The Signal Transduction Mechanisms Of Vasorelaxation By SO₂ And Its Combined Effects With Several Endogenous Substances

Sulfur dioxide (SO2) and its derivatives sulfite and bisulfite are common gaseous pollutants in the atmosphere. The previous studies have revealed that SO2 and its derivatives are systemic toxins, which may cause many kinds of toxicological effects in multiple organs of mammals. The epidemiological studies found that SO2 and its derivatives are correlative with the cardiovascular diseases. SO2 and its derivatives are also, however, endogenously generated from sulfur-containing amino acids and the intracellular hydrogen sulfide (H2S) in vivo. SO2 derivatives sulfite and bisulfite can be oxidized by sulfite oxidase to sulfate and excreted in urine.Since the early 1980s, Nitric oxide (NO), carbon monoxide (CO) and H2S are discovered to be biological gas transmitters, which play a number of important physiological and toxicological roles. By analogy to these gases, SO2 is hypothesized to have physiological and toxicological role in cardiovascular system in human and mammal. In a previous study, it was showed that rat blood pressure could be lowered by SO2 and its derivatives bisulfite and sulfite, SO2 derivatives caused the vasodilatory effect of isolated rat aortic rings in vitro. To date, however, the cardiovascular effects of SO2, a known precursor of bisulfite and sulfite have not been studied. The mechanisms underlying the vascular effect of SO2 are not understood yet. The effect of SO2 on isolated vascular rings in vitro remains to be elucidated. Whether the vascular effects of SO2 are mediated by vasoactive factors released from the endothelium or peripheral nerve endings, e.g., nonadrenergic and noncholinergic (NANC) nerves, is not clear. The interaction of SO2 with different cellular signaling pathways in vascular tissues, including cGMP and oxygen-derived free radicals, has not been examined. The combined effects of endogenous small molecular chemicals and SO2 on the ring tension were rarely studied.The objective of the present study is to explore the physiological and toxicological role of SO2 on vascular contractility and its underlying cellular and molecular mechanisms, we systematically examined the roles of the endothelium, and vasodilation experiment of isolated rat thoracic aortic rings by SO2 was carried out and the signal transduction pathways involved in the vascular effects of SO2 were investigated. Effects of the in vivo small molecular chemicals ammonia, lactic acid, or pyruvic acid on the ring tension and the combined effects of SO2 with these chemicals were studied in order to evaluate correctly the toxicological role of SO2 on human health.In the present studies, isolated rat thoracic aortic rings were exposed directly to SO2 gas or SO2 gas-bubbled saline (SO2 stock solution), and effects of inhibitors of different signal transduction pathways and different ion channels for vasorelaxation of SO2 on the rings were investigated. At one time, relationship between vasorelaxation and pH decrease of Krebs solution caused by SO2 was studied using anology method. The vasodilatory effects of ammonia, lactic acid and pyruvic acid on the rings and its mechanisms were also studied with same methods mentioned above. Moreover, effects of SO2 on eNOS-NO-cGMP pathway in rat vascular tissues were studied with histopathological and immunohistochemical analysis, and biochemical and molecular biological technique and methods, for example, radioimmunoassay, real-time quantitative RT-PCR and so on.The results show:(1) The venous transfusion of SO2 (20,60μmol·kg-1w.t.) lowered rapidly blood pressure in a dose-dependent manner in rats; (2) SO2 relaxed rat thoracic aortic rings in a dose-dependent manner (from 1 to 2000μmol·L-1). The vasorelaxant effect of SO2 at low concentrations (<450μmol·L-1) was endothelium-dependent, and at high concentrations (>500μmol·L-1) was endothelium-independent. (3) The vasorelaxation by addition of SO2 stock solution (final concentrations≤2 mM) was actually caused by SO2 molecules, not by sulfite or bisulfite, and the characteristic of vasorelaxation by SO2 were different from that of sulfite and bisulfite. (4) The vasorelaxant effect of SO2 was not due to the altered neurotransmitter release from the autonomous or nonadrenergic and noncholinergic (NANC) nerve