GYY4137 and Sodium Hydrogen Sulfide Relaxations Are Inhibited by L-Cysteine and KV7 Channel Blockers in Rat Small Mesenteric Arteries

Figures

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  FIGURE 1. Role of H2S for GYY4137 relaxations small mesenteric arteries. (A) Sulfide release from GYY4137 in the absence and the presence of small mesenteric arteries (B) Original recordings in small mesenteric arteries showing contraction to 3 µM U46619 and addition of increasing concentrations of GYY4137 (1–500 µM) and the hydrolyzed form of GYY4137H (1–500 µM) (C) Average relaxations induced by the parent compound and the hydrolyzed form of GYY4137. (D) Effect of the substrate for H2S synthesis, L-cysteine, on vascular tone in small mesenteric arteries. The arteries were incubated with an inhibitors of H2S biosynthesis using the cystathionine-gamma-lyase (CSE) inhibitor, PPG (1 mM), and the cystathionine-beta-synthase (CBS)/cystathionine-gamma-lyase (CSE) inhibitor aminooxyacetic acid (AOAA, 1 mM). L-cysteine was added in increasing concentrations (1 µM-3 mM). Data are means ± SEM (n = 6).

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  FIGURE 2. Effect of L-cysteine on Na2S and GYY4137 relaxations in rat small mesenteric arteries. Mixture of Na2S (300 µM) or GYY4137 (1 mM) with and without L-cysteine (1 mM) was added to air-tight bottles and relaxations measured to time = 0 and 10 h for (A) Na2S and (B) GYY4137, and the amount of sulfides measured at 10 h for (C) Na2S and GYY4137 in the absence, and for (D) Na2S and GYY4137 in the presence of L-cysteine, where the concentration of sulfides generated by L-cysteine were subtracted. Please note the difference in scale comparing data in (C and D). Data are means ± SEM (n = 6). *p < 0.05, Students t-test.

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  FIGURE 3. Effect of L-cysteine and L-NAME incubation on Na2S and GYY4137 relaxations in rat small mesenteric arteries. Original traces showing the effect of increasing concentrations of Na2S in U46619-contracted preparations in (A) control conditions (B) in the presence of L-cysteine (1 mM), and (C) in the presence of L-cysteine and the nitric oxide synthase inhibitor, L-NAME (100 µM). Original traces showing the effect of increasing concentrations of GYY4137 in U46619-contracted preparations in (D) control conditions (E) in the presence of L-cysteine (1 mM), and (F) in the presence of L-cysteine and the nitric oxide synthase inhibitor, L-NAME (100 µM). The horizontal bar indicates time in min and the vertical bar increase in force in mN (G) Average effect of L-cysteine without and with L-NAME on Na2S-induced vascular relaxation (H) Average effect of L-cysteine without and with L-NAME on GYY4137-induced vascular relaxation. Data are means ± SEM (n = 6). *p < 0.05, two-way ANOVA.

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  FIGURE 4. Effect of the cystathionine γ-lyase (CSE) inhibitor, PPG, and DTT on Na2S and GYY4137 relaxations in rat mesenteric arteries (A) Effect of PPG (1 mM) on Na2S induced vascular relaxation (B) effect of PPG on GYY4137 induced vascular relaxation (C) effect of DTT (1 mM) on Na2S induced vascular relaxation (D) effect of DTT on GYY4137 induced vascular relaxation. Where no error bars are indicated, error lies within dimensions of the symbol. *p < 0.05, two-way ANOVA. All data is represented as mean ± SEM (n = 5–6).

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  FIGURE 5. Role of the endothelium and NO for Na2S and GYY4137 relaxation (A) Average relaxations induced by acetylcholine (ACh) in the absence (n = 7) and the presence of either L-NAME (100 µM) (n = 6) or L-cysteine (1 mM) (n = 7) in rat mesenteric arteries (B) Na2S induced vascular relaxation in vessels with (n = 11) and without endothelium (n = 5), or with endothelium in the presence of an NO synthase inhibitor, L-NAME (n = 6) (C) GYY4137 induced vascular relaxation in vessels with (n = 12) and without endothelium (n = 6) or with endothelium in the presence L-NAME (n = 6). Where no error bars are indicated, error lies within dimensions of symbol. All data are means ± SEM. *p < 0.05, two-way ANOVA.

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  FIGURE 6. Effect of Na2S and GYY4137 on relaxations induced by the NO donor, sodium nitroprusside (SNP) in small mesenteric arteries. The arteries were incubated with and inhibitor of NO synthase, L-NAME (10 µM), and Na2S (300 µM) (n = 6), or GYY4137 (1 mM) (n = 5), contracted with U46619 (0.3 µM) and concentration-response curves constructed for SNP. *p < 0.05, two-way ANOVA. All data are means ± SEM.

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  FIGURE 7. Effect of high extracellular potassium (KPSS) on Na2S and GYY4137 relaxation. Original traces showing contractions induced by 3 µM U46619 or 60 mM KPSS and effect of increasing concentrations of (A,B) Na2S (1–1,000 µM) or (C,D) GYY4137 (1–1,000 µM). (E) Average Na2S induced vascular relaxations in vessels contracted with U46619 or KPSS; (F) Average GYY4137 induced vascular relaxations in vessels contracted with U46619 or KPSS. Where no error bars are indicated, error lies within dimensions of symbol. All data are means ± SEM (n = 5–10). *p < 0.05, two-way ANOVA.

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  FIGURE 8. Effect of K channels blockers on Na2S and GYY4137 relaxation. (A) Effect of inhibition of KATP channels by glibenclamide (1 µM) on Na2S-induced vascular relaxation; (B) effect of inhibition of KATP channels by glibenclamide on GYY4137-induced vascular relaxation; (C) effect of selective blockade of BKCa channels by iberiotoxin (IBTX, 100 nM) on Na2S-induced vascular relaxation; (D) effect of selective blockade of BKCa channels by IBTX on GYY 4137-induced vascular relaxation; (E) effect of selective blockade of Kv7 channels by XE991 (10 µM) on Na2S-induced vascular relaxation; (F) effect of selective blockade of KV7 by XE991 on GYY4137-induced vascular relaxation. Where no error bars are indicated, errors lies within dimensions of symbol. All data are means ± SEM (n = 8–10). *p < 0.05, two-way ANOVA.

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  FIGURE 9. Schematic figure proposing the mechanisms involved in (A) contraction in small mesenteric arteries induced by Na2S and GYY4137 in the presence of L-cysteine. This involve a formation of a sulfide product (XSn) that may modulate guanylate cyclase leading to contraction of the vascular smooth muscle cells (VSMC). The effect is partly reversed by the NO synthase inhibitor, L-NAME (B) Relaxation induced by Na2S and GYY4137 leads to, respectively, fast and slow release of H2S and followed by activation of subsets of different potassium channels: ATP-sensitive K (KATP) channels by Na2S, and of large-conductance calcium activated (BKCa) and voltage-gated type 7 (KV7) channels by GYY4137.