Role of NO and K+ channels in vascular hyporeactivity in rats with endotoxin shock

• Main Authors: Chen Shin-Jen, 陳秀珍
• Other Authors: Yen Mao-Hsiung
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/00224109724135424989

博士 === 國防醫學院 === 生命科學研究所 === 88 === Septic shock-like syndromes are usually characterized by hypotension and hyporeactivity to vasoconstrictor agents. The hypotension and vascular hyporeactivity are associated with the development of multiple organs dysfunction which causes death. Thus, it is important to investigate the mechanisms of vascular hyporeactivity in septic shock. Several studies (in vivo and in vitro) showing that the overproduction of nitric oxide (NO) is associated with vascular hyporeactivity in animals with septic shock. Inhibitors of NO synthase are able to improve the vascular hyporeactivity to vasoconstrictors. Our previous results have shown that the hyporeactivity to norepinephrine (NE) in aortic rings from rats treated with endotoxin (E. coli lipopolysaccharide, LPS) is because of the activation of soluble guanylate cyclase (sGC), which is partially mediated by NO, indicating that LPS induces the production of other mediator(s) that activates sGC in the vascular smooth muscle. Recent studies show that selective inhibitors of inducible NOS (iNOS or NOS II) only partially reverse the vascular hyporeactivity elicited by endotoxin, suggesting that other mechanisms may contribute to endotoxin-induced vascular hyporeactivity. Therefore, my project was to examine the possible mechanisms or mediators (eg. NO, K+ channels, etc) that contributed to vascular hyporeactivity in animals with septic shock. Firstly, whether a complete inhibition of NO formation caused by bacterial endotoxin (LPS) in vivo prevents the hypotension and restores the vascular hyporeactivity to normal in vivo and ex vivo was investigated. The combination of dexamethasone (DEX; 3 mg/kg at 30 min before LPS) plus aminoguanidine (AG; 15 mg/kg at 2 h after LPS) not only inhibited the overproduction of nitrate (an indicator of NO) in the plasma and aortic smooth muscle, but also prevented the development of the delayed hypotension in Wistar-Kyoto rats treated with LPS for 6 h. However, the vascular hyporeactivity to NE was only partially improved either in vivo or ex vivo in endotoxemic rats treated with DEX plus AG. Pretreatment of aortic rings with Nw-nitro-L-arginine methyl ester (L-NAME) or 1H-[1,2,4]oxidazolo[4,3-a]quinoxalin-1-one (ODQ) enhanced the contraction to NE in rings obtained from LPS-treated rats, but not in those from DEX plus AG-treated endotoxemic rats. Methylene blue (MB), an inhibitor of soluble guanylate cyclase (sGC), completely restored contractions to NE and aortic cGMP levels to normal either in LPS-treated rats or in DEX plus AG-treated endotoxemic rats, whereas the cGMP level was partially inhibited by ODQ in LPS-treated rats only. These results suggest that non-NO mediator(s) also activates sGC during endotoxemia. Interestingly, I found that in the presence of tetraethylammonium (TEA, an inhibitor of non-selective K+ channels) plus L-NAME or charybdotoxin [CTX, a specific inhibitor of large-conductance Ca2+-activated K+ (BKCa) channels] plus ODQ, the vascular hyporeactivity to NE in the LPS-treated group was completely restored to normal. In addition, in the presence of L-NAME or ODQ, the vascular hyporeactivity to high K+ was abolished in rings from the LPS-treated group. These results suggest that LPS causes the production of other mediator (s), in addition to NO, which also stimulates sGC (i.e. increases the formation of cGMP) and then activates the BKCa channels in the vascular smooth muscle causing vascular hyporeactivity. Secondly, the possible mechanisms associated with vascular hyporeactivity to vasoconstrictor