Modulation Of Hydrogen Sulfide On Tension Of Isolated Rabbit Renal Artery

Hydrogen sulfide (H2S), the colorless gas with a strong odor of rotten eggs, has been generally considered as a toxic gas. The main toxicity effect of H2S is a potent inhibition of mitochondrial cytochrome c oxidase and then inhibits mitochondrial respiration. But recently H2S has been proposed to be the third endogenous gasotransmitter which was similar to two other vasoactive gases, nitric oxide (NO) and carbon monoxide (CO). Some study suggested that heart tissues could endogenously produce H2S as a physiological and pathological cardiac function regulator. It also has an important modulation function on the pathogenesis of spontaneous hypertension, hypoxic pulmonary hypertension, high pulmonary blood hypertension and renovascular hypertension. H2S relaxed volume vascular tissues of rat aortic directly through opening ATP-sensitive potassium channels (KATP) in vascular smooth muscle cells (SMCs), but relaxed resistance vascular tissues of mesentery artery through two targets: opening KATP channels in vascular SMCs and KCa channels which can inhibited by ChTX/apamin in vascular endothelial cells, the target of EDHF. It is thus evident that the pathway of H2S relaxing vascular vessels is different from NO and CO (they mainly activate soluble guanylate cyclase and increase intracellular cGMP concentration). H2S also inhibits SMCs proliferation, enhances SMCs apoptosis and improves pulmonary vascular structural remodeling. The recent study suggested that heart tissues could endogenously produce H2S as a physiological and pathological cardiac function regulator. The pathological renal arteries produced lumens stenosis and then activate rennin-angiotensin system to result in renovascular hypertension. The increscent resistance of renal artery is important character of renal haemodynamics in renovascular hypertension. It is thus evident that renal artery tension has important roles in the pathogenesis of hypertension and renovascular hypertension. The effects of H2S on renal artery have not been reported yet.AimThe purpose of the present research was to measure the H2S concentration in normal rabbit plasma, to assay CSE activity in renal artery smooth muscle homogenatestissue, to investigate the effects of H2S on rabbit renal artery and to explore the underlying mechanism.Methods1 Measurement of plasma H2S concentration in rabbit.A sample of plasma (0.1 ml) was added to a test tube containing 0.5 ml of 1% zinc acetate and 2.5 ml of distilled water, then 0.5 ml of 20 mmol/L N,N-dimethyl-p-phenylenediamine dihydrochloride in 7.2 mol/L HCl and 0.4 ml of 30 mmol/L FeCl3 in 1.2 mol/L HCl were also added to the same test tube for 20 min of incubation at room temperature. The protein in the plasma was removed by adding 1 ml of 10% trichloroacetic acid to the solution and centrifuging it. The optical absorbance of the resulting solution at 665 nm was measured with a spectrometer. H2S concentration in the solution was calculated against the calibration curve of the standard NaHS solution.2. Assay of renal artery tissue CSE activity.The renal artery tissues were homogenized in ice-cold 50 mmol/L potassium phosphate buffer (PH 6.8). Reactions were performed in 25 ml Erlenmeyer flasks. The reaction mixture contained (mmol/L): 10 L-cysteine, 2 pyridoxal 5'-phosphate, 100 potassium phosphate buffer (PH 7.4), and 10% (w/v) homogenates. The total volume of the reaction mixture was 1 ml. A small piece of filter paper was put into the central well of the flask and 0.5 ml of 1% zinc acetate was also added in the central well for trapping evolved H2S in the mixture. The flasks were then flushed with N2 before being sealed with a double layer of parafilm. The catalytic reaction was initiated by transferring the flasks from an ice bath to a 37℃shaking water bath. After 90 min at 37℃, the reactions were stopped by injecting 0.5 ml of 50% trichloroacetic acid. Flasks were incubated in the shaking water bath for an additional hour at 37℃to complete trapping of H2S. The content of the central well was transferred to test tubes and mixed with 3.5 ml of distilled water and 0.5 ml of 20 mmol/L