Involvement of 3′,5′‐cyclic inosine monophosphate in cystathionine γ‐lyase‐dependent regulation of the vascular tone

Figures

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  SCHEME 1. Reagents and conditions: Butyric anhydride, triethylamine, anhydrous pyridine, rt, 48 h. all reactions were followed by TLC carried out on Merk silica gel 60 F254 plates with a fluorescent indicator on the plates were visualized with UV light (254 nm). Preparative chromatographic purifications were performed using a silica gel column (Kieselgel 60). Solutions were concentrated with a Buchi R‐114 rotary evaporator at low pressure. Melting points, determined using a Buchi melting point B‐540 instrument, are uncorrected and represent values obtained on chromatographically purified material. RP‐HPLC preparative purification was performed on a Shimadzu prominence LC‐20 AP system equipped with a multiwavelength prominence SPD‐20A UV–vis detector on a Phenomenex Kinetex XB‐C18 column (5 mm, 21.2 × 250 mm) employing the following solvents: A: 100% acetonitrile in 0.1% TFA, B: 100% H2O in 0.1% TFA. The operational conditions were as follows: Linear‐gradient of 5%–70% acetonitrile + 0.1% TFA in 30 min, using a flow rate of 30 ml·min‐1. The purity of the products was assessed by analytical RP‐HPLC using a Phenomenex Kinetex XB‐C18 column (5 μm, 4.6 × 250 mm). The column was connected to a…

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  FIGURE 1. l‐cysteine and NaHS cause a further increase in tension of phenylephrine (PE)‐contracted mouse aortic rings. Representative traces of concentration–response curves induced by (a) l‐cysteine (10–300 nM), (b) NaHS (10–300 nM) or (c) vehicle (H2O) on a stable tone of PE (1 μM). The increase in contraction was blunted by the mechanical endothelium removal for both (d) l‐cysteine (n = 10 mice) and (e) NaHS (n = 7 mice). Values shown are means ± SEM and expressed as δ increase in tension over PE contraction (dyne/mg tissue). *P < 0.05 significantly different as indicated; two‐way ANOVA with Bonferroni's post hoc test

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  FIGURE 2. l‐cysteine and NaHS cause a further increase in tension of phenylephrine (PE)‐contracted mouse carotid artery rings. Concentration–response curve of (a) l‐cysteine (10 nM to 300 μM) and (b) NaHS (10 nM to 300 μM) on a stable tone of PE (1 μM). The increase in contraction was blunted by the mechanical endothelium removal for both l‐cysteine (n = 6 mice) and NaHS (n = 6 mice). Values shown are means ± SEM and expressed as δ increase in tension over PE contraction (dyne/mg tissue). *P < 0.05 significantly different as indicated; two‐way ANOVA with Bonferroni's post hoc test

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  FIGURE 3. l‐cysteine‐ and NaHS‐induced increase in tension involves the eNOS/sGC/NO pathway. (a) l‐cysteine‐ and (b) NaHS‐induced contraction on phenylephrine (PE)‐precontracted aortic rings was abolished in aorta harvested from eNOS−/− (n = 7 mice l‐cysteine, n = 6 mice NaHS) and sGCα1 −/− mice (n = 7 mice l‐cysteine, n = 9 mice NaHS) compared to WT mice (n = 7 mice l‐cysteine, n = 9 mice NaHS). (c) l‐cysteine‐induced contraction is abolished in PE‐precontracted aortic rings incubated with l‐NIO (10 μM, 20 min; n = 5 mice) or ODQ (10 μM, 15 min; n = 8 mice) compared to vehicle (n = 8 mice). (d) NaHS‐induced contraction is abolished in PE‐precontracted aortic rings incubated with l‐NIO (10 μM, 20 min; n = 6 mice) or ODQ (10 μM, 15 min; n = 6 mice) compared to vehicle (n = 8 mice). Values shown are means ± SEM and are expressed as δ increase in tension over PE contraction (dyne/mg tissue). *P < 0.05 significantly different as indicated; two‐way ANOVA with Bonferroni's post hoc test

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  FIGURE 4. l‐cysteine‐ and NaHS‐induced increase in tension do not involve endothelin‐1. (a) l‐cysteine and (b) NaHS concentration–response curves are performed on the stable tone induced by phenylephrine (PE) in the presence of selective ET receptor subtype antagonists. Selective inhibition of ETA (mainly expressed on smooth muscle component) by FR 139317 (1 nM, 20 min) does not affect l‐cysteine‐ and NaHS‐induced contraction compared to vehicle (n = 8 mice). Selective inhibition of ETB receptor (expressed mainly on endothelium whose activation induces NO release) by BQ788 (10 μM, 20 min), significantly reduces l‐cysteine‐ and NaHS‐induced contraction compared to vehicle (n = 8 mice). Values shown are means ± SEM and are expressed as δ increase in tension over PE contraction (dyne/mg tissue). *P < 0.05 significantly different compared to vehicle, two‐way ANOVA with Bonferroni's post hoc test