endings, also not due to superoxide and hydrogen peroxide produced in the vascular tissues, also disapproving the involvement of prostaglandin, PKC,β-adrenoceptor and cAMP pathways. (5) The vasorelaxant effect of SO2 at low concentrations was mediated by the cGMP pathway. (6) There was the synergistic effect on smooth muscle relaxation between much lower concentrations of SO2 (3μM) and NO (3 or 5nM). (7) SO2 could increase activity of endothelial nitric oxide synthase (eNOS), but not of induced NOS (iNOS); Also it potentiated expression of eNOS gene on the transcription and translation levels in rat aorta; (8) SO2 enhanced nitric oxide (NO) formation in aortic tissue, level of cGMP in rat aorta, but no change of cAMP. (9) The venous transfusion of lactic acid or pyruvic acid (120,300μmol·kg-1w.t.) could rapidly cause a decrease of rat blood pressure in a dose-dependent manner in vivo; The venous transfusion of ammonia at 8μmol·kg-1w.t. caused obviously an increase of rat blood pressure, but at 20μmol·kg-1w.t. caused rapidly a decrease of rat blood pressure; (10) The role of ammonia on rat thoracic aortic rings was complexity, effect of ammonia at 100μmol·L-1 on the vascular tension was not notable, at 0.5-3mmol·L-1 caused a vasoconstriction response and the antagonistic effect to vasorelaxation by SO2 was observed; at higher concentrations(5-8mmol·L-1)caused vasorelaxation and the independent joint action between the vasorelaxations by higher ammonia and SO2 was found; (11) Lactic acid relaxed rat thoracic aortic rings in a dose-dependent manner, the vasorelaxation was partially mediated by the NO/cGMP pathway and ATP-Sensitive K+(KATP) Channels. The mechanisms of vasorelaxation by lactic acid were not whole similar to that of SO2, and the joint action of their vasorelaxation might be the independent joint action; (12) Effect of pyruvic acid at physiological concentrations on rat vascular ring tension was not found. Pyruvic acid at both lower and higher concentrations caused vasorelaxation, which was partially mediated by by L-type calcium-channels. However, the vasorelaxation by pyruvic acid at lower concentrations was also mediated by the NO/cGMP pathway and small-conductance Ca2+-activated K+(SKCa) channels, the vasorelaxation by pyruvic acid at higher concentrations was also mediated by KATP Channels. The mechanisms of vasorelaxation by pyruvic acid were not whole similar to that of SO2, and the joint action of their vasorelaxation might be the independent joint action.These findings led to the conclusions:(1) SO2 at low concentrations might cause the endothelium-dependent vasodilation, at high concentrations caused the endothelium-independent vasodilation, which might be a toxic effect on vascular tissues. Endogenous SO2 at physiological concentrations was a vasoactive factor, which might regulate vascular smooth muscle tone in synergy with NO, suggesting there was some forms of "cross-talks" between SO2 and NO in vascular tissue; (2) The vasorelaxation effect was caused by SO2 (1-2000μmol·L-1) in a dose-dependent manner. Not only the effect by SO2 was much greater than that of sulfite and bisulfite, and that the mechanisms of their vasorelaxations were also different, suggesting sulfite and bisulfite were metabolic products in inactivity process of SO2 as a vasoactive substance; (3) There were acute and prolonged effects of SO2 on the NO/cGMP signalling pathway; SO2 could upregulate the eNOS-NO-cGMP pathway and at least partly by which the SO2 might cause vasodilation and inhibition to vasoconstriction; (4) The effect of ammonia on rat thoracic aortic rings was complicated, the vascular tension might be regulated together by the antagonistic effect between vasoconstriction by ammonia at lower concentrations and vasorelaxation by SO2, but ammonia at high concentrations could cause vasorelaxation and potentiate the vasorelaxation by SO2; (5) The vasorelaxation was also caused by lactic acid and pyruvic acid in a dose-dependent manner, the both endogenous chemicals at lower concentrations might execute a protective role on blood vessels, and at high concentrations might potentiate the vasorelaxation by SO2. Therefore, the combined toxic effects of SO2 and endogenous small molecular chemicals on the cardiovascular system should be considered when the influence of SO2 on human health is evaluated.