N,N-dimethyl-p-phenylenediamine dihydrochloride in 7.2 mol/L HCl. To each tube, 0.4 ml of 30 mmol/L FeCl3 in 1.2 mol/L HCl was added immediately. After 20 min of incubation at room temperature, the optical absorbance of the resulting solution at 665 nm was measured with a spectrometer. The H2S concentration in the solution was calculated against the calibration curve of the standard H2S solution. For each sample, the measurement was done in duplicate. Protein was determined using the Bradford technique with bovine serum albumin (BSA) as a standard. The H2S production was expressed in a unit of pmol/mg protein/minute.3. Modulation of hydrogen sulfide on tension of isolate rabbit renal artery and its mechanism.The functional curve of hydrogen sulfide on rabbit renal artery was measured by recording the changes of H2S concentration and drugs with organ bath system.Results1. The plasma level of H2S in normal rabbit was 44.39±4.93μmol/L.2. The activity of enzymes in renal artery tissue was 143.94±4.80 pmol/mg protein/minute.3. (1) NaHS (50, 100, 200, 400 and 800μmol/L) induced significant relaxation of renal artery rings with intact endothelium in a concentration-dependent manner preshrunk by KCl (50 mmol/L) as a control. The IC50 of the concentration-response relaxation curve was 281.46±17.26μmol/L. A maximum relaxation of 89.97±2.40% was attained at 800μmol/L of NaHS. (2) Pretreatment with KATP channel blocker glibenclamide (Gli, 20μmol/L), calcium channels agonist Bay K8644 (500 nmol/L), NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 100μmol/L), prostaglandin (PGI2) inhibitor indomethacin (10μmol/L) and removal of the endothelium significantly inhibited H2S-induced relaxation. The concentration-response relaxation curve shifted right obviously and the IC50 changed from 281.46±17.26μmol/L to 299.69±8.64μmol/L (P < 0.05), (405.32±2.84μmol/L,P < 0.05), (369.25±0.85μmol/L,P < 0.05), (387.18±26.26μmol/L,P < 0.01) and (331.15±4.13μmol/L,P < 0.05) respectively. Furthermore, pretreatment with glibenclamide, Bay K8644 and removal of the endothelium significantly decrease H2S-induced maximum relaxation from (89.97±2.40%) to (68.65±4.31%, P < 0.01), (80.10±3.11%, P < 0.05) and (76.03±2.35%, P < 0.01) respectively, but L-NAME and indomethacin did not change the maximum relaxation. (3) Pretreatment with the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3a]quinoxalin-1-one (ODQ, 10μmol/L) and co-application of charybdotoxin and apamin (Ca2+-dependent K+ channel blocker, KCa, 50 nmol/L) have no effect on the action of H2S; the IC50 of the concentration-response curve was (295.31±16.07μmol/L,P > 0.05) and (261.47±36.57μmol/L,P > 0.05). (4) After 1 hour equilibration, increasing concentrations of KCl (5, 15, 25, 35, 45, 55 and 65 mmol/L) to study the contraction effect of KCl as compare. KCl induced significant vasoconstriction of renal artery rings in a concentration-dependent manner. The IC50 of the concentration-response curve was 14.91±0.16 mmol/L. (5) Pretreatment with DL-propargylglycine (PPG, CSE inhibitor, 200μmol/L), to inhibit endogenously H2S, for 30 min before the rings were administration with KCl. The vasoconstriction was markedly enhanced; the concentration-response curve shifted to the left and upward, and the IC50 was decreased to 11.65±1.08 mmol/L (P < 0.05). (6) Pretreatment with NaHS (200μmol/L) distinguishably suppressed the vasoconstriction by KCl, the concentration-response curve shifted to the right and downward, and the IC50 was increased to 19.16±1.09 mmol/L (P < 0.05).Conclusion1. The plasma concentration of H2S in rabbit was similar to the level of rat (45.6±14.2μmol/L).2. The activity of enzymes in renal artery tissue was lower than rat pulmonary artery (831.8±70.5) pmol/mg protein/minute.3. Exogenous H2S could endothelium-dependently relax rabbit renal artery through opening of KATP channels further closing the calcium channels in vascular smooth muscles. NO and PGI2 possible have a synergism with H2S on the effect of vasodilation. After inhibited endogenously H2S induced an increasing contraction effect of the vascular. While H2S relaxed vascular tissue is not dependent on the activation of the cGMP pathway or KCa channels.