolved H2S followed by trichloroacetic acid (TCA, 10 %, 250 µl Sigma-Aldrich, Milan, Italy). Subsequently, N,N-

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dimethyl-p-phenylenediamine sulfate (DPD, 20 µM, 133 µl, Sigma-Aldrich, Milan, Italy) in 7.2 M HCl and FeCl3 (30 µM, 133 µl) in 1.2 M HCl were added. After 20 minutes all samples were assayed in duplicate and H2S release was evaluated through absorbance values measured at a wavelength of 668 nm. A calibration curve of NaHS (3.12-250 µM, Sigma-Aldrich,

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Milan, Italy) was performed as positive control.

2.1.2. H2S release in cell-free assay: amperometric assay The amperometric assay was performed in a cell-free assay by using an Apollo-4000 Free

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Radical Analyzer detector (World Precision Instruments, Sarasota, FL, USA) and 2 mm H2Sselective mini electrodes [28]. Assay buffer was prepared according to the manufacturer's

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instructions. The electrode was equilibrated in 10 mL of assay buffer until a stable baseline was achieved. Then, 100 µL of an aqueous solution of MPT was added (final concentration 1 mM). The H2S generation was monitored for 20 min. When required by the experimental design, L-cysteine (4 mM, Sigma-Aldrich, Milan, Italy) was added 10 min before MPT. An aqueous solution containing L-cysteine (4 mM) alone, was used as control. H2S concentration was determined by opportune calibration curves, previously obtained plotting amperometric

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ACCEPTED MANUSCRIPT currents (recorded in nA) vs. increasing concentrations of NaHS (1, 3, 5, and 10 µM) at pH 4.0. 2.1.3. H2S release in cell-free assay: fluorescence assay H2S release by MPT was also tested in a fluorescence-based assay by using the fluorescent

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probe sulfidefluor-7-acetoxymethyl ester (SF7-AM, Sigma-Aldrich, Milan, Italy) [29]. The SF7-AM was dissolved in dimethylsulfoxide (DMSO) and used at final concentration of 10µM. MPT solution was prepared in PPB pH 7.4 within the range 3µM-10mM. SF7-AM

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was added to MPT solution and the mixture was incubated in black 96-well plate in the dark at 37°C. Fluorescence was measured after 90 minutes of incubation in order to achieve

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plateau in response (ex 475-em 500-550). The concentration of H2S released were determined through a calibration curve of Na2S (50nM-200µM) dissolved in PPB pH 7.4.

2.2. Animals

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All animal care and experimental procedures in this study followed specific guidelines of the Italian and the European Council law. These procedures were also approved by the Animal Ethics Committee of the University of Naples “Federico II” (Italy). Male C57BL/6 mice (WT

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mice), (Charles River, Italy, 22–25 g) and 3-MST-ablated mice (3-MST-/-) [30] were used. Mice were kept at temperatures of 23 ± 2°C, humidity range 40–70% and 12 h light/dark

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cycles. Food and water were provided ad libitum. Mice were euthanized by cervical dislocation prior sedation.

2.3. Isolated organ bath studies The aorta was rapidly harvested, and adherent connective and fat tissue were removed. Rings of 1–1.5 mm length were cut and placed in organ baths (3.0 mL) filled with oxygenated (95% O2–5% CO2) Krebs solution (NaCl 118 mM, KCl 4.7 mM, MgCl2 1.2 mM, KH2PO4 1.2 mM,

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ACCEPTED MANUSCRIPT CaCl2 2.5 mM, NaHCO3 25 mM and glucose 10.1 mM, Carlo Erba, Milan, Italy) and kept at 37°C. The rings were connected to an isometric transducer (7006, Ugo Basile, Comerio, Italy) and changes in tension were continuously recorded with a computerized system (DataCapsule-17400, Ugo Basile, Comerio, Italy). The rings were initially stretched until a

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resting tension of 1.5 g was reached and then were equilibrated for at least 30 min; during this period the tension was adjusted, when necessary, to 1.5 g and the bath solution was periodically changed [31]. In each set of experiments, rings were firstly challenged with

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phenylephrine (PE, 1 µM, Sigma-Aldrich, Milan, Italy) until the responses were reproducible. In order to verify the integrity of the endothelium, acetylcholine (Ach, Sigma-Aldrich, Milan,

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Italy) cumulative concentration-response curve (10 nM-30 µM) was performed on PE precontracted rings [32]. Moreover, in order to assess the contribution of endothelium, in another setting of experiments, it was mechanically destroyed and the absence of the endothelial activity was proved by adding Ach in pre-contracted ring. Thereafter, tissues were then