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  FIGURE 5. Aortas of CSE−/− mice displays vascular hyporeactivity to phenylephrine (PE) and an unmodified NO signalling. Representative traces of PE‐induced contraction in aortic rings harvested from (a) WT mice and (b) CSE−/− mice. CSE−/− mice display a reduced level of contraction compared to WT mice. (c) the cumulative concentration–response curve to PE on aorta of CSE−/− mice (n = 10 mice) is significantly reduced compared to WT mice (n = 7 mice). (d) Concentration–response curve to PE performed on aortic rings harvested from WT mice. The incubation with l‐NIO (10 μM, 20 min), the eNOS selective inhibitor, does not affect PE‐induced contraction (n = 7 mice). (e) Concentration–response curve to ‐ performed on aortic rings harvested from CSE−/− mice. The incubation with l‐NIO (10 μM, 20 min), the eNOS selective inhibitor, does not affect PE‐induced contraction (n = 7 mice). Values shown are means ± SEM and are expressed as a contraction (dyne/mg tissue). *P < 0.05 significantly different as indicated; two‐way ANOVA with Bonferroni's post hoc test. (f) Representative western blot analysis (upper) and densitometric analysis (lower) for eNOS expression in aorta of WT and CSE−/− mice (n = 6…

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  FIGURE 6. Aortas of CSE−/− mice display a generalized hyporeactivity to different agonists. Cumulative concentration–response curves performed with (a) 5‐HT (1 nM to 3 μM; n = 5 WT and n = 5 CSE−/− mice), (b) U46619 (1 nM to 0.3 μM; n = 6 WT and n = 6 CSE−/− mice) or (c) KCl (10, 20, 40, 60 mM; n = 9 WT and n = 12 CSE−/−mice) were significantly reduced in CSE−/− mice compared to WT mice. Values shown are means ± SEM and are expressed as contraction (dyne/mg tissue). *P < 0.05 significantly different compared to WT mice; two‐way ANOVA with Bonferroni's post hoc test

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  FIGURE 7. Carotid arteries of CSE−/− mice display a generalized hyporeactivity to different agonists. Cumulative concentration–response curves performed with (a) phenylephrine (PE; 1 nM to 3 μM; n = 7 WT and n = 8 CSE−/− mice), (b) 5‐HT (1 nM to 3 μM; n = 5 WT and n = 9 CSE−/− mice), (c) U46619 (1 nM to 0.3 μM; n = 5 WT and n = 8 CSE−/− mice) were significantly reducted in CSE−/− mice compared to WT mice. Values shown are means ± SEM and are expressed as contraction (dyne·mg‐1 tissue). *P < 0.05 significantly different compared to WT mice; two‐way ANOVA with Bonferroni's post hoc test

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  FIGURE 8. The relative content of cyclic nucleotides in the aorta of CSE−/− mice and WT mice. The cyclic nucleotides levels are detected simultaneously in homogenates of mouse aortas using UPLC‐MS/MS. typical trace analysis of the determination of the cyclic nucleotides in (a) WT and (b) CSE −/− mice. The cyclic nucleotide levels in WT are 261.99 ± 9.36 (cAMP), 9.13 ± 0.89 (cGMP), 19.87 ± 6.43 (inosine 3′,5′‐cyclic monophosphate/cIMP) pg/mg tissue. The cyclic nucleotides levels in CSE−/− mice are 156.38 ± 5.51 (cAMP), 13.31 ± 0.63 (cGMP), 0.18 ± 0.02 (cIMP) pg/mg tissue. (c) the data are expressed as a relative change compared to the levels of cyclic nucleotides detected in WT aorta, taken as 1. Values shown are means ± SEM; n = 5 determinations (three mouse aortas are pulled in each sample). *P < 0.05 significantly different as indicated; one‐way ANOVA with Bonferroni's post hoc test

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  FIGURE 9. NaHS (1 μM) modulates cyclic nucleotides through PDEs activity in WT mice aorta. Following 5 and 15 min of incubation, NaHS induces (a) significant reduction of cGMP content of 18.7% and 20.3%, compared to the respective vehicle that is 18.58 ± 2.43 pg·mg‐1 tissue (n = 9 determinations), (b) significant reduction of cAMP content of 13.9% and 21.7%, compared to the respective vehicle that is 153.87 ± 6.02 pg·mg‐1 tissue (n = 9 determinations) and (c) trend of increase in inosine 3′,5′‐cyclic monophosphate (cIMP) levels of 16.4% and 15.7%, compared to the respective vehicle that is 5.41 ± 1.05 pg·mg‐1 tissue (n = 8 determinations). Values shown are means ± SEM n = 9 determinations or 8 determinations (three mouse aortas are pulled in each sample) and are expressed as the percentage of change compared to the level measured in the vehicle. *P < 0.05 significantly different; one sample t‐test. (d) Upper: Representative western blot for pPDE4A and PDE4A expression in homogenates of aorta incubated with NaHS (1 μM) for 5 or 15 min or vehicle (V). Lower: Densitometric analysis of pPDE4A/PDE4A ratio. The ratio pPDE4A/PDE4A is significantly enhanced following NaHS incubation at 5 min…

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  FIGURE 10. Inosine 3′,5′‐cyclic monophosphate (cIMP) butyrate causes an increase in tension in phenylephrine (PE)‐contracted mouse aortic rings. (a) Chemical structure of cIMP butyrate, (b) representative trace of concentration–response curves induced by cIMP butyrate (0.1 nM to 1 μM) on basal tone, (c) cIMP butyrate (0.1 nM to 1 μM, n = 8 mice) causes an increase in contraction on PE stable tone (n = 10 mice). Values shown are means ± SEM and expressed as δ increase in tension over PE contraction (dyne/mg tissue). *P < 0.05 significantly different as indicated; two‐way ANOVA with Bonferroni's post hoc test