Effects Of Spironolactone On Isolated Rat Thoracic Aortic Rings And Vascular Smooth Muscle Cell Currents

Objective:Spironolactone (SPT), also known as spironolactone, has been used as a potassium-sparing diuretics for more than30years, it is a synthetic17-lactone steroid aldosterone antagonist. Clinical antihypertensive effect is relatively weak, but its antihypertensive effect has been fully affirmed; the SPT also has been shown to reduce mortality in patients with heart failure (HF), improving HF patients with atrial conduction and remodeling, SPT also has good protective effect on the cardiovascular system. However, isolated vascular smooth muscle studies have not been reported. Studies also have reported that SPT affect Kv channel of fibroblasts, also observed SPT manner similar to calcium blockers act on slow calcium channels, but the impact of SPT on thoracic aorta vascular smooth muscle potassium current and calcium current has not been reported.The experiments were respectively from the isolated organ and molecular level to study the mechanism of the vasodilatory effects of SPT.Method:Use the isolated vascular smooth muscle tension Records Method:First the rats were sacrificed with the dislocation, chest was opened and separated the thoracic aorta quickly, taken down the rat thoracic aorta into4℃physiological salt solution prepared beforehand, pruning aorta surrounding tissue, and cut into2-3mm vascular ring. Vascular ring lumen was with stainless steel hooks through,the level of vascular ring suspended in10ml of the PSS liquid bath tube, connected to tension transducer with a thin wire,use the Powerlab biological signal measurement system to record vascular ring tension changes. The bath need oxygen supply (95%oxygen and5%carbon dioxide mixture), preload2g, in37℃of the PSS liquid medium was changed every20minutes once,after equilibrating for1hour, use SPT, observed its effect on isolated thoracic aortic rings of Sprague Dawley (SD) rats and different tools drugs. First the rats were sacrificed with the dislocation, chest was opened and separated the thoracic aorta quickly, blunt dissection thoracic aorta, separate suspension liquid containing the vascular smooth muscle cells, choose the clear edge of cell do the patch-clamp experiments. Use the Whole-cell patch clamp recording method to record membrane currents, explore the effect of spironolactone (SPT) on thoracic aorta vascular smooth muscle potassium current and calcium current of normal rat. The results of the experiment except the special stated data with Mean±SE, the data obtained using SPSS11.0statistics software for two independent samples t-test.Result:(1) Under the resting state, SPT (1×10-5～2×10-4mol·L-1) has no significant effect on vascular rings isolated thoracic aorta.(2) SPT (1×10-5～2×10-4mol·L-1) has concentration-dependent relaxant effect on KCl (30mmol·L-1) or NE (1×10-6mol·L-1)-induced vascular ring contraction, compared with the control group, the difference was statistically significant(P<0.01).(3)SPT(1×10-5-2×10-44mol·L-1) has concentration-dependent relaxation role on KCl (30mmol-L-1) or NE (1×10-6mol·L-1) induced endothelium-denuded vascular contraction, with the endothelium intact group it showed no significant difference (P>0.05).(4) On the basis of the vascular ring with KCl (30mmol·L-1) pre-contracted, added in the Na+/H+exchange blocker amiloride (AM,1umol·L-1), the vasodilating effect of SPT(1×10-5～2×10-4mol·L-1) significantly inhibited by AM, compared with the control group,the difference was statistically significant (P<0.01).(5) On the basis of the vascular ring with KCl (30mmol·L-1) pre-contracted, added in potassium channel blocker barium chloride (BaCl2,2u mol·L-1),tetraethylammonium(TEA,0.2mol·L-1),four-aminopyridine(4-AP,1m mol·L-1),they had no significant inhibition on the SPT relaxation of vascular compared with the control group, the difference was not statistically significant(P>0.05). But ATP-sensitive potassium channels (KATP) glibenclamide phenylurea (Gli,10μmol·L-1) significantly inhibited the relaxant effect of SPT vasodilating, compared with the control group, the difference was statistically significant (P<0.01).(6) with SPT(2×10-4mol·L-1) pre-incubation20minutes, added in1μmol·L-1of NE, then found vascular brief contraction, wait it until smooth, then added2.5m mol·L-1of CaCl2, found vessels continue to shrink. The control group pre-incubation with solvent. The results show that:in calcium-free physiological salt solution, SPT can not significantly inhibit vascular contraction caused by NE compared with the control group, the difference was not statistically significant(P> 0.05); SPT significantly inhibited contraction of blood vessels caused by compared with control group. The difference was statistically significant (P<0.01).(7) SPT (2×10-4mol·L-1) has no significant effect on thoracic aorta vascular smooth muscle potassium current, compared with the control group, the difference was not statistically significant (P>0.05).(8) SPT(2×10-4mol·L-1) has significant effect on thoracic aorta vascular smooth muscle calcium current, compared with the control group, the difference was statistically significant(P<0.01).Conclusion:1. On resting state, SPT has no effect on isolated thoracic aortic rings.2. SPT (1×10-5～2×10-4mol·L-1) has concentration-dependent relaxant effect on KCl (30m mol·L-1) or NE (1×10-6mol·L-1)-induced vascular ring contraction with or without endothelium.3. The relaxant effect on isolated thoracic aorta vascular ring of SPT has related to the Na+/H+exchanger.4. The relaxant effect on isolated thoracic aorta vascular ring of SPT has nothing to do with KiR channel, Kca channels and Kv channel, but the KATP channel.5. SPT can make blood vessels diastolic through inhibition of calcium influx.6. SPT (2×10-4mol·L-1) has no significant effect on thoracic aorta vascular smooth muscle cell potassium current.7. SPT(2×10-4mol·L-1) has significant effect on thoracic aorta vascular smooth muscle cell calcium current.