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washed and contracted with PE (1 µM) and, once plateau was reached, a cumulative concentration-response curve of MPT (0.1 µM-1 mM, Sigma-Aldrich, Milan, Italy) was performed in presence or in absence of endothelial activity. In order to investigate the

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mechanism of MPT-induced effect, we operated a pharmacological modulation. To investigate the endothelial nitric oxide synthase (eNOS) involvement in MPT relaxation, aorta were

incubated

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rings

for

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minutes

with

either

N5-(1-Iminoethyl)-L-ornithine

dihydrochloride (L-NIO, 10 µM, Tocris, UK), an inhibitor of eNOS, or with 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 µM, Tocris, UK), a soluble guanylyl cyclase (sGC) inhibitor. A cumulative concentration-response curve of MPT (0.1 µM-1 mM) was performed. In a separate set of experiments, in order to highlight the exclusive H2S contribution, we tested the effect of MPT (0.1 µM-1 mM) in presence of a combination of hemoglobin (Hb, 30 µM Sigma-Aldrich, Milan, Italy), as scavenger of both H2S and NO, plus

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ACCEPTED MANUSCRIPT L-NIO compared to L-NIO alone. Glybenclamide (10µM, Sigma-Aldrich, Milan, Italy), a selective ATP-dependent potassium (KATP) channel blocker, in absence of endothelium was used, too. Moreover, to further investigate the effect of MPT, a cumulative concentrationresponse curve (0.1 µM - 1 mM) was also operated in 3-MST-/- mice in presence or in absence

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of endothelium.

2.4. Western Blot analysis

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Aorta harvested from WT and 3-MST-/- mice were lysed in modified RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1

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mM EDTA, 1% Igepal) (Roche Applied Science, Italy) and protease inhibitor cocktail (Sigma-Aldrich, Milan, Italy). Protein samples (40 µg) were resolved by 8% SDS-PAGE and analyzed as previously describe [33,34]. The membranes were incubated with rabbit polyclonal anti-3-MST (1:500, Novus Biologicas, Cambridge, UK). Membranes were

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extensively washed in PBS containing 0.1% v/v Tween-20 prior to incubation with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Following incubation, membranes were washed and developed using ImageQuant-400 (GE

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Healthcare, USA). The target protein band intensity was normalized over the intensity of the

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housekeeping β-actin (1:5000, Sigma-Aldrich, Milan, Italy).

2.5 H2S determination in aorta tissue Aortas were homogenized in ice-cold 100 mM potassium phosphate buffer, pH = 7,4 supplemented with 1% protease and phosphatase inhibitor cocktail (Calbiochem-Novagen; Lab Supplies, Athens, Greece). Protein amount was quantified using the Bradford assay and concentration was normalized before methylene blue analysis. All reagents for the colorimetric assay were from Sigma-Aldrich, Life-Science Chemilab, Attica, Greece. Samples

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ACCEPTED MANUSCRIPT were prepared in parafilm-sealed eppendorf tubes containing tissue homogenates and MPT (1mM). After 30 min of incubation at 37◦C in a shaking water bath the reaction was terminated by adding 1% zinc acetate to trap H2S, followed by 10% trichloroacetic acid to precipitate proteins. Subsequently, N,N-dimethyl-p-phenylenediamine-sulfate in 7.2 M HCl

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was added followed by FeCl3 in 1.2 M HCl. Absorbance was measured at 650 nm and H2S content was calculated against a calibration curve of Na2S (0–250 µM) [35]. Results were

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expressed as concentration of H2S (µM) formed per µg of protein.

2.5 Statistical analysis

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The data were analyzed statistically with GraphPad Prism software. Results were analyzed by using one way ANOVA or two way ANOVA following by Bonferroni as post test, as needed.

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p<0.05 was considered significant.

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ACCEPTED MANUSCRIPT 3. Results 3.1. MPT releases H2S To assess whether MPT can act as an H2S donor, we evaluated the ability of MPT to release H2S in solution by using three different methods. The results obtained with a colorimetric

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assay are reported in figure 1A. MPT (3 µM-10 mM) releases H2S in a concentrationdependent manner (Fig. 1A; ***P<0.001versus all concentrations;

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P<0.01 versus 30 µM

and 100 µM;°P<0.05 versus 30 µM). In order to confirm this data, we carried out an

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amperometric technique to measure H2S. In this experimental set up, the addition of MPT (1 mM) into the assay buffer at pH 7.4, led to a slow and significant (Fig. 1B; ***P<0.001