agents in rats with endotoxemia were examined. Rats were anesthetized and injected with endotoxin for 4 h. Pressor responses to NE (1 mg/kg, i.v.) were determined prior to and at every hour after LPS injection. After the in vivo experiment, the rat thoracic aorta was excised and prepared as rings 3-4 mm in width. The endothelium was mechanically removed to evaluate K+ channels activity and the effects of nitric oxide (NO) on the vascular smooth muscle. The results demonstrated that: (1) injection of LPS caused a significant fall in blood pressure and a severe vascular hyporeactivity to NE in the anesthetized rat; (2) the relaxation induced by the K+ channels opener cromakalim was greater in rings obtained from endotoxemic rats and this enhanced relaxation was partially inhibited by pretreatment of these rings with ODQ, an inhibitor of the NO/cGMP pathway; and (3) endotoxemia for 4 h was also associated with a profound vascular hyporeactivity to NE ex vivo and this vascular hyporesponsiveness was partially inhibited by ODQ, TEA, and CTX, but not by apamin, a selective inhibitor of small conductance Ca2+-activated K+ (SKCa) channels. The combination of TEA or CTX with ODQ completely restored vascular hyporesponsiveness to normal. These results suggest that activation of BKCa and overproduction of NO in the vascular smooth muscle simultaneously contribute to vascular hyporeactivity to vasoconstrictor agents in endotoxemia. Thirdly, the role of membrane hyperpolarization in mediating vascular hyporeactivity induced by bacterial LPS in endothelial-denuded strips of rat thoracic aorta ex vivo was examined. The injection of rats with LPS caused a significant fall of blood pressure and a severe vascular hyporeactivity to NE. The membrane potential recording showed that endotoxemia caused a hyperpolarization when compared to the control. This hyperpolarization was fully restored by MB (10 mM) and partially reversed by L-NAME (0.3 mM), ODQ (1 mM), TEA (10 mM), CTX (0.1 mM), or glibenclamide (GB; 10 mM), however, this hyperpolarization was not significantly affected by apamin (0.1 mM), 4-aminopyridine (4-AP; 1 mM), or Ba2+ (50 mM). In addition, the basal tension of the tissues obtained from endotoxemic rats was enhanced by the following order: MB > ODQ > TEA > L-NAME > CTX > GB; whereas apamin, 4-AP or Ba2+ has no significant effects on these tissues. In contrast, none of these inhibitors had significant effects on the membrane potential or the basal tension in control tissues. Our electrophysiological results further confirmed previous studies showing that in addition to NO, the BKCa channels and ATP-sensitive K+ (KATP) channels are, most likely, responsible for endotoxin-mediated hyporeactivity to vasoconstrictor agents in vascular smooth muscle. Fourthly, the role of K+ channels in vascular hyporeactivity of rats with endotoxic shock ex vivo was examined. Rats were anesthetized and injected with LPS for 6 h. Endotoxemia for 6 h resulted in a reduction in blood pressure, an increase in heart rate and a severe vascular hyporeactivity to NE. At the end of the in vivo experiments, thoracic aortas were removed from endotoxemic and control rats. After removal of the endothelium, aortic segments were mounted in myographs for recording of isometric tension and smooth muscle membrane potential. Aortic rings without the endothelium were used to examine the relaxation induced by two agonists of K+ channels, NS1619 and pinacidil. Smooth muscle membrane potentials recorded from endotoxemic rats were hyperpolarized when compared to those of the controls. This hyperpolarization was partly reversed by TEA, CTX or GB, but not apamin. The hyperpolarization was also partly attenuated by L-NAME or ODQ. In addition, NS1619 induced a greater hyperpolarization in aortic