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versus L-Cys) generation of H2S, which reached a concentration of approximately 2 µM (after 20 min of incubation). The presence of L-cysteine (4 mM) significantly (***P<0.001 versus L-Cys) increased the H2S-releasing activity of MPT, leading to the generation of higher amounts of H2S (Fig. 1B). On the other hand the administration of L-cysteine (4mM) alone

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into the buffer did not cause any generation of H2S (Fig 1B). In addition, to further confirm our hypothesis we also used a fluorescence method based on the sulfide selective detector SF7-AM. In this setting, MPT was again found to release H2S in a concentration-dependent

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manner, with a maximum of ̴ 11µM H2S corresponding to 10mM MPT (Fig. 1C; **P<0.01

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and ***P<0.001 versus 3µM).

3.2. MPT relaxes aortic rings in WT mice MPT (0.1 µM-1 mM) relaxed mouse aortic rings in a concentration-dependent and endothelium-independent manner (Fig. 2A). In order to test the contribution of NO to the 3MPT effect, we incubated intact aortic rings with either L-NIO (10 µM), a selective inhibitor of eNOS, or ODQ (10 µM), a selective inhibitor of NO-sensitive guanylyl cyclase. Neither LNIO, nor ODQ, affected MPT induced relaxation (Fig. 2B and 2C, respectively). Incubation

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ACCEPTED MANUSCRIPT of aortic rings with a combination of Hb (30 µM) plus L-NIO significantly (***P<0.001) reduced MPT relaxation (Fig. 3A). In addition, in denuded aortic tissue, the MPT relaxant effect was significantly decreased by glybenclamide (10 µM), a KATP channel blocker

3.3. MPT relaxes mouse aortic rings in 3-MST-/- mice

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(***P<0.001, Fig. 3B).

We next used 3-MST-/- mice to test whether MPT could still elicit relaxation acting as a H2S

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donor rather than a substrate. Western blot analysis confirmed the complete absence of 3MST in aortic homogenates of 3-MST-/- mice (Fig. 4A). In the organ bath studies, we

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observed that there were no differences in the MPT-stimulated vasorelaxation effect induced by MPT (0.1 µM-1 mM) in aortic rings harvested from WT and 3-MST-/- mice either in presence (Fig. 4B) or in absence of endothelium (Fig. 4C). In addition, as also seen in WT mice, MPT-induced relaxation (0.1 µM-1 mM) in 3-MST-/- mice is not affected by

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endothelium removal.

3.4. MPT-induced H2S production in aortic homogenates of 3-MST-/- and WT mice

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As expected, the addition of MPT (1mM) in aortic homogenates of WT mice significantly (Fig. 5 ***p<0.001) increased H2S production compared to basal (vehicle) condition. MPT

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(1mM) still caused a significant increase in H2S production in aortic homogenates of 3-MST-/mice compared to basal (vehicle) condition (***p<0.001). However, the amount of H2S produced was significantly lower in 3-MST-/- compared to WT (Fig. 5 °°p<0.01).

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ACCEPTED MANUSCRIPT 4. Discussion 3-MST is the only H2S-generating enzyme that is not directly dependent on pyridoxal phosphate [16,36]. However, the formation of H2S from 3-MST depends on tandem reactions involving CAT and 3-MST. CAT is a transaminase and as such depends on pyridoxal

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phosphate for its activity. The MPT formed is then used as a substrate by 3-MST which transfers a sulfur atom from MPT to cysteine 247 in the active site of 3-MST. H2S is then released from 3-MST persulfide by reducing agents or enzymatically by thioredoxin [37-40].

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Even though 3-MST is widely distributed in various cells and tissues, studies on its potential pathophysiological role have only recently emerged. Highest expression/activity of 3-MST is

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encountered in the kidney, liver, cardiac and neuroglial cells. Within the cell, 3-MST is located in both the cytoplasm and in the mitochondria. However, because the concentration of cysteine is much higher in the mitochondria, as compared to the cytoplasm, it is plausible that most of the H2S generated by 3-MST occurs in mitochondria [41,42]. Exogenous addition of

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MPT to cells elicits a biphasic effect: at low concentrations it enhances mitochondrial electron transport and stimulates cellular bioenergetics while at high concentrations it inhibits cellular bioenergetics [43]. Moreover, it has been suggested that 3-MST is unable to produce H2S in

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normal physiological conditions as it exhibits activity at higher alkaline pH level [44]. Based on the above, the effects of exogenously added MPT will depend on the microenvironment, as