segments from endotoxemic rats and this increase was completely inhibited by CTX, L-NAME or ODQ, and partially inhibited by TEA, but not significantly affected by apamin. Similarly, pinacidil also caused a greater hyperpolarization in arteries from endotoxemic rats and this increase was completely inhibited by GB, L-NAME or ODQ. In phenylephrine-contracted aortic rings, both NS1619 and pinacidil induced greater relaxation in the preparations obtained from endotoxemic rats. The NS1619-induced relaxation in arteries from endotoxemic rats was partially inhibited by TEA and completely inhibited by CTX, L-NAME or ODQ, but was not significantly affected by apamin. These inhibitors had significant effect on that ralaxation in arteries from controls. In addition, the difference of pinacidil-induced relaxation between endotoxemic and control rats was antagonized by GB to a greater extent and by L-NAME to a less extent, whereas ODQ had a greater inhibition on that relaxation in preparations from endotoxemic rats than those from controls. In conclusion, this study provides the electrophysiological and functional evidence showing an abnormal activation of K+ channels in vascular smooth muscle in animals with endotoxic shock. Our observations suggest that overproduction of NO causes an activation of BKCa channels and KATP channels which contributes to endotoxin-mediated vascular hyporeactivity. 目錄 I 表目錄 II 圖目錄 III 縮寫對照表 VII 中文摘要 IX 英文摘要 XV 第一章、 緒論 1 第二章、 非一氧化氮/鳥核苷單磷酸在內毒素性休克鼠中 所扮演的角色 25 第三章、 一氧化氮與鉀離子通道在內毒素所誘發血管低反應性 中所扮演的角色 59 第四章、 過度極化作用參與內毒素性休克鼠的血管低反應性 77 第五章、 電生理及功能性的證據證明有不正常鉀離子通道的 活化參與內毒素性休克鼠的血管低反應性 92 第六章、 總結與討論 119 第七章、 未來的研究方向與展望 124 第八章、 參考文獻 126 表 目 錄 頁數 Table II-1. Effects of L-NAME, ODQ, and MB on NE-induced contractions in endothelium-denuded aortic rings 47 Table IV-1. Effects of lipopolysaccharide (LPS) on mean arterial blood pressure (MAP), heart rate (HR), and pressor esponses to norepinephrine (NE) in the anesthetized rats 89 Table V-1.Hemodynamic changes in rats treated with E. coli LPS (5 mg/kg i.v.) or saline (i.e. SOP) for 6 h 110 Table V-2. Changes of tension before (i.e. basal) and after phenylephrine (0.1 - 0.3 mM) and NS1619 (0.1 mM) in aortic segments obtained from SOP rats and LPS rats in vitro treated with or without (i.e. none) TEA, CTX, APM, L-NAME, and ODQ in electrophysiological recording experiments 111 Table V-3. Changes of tension before (i.e. basal) and after phenylephrine (0.1 - 0.3 mM) and pinacidil (10 mM) in aortic segments obtained from SOP rats and LPS rats in vitro treated with or without (i.e. none) GB, L-NAME, and ODQ in electrophysiological recording experiments 112 圖 目 錄 頁數 Fig. I-1a. Signal transduction pathway for LPS, TNF-a and IL-1b 16 Fig. I-1b. Schematic diagram of septic shock 17 Fig. I-2. Schematic representation of lipids and proteins in the E. Coli cell envelope 18 Fig. I-3. Diagrammatic representation of the structure of endotoxin (LPS) 19 Fig. I-4. The L-arginine/NO pathway in the blood vessel wall 20 Fig. I-5. Vascular relaxation mediated by NO in physiological, pharmacological and pathophysiological conditions 21 Fig. I-6. Membrane potential (Vm) in the regulation of arterial smooth muscle tone 22 Fig. I-7. Four types of K+ channels in arterial smooth muscle 23 Fig. I-8. Chemical structures of cromakalim, pinacidil, NS1619, methylene blue, ODQ, L-arginine, L-NAME and glibenclamide (GB) 24 Fig. II-A. Schematic diagram of the instrumental setup of nitric oxide analyzer (NOA) 48 Fig. II-1. Effects of DEX plus AG on the production of plasma nitrate in lipopolysaccharide (LPS)- treated rats 49 Fig. II-2. Effects of DEX plus AG on the production of nitrate in aortic smooth muscle obtained from LPS-treated rats 50 Fig. II-3. Effects of DEX plus AG on the blood pressure and heart rate in LPS-treated rats 51 Fig. II-4. Effects of DEX plus AG on pressor responses to NE in LPS-treated rats 52 Fig. II-5. Effects of DEX plus AG on the vascular tone induced by NE in LPS-treated rats 53 Fig. II-6. Effects of ODQ and methylene blue on the vascular tone induced by NE in presence of NAC in LPS rats pretreated with DEX plus AG 54 Fig. II-7. Effects of L-NAME, ODQ, and methylene blue on aortic cGMP level in normal rats, LPS rats, and LPS rats pretreated with DEX plus AG 55 Fig. II-8. Effect of tetraethylammonium (TEA) or harybdotoxin (CTX) on NE-induced contraction in rats treated with LPS alone 56 Fig. II-9. Effect of TEA or CTX on NE-induced contraction in LPS rats pretreated with DEX plus AG 57 Fig. II-10 Effects of L-NAME or ODQ on the vascular tone induced by high K+ in LPS-treated rats 58 Fig. III-1.Effects of E. coli LPS (10 mg/kg, i.v. for 4 h) on (a) mean arterial blood pressure (MAP) and (b) pressor responses to NE (1 mg/kg, i.v.) in the anesthetized rat 72 Fig. III-2.Effects of ODQ on (a) cromakalim-induced relaxation and (b) NE-induced contraction in endothelium-denuded aortic rings obtained from sham-operated (SOP) rats and rats treated with LPS 73 Fig. III-3.Effects of TEA with or without ODQ on NE-induced contraction in endothelium-denuded aortic rings obtainedfrom (a) SOP rats and (b) rats treated with LPS 74 Fig. III-4.Effects of CTX with or without ODQ on NE-induced contraction in endothelium-denuded aortic rings obtained from (a) SOP rats and (b) rats treated with LPS 75 Fig. III-5.Effects of apamin (APM) with or without ODQ on NE-induced contraction in endothelium-denuded aortic rings obtained from (a) SOP rats and (b) rats treated with LPS 76 Fig. IV-1. Effects of (a) L-NAME (0.3 mM), (b) ODQ (1 mM), and (c) methylene blue (MB; 10 mM) on the membrane potential and the basal tension in endothelium- denuded aortic strips obtained from SOP rats or rats treated with LPS for 6 h 90 Fig. IV-2. Effects of (a) TEA (10 mM), (b) CTX (0.1 mM), (c) GB (10 mM), (d) APM (0.1 mM), (e) 4- aminopyridine (4-AP; 1mM) and (f) Ba2+ (50 mM) on the membrane potential and the basal tension in endothelium-denuded aortic strips obtained from SOP rats or rats treated with LPS for 6 h 91 Fig. V-1. Membrane potential and NS1619-induced repolarization in endothelium-denuded aortic segments from rats treated with E. coli LPS or saline 113 Fig. V-2. Effects of (a,d) TEA (10 mM), (b,e) CTX (0.1 mM) and (c,f) APM (0.1 mM) on (left panels) membrane potential and NS1619-induced repolarization and (right panels) NS1619-induced relaxation in endothelium-denuded aortic preparations obtained from rats treated with saline (i.e. SOP) or LPS for 6 h 114 Fig. V-3. Effects of (a,c) L-NAME (0.3 mM) and (b,d) ODQ (1 mM) (left panels) membrane potential and NS1619-induced repolarization and (right panels) NS1619-induced relaxation in endothelium-denuded aortic preparations obtained from rats treated with saline (i.e. SOP) or LPS for 6 h 115 Fig. V-4. Membrane potential and pinacidil-induced repolarization in endothelium-denuded aortic segments from rats treated with E. coli LPS or saline 116 Fig. V-5. Effects of GB (10 mM) on (a) membrane potential and pinacidil-induced repolarization and (b) pinacidil-induced relaxation in endothelium-denuded aortic preparations obtained from rats treated with saline (i.e. SOP) or LPS for 6 h 117 Fig. V-6. Effects of (a,c) L-NAME (0.3 mM) and (b,d) ODQ (1 mM) (left panels) membrane potential and pinacidil-induced repolarization and (right panels) pinacidil-induced relaxation in endothelium-denuded aortic preparations obtained from rats treated with saline (i.e. SOP) or LPS for 6 h 118 Fig. VI-1. Drugs (studied in the thesis) affect iNOS/CGMP pathway 122 Fig. VI-2. A hypothesis of possible mechanisms in vascular hyporeactivity in septic shcok 123