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well as the concentration used and the tissues/organs examined. Indeed, similarly to the case of authentic H2S, the vasoactive effects of MPT varies among vascular beds. In the rat mesenteric bed, MPT is a vasodilator while in mice coronary artery it promotes contraction [17,45]. This pathway also plays a role in angiogenesis and its impairment may contribute to the pathogenesis of hyperglycemia and endothelial dysfunction [17]. Changes in H2S signaling have been associated with several others pathologies such as pulmonary hypertension [46], ischemia and reperfusion [47], diabetes [48] and urogenital

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ACCEPTED MANUSCRIPT tract disease [49-51]. Thus, the discovery or development of molecules able to release H2S with well-controlled release rates is an important point to consider when attempting to restore reduced H2S levels in several disorders. In this study, we have demonstrated that MPT can also release H2S independently of 3-MST enzymatic activity. Although, a non-enzymatic

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release of H2S has been already suggested [17], the scientific literature refers to MPT exclusively as a substrate of 3-MST neglecting non-enzymatic generation of H2S by MPT. Herein, we thoroughly investigated this hypothesis using different approaches. Initially, we

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used three different cell-free assays to determine the ability of MPT to act as a H2S donor. The colorimetric assay showed that MPT released H2S in a concentration-dependent manner

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in an enzyme-free buffer. Similarly, the H2S-selective amperometric assay confirmed these results highlighting the accumulation of H2S at a slow and constant rate. The release of H2S from MPT was further increased by adding a nucleophile, i.e L-cysteine, as already observed for other well-established H2S-donors including GYY4137, thioamide or isothiocyanate

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derivatives [20]. This observation might have an important impact in vivo where MPT would react with L-cysteine (abundant within the body) or other nucleophiles thus amplifying or prolonging H2S release. In addition, our findings were further confirmed using a fluorescent

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probe that is highly selective for H2S, namely SF7-AM [29]. Once again we observed that MPT releases H2S in a concentration-dependent manner. However, we cannot exclude the

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hydrogen polysulfides release from MPT as it has been reported by Kimura and co-workers [52]. We next moved to a functional study employing mouse aortic rings to evaluate whether the biological effects of MPT involve 3-MST or occur independently of its enzymatic activity. We found that MPT relaxed aortic rings in vitro, in an endothelium- and eNOS/NO independent manner. This latter conclusion was based on experiments using pharmacological inhibitors; when rings with the endothelium were incubated with either the eNOS or the sGC inhibitor, the ability of MPT to relax preconstricted arteries was unaffected ruling out the

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ACCEPTED MANUSCRIPT involvement of the eNOS/NO pathway. To further characterize the vasodilatory effect of MPT we used Hb to bind and remove H2S. Since Hb is also a scavenger of NO we performed the experiments in presence of the combination Hb plus L-NIO. Under these conditions, we observed an impairment in the relaxation response to MPT. It is known that some H2S donors

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exert their actions in a NO-dependent manner, while others work independently of eNOS [52]. Based on our findings MPT acts as in a NO-independent manner, similarly to AP-39 and GYY-4137. Since it is well established that the actions of H2S on vascular smooth muscle

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involves opening of ATP-sensitive K+ channel, we tested also the effect of glybenclamide, a selective blocker of these channels [54,55]. Glybenclamide significantly reduced MPT-

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induced relaxation. In order to provide direct evidence that MPT exerts its effects through non-enzymatic H2S production, we performed identical functional studies using aortic rings harvested from 3-MST-/- mice. In these experiments, MPT caused a concentration-dependent relaxation in vascular tissue lacking 3-MST that, overlapped with that seen in WT mice. Thus,

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the functional and the biochemical data taken together indicate that exogenously supplied MPT acts as H2S donor, rather than a 3-MST substrate causing relaxation in the experimental conditions used. To confirm that the 3-MST enzyme in aorta is able to convert MPT to H2S

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we used aortic tissue homogenates from WT and 3-MST-/- mice. As expected the addition of the substrate (MPT 1mM) causes a significant increase of H2S generation in WT aorta mice

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homogenates. In homogenates from 3-MST-/- mice we still observed a significant increase in H2S production compared to basal condition that reached approximately 80% of that seen in WT mice. These results support the notion that non-enzymatic release of H2S from MPT accounts for the majority of H2S released by MPT, compared to enzymatically-generated H2S mediated by 3-MST. In line with our results, a feeble increase in H2S generation followed MPT (500µM) has been observed also in MST-/- mice whole brain cells [45]. This weak increase in MPT-derived H2S may be explained by the different concentration and tissue used

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ACCEPTED MANUSCRIPT containing different types and amounts of nucleophiles. However, Kuo et al., provided no functional in vitro or in vivo evidence to demonstrate the biological importance of nonenzymatically generated H2S by MPT as was done in our study. Enzymatically-derived H2S from MPT/3-MST might have a more pronounced role in the

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microcirculation especially under pathological conditions as reported by Coletta and coworkers [17]. Alternative explanations exist: tissues might contain saturating concentrations of MPT as a 3-MST substrate or exogenous supplementation might not increase appreciably

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function as a donor would relax blood vessels.

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intracellular MPT concentrations. Still H2S released from MPT extracellularly through it

5. Conclusions

Based on the data presented herein, MPT might represent a suitable H2S therapeutics candidate. It could be considered in the future for clinical studies, given the potential

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therapeutic value of H2S in several conditions such as cardiovascular disease, diabetes, inflammation and oxidative stress status, and down regulation of cellular metabolism under stress [18,56,57]. Use of endogenously existing molecules as drug candidates has a theoretical

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advantage since these molecules should not have significant side effects at least at doses close to their endogenous levels. The possibility to deliver H2S to tissues and cells has a theoretical

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advantage over delivering NO. Although H2S shares several features with NO at vascular level unlike NO, H2S donors, when given intravenously to human volunteers, do not cause appreciable changes in blood pressure and hemodynamic parameters [58 Thus, H2S donors would be preferred in subjects where no reduction in blood pressure is desired to exert this beneficial effects.

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ACCEPTED MANUSCRIPT Acknowledgments We thank the veterinarian Dr Antonio Baiano, and Giovanni Esposito and Angelo Russo for animal care assistance.

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Founding sources

2015ZT9HXY_006.

Conflict of interest

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The authors declare no conflict of interest.

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This work was supported by Ministero della Università e della Ricerca (MIUR) PRIN

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ACCEPTED MANUSCRIPT Figure 1. H2S measurement (A) MPT (3 µM-10 mM) released H2S in concentration-dependent manner (***P<0.001 ##

versus all concentrations;

P<0.01 versus 30µM and 100µM;°P<0.05 versus 30µM) in

aqueous solution measured by a methylene blue assay and (B) MPT (1 mM) slowly released

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H2S in absence (***P<0.001 versus L-Cys) or in presence of an excess of L-cysteine (4 mM) (***P<0.001 versus L-Cys) measured by amperometric assay. (C) MPT released H2S, measured by SF7-AM-based fluorescence assay, in a concentration-dependent manner (**

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P<0.01 and ***P<0.001 versus 3µM). Data were expressed as mean ± SE (8, 6 and 4,

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Figure 2. MPT effect on aortic rings harvested from WT mice

(A) MPT (0.1µM-1mM) relaxed in an endothelium independent manner aortic rings. (B) LNIO (10µM) and (C) ODQ (10µM) did not affect the relaxation induced by MPT in presence

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of endothelium. Data were reported as % of relaxation and expressed as mean ± SE (n=5)

Figure 3. MPT effect on aortic rings harvested from WT mice

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(A) Hb (30µM µM) plus L-NIO (10 µM) significantly (***P<0.001) reduced the relaxation induced by MPT (0.1µM-1mM) in presence of L-NIO alone. The relaxant concentration

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response curve induced by MPT was not affected by L-NIO (B) Glybenclamide (10µM) significantly (***P<0.001) reduced the relaxation induced by MPT in absence of endothelium. Data were reported as % of relaxation and expressed as mean ± SE (n = 5).

Figure 4. MPT effect on aortic rings harvested from MST-/- mice (A) Representative western blot for 3-MST in aortic homogenates in 3-MST-/- and in WT mice. MPT relaxing effect was not statistically different between WT and MST-/- mice either

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Figure 5. H2S production in aortic homogenates of MST-/- and WT mice

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The addition of MPT (1mM) in aortic homogenates of WT or MST-/- mice caused a significant increase in H2S production (***P<0.001) compared to basal condition (vehicle). The MPT-induced increase in H2S production was significantly higher in WT compared to

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MST-/- (°°P<0.01). Data were reported as µM/µg protein and expressed as mean ± SE (n = 6).

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ACCEPTED MANUSCRIPT 3-mercaptopyruvate is able to release H2S in solution 3-mercaptopyruvate relaxes mouse aortic rings in endothelium-independent manner 3-mercaptopyruvate relaxation is not modified by eNOS or guanylyl cyclase inhibition 3-mercaptopyruvate relaxation is reduced by hemoglobin or glybenclamide

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3-mercaptopyruvate relaxes aortic rings of WT or 3-MST-/- mice in the same extent