Regulatory Effects Of Myo-endothelial Gap Junction On Vascular Reactivity And Its Mechanisms Following Hemorrhagic Shock In Rats

After severe trauma or shock, including hemorrhagic,endotoxic and septic shock, vascular reactivity to vasoconstrictors and vasodilators is greatly reduced.Previous studies showed that it may be related to the increased level of nitric oxide(NO),the functional disorder of the K+ and Ca2+ channels on the vascular smooth muscle cell (VSMC) membrane and the hyperpolarization of cell membrane,or calcium desensitization of VSCM. However,suppressing the level of NO,recovering the function of the K+ and Ca2+ channels and polarization state of the cell membrane,increasing the calcium sensitivity of VSMC can not restore the vascular reactivity completely.Vascular endothelial cell(VEC) is a monolayer squamous cell lining the luminal surface of entire vascualr system.Except providing a structural and metabolic barries between the blood and the underlying tissues, it also secretes,synthesizes and degrades a lot of vascular active compounds to regulate vascular tone.Gap junction(GJ) comprises of connexins and forms transmembrane channels connecting cytoplasms of adjacent cells,which allows electronic signal,ions,small metabolites(<1kDa), and second messengers to translocate from cell to cell.Its functional deficit and abnormality have an intimate relation to many cardiovascular disease, e.g. hypertension and atherosclerosis. GJ between VEC and VSMC is named myo-endothelial gap junction(MEGJ),which allows direct and bidirectional electronic and chemical communication among cells and plays central roles in the regulation of vascualr tone. However,whether MEGJ is involved in the regulation of vascular hyporeactivity following hemorrhagic shock is still unknown.Therefore,with hemorrhagic shock rats,we studied the regulatory roles of Cx37,Cx40 and Cx43 in the changes of vascular contractile and relaxant response and their possible mechanism. The experiments were conducted in four parts, including:(1)observing the regulatory role of MEGJs in development of vascular hyporeactivity following hemorrhagic shock;(2) studying the connexins' type that involved in the regulation of vascular reactivity following hemorrhagic shock;(3) exploring the possible mechanism that MEGJs regulate the endothelium-dependent vasoconstrictor reactivity;(4) analysing the possible mechanism that MEGJs regulate the endothelium-dependent vasodilator reactivity.The main results as follows:1. The regulatory role of MEGJs on the vascular reactivity following hemorrhagic shock.18α-GA had no effect on the endothelium-independent vasoconstrictor (NE) and vasodilator (SNP) induced vascular contraction and relaxation.The endothelium-dependent vasoconstriction of SMA to myricetin gradually increased from 0h to 2h after shock and obviously decreased at 3h and 4h after shock. 18α-GA can abolish this tendence induced by myricetin in all of groups.This result indicates that MEGJs play important roles in endothelium-dependent vasoconstrictor response of SMA following hemorrhagic shock.The response of SMA at different time after shock to 10-5mol/L Ach had a trendency of decrease,increase and decrease. It decreased at the first 0.5h after shock ,and began to increase at 1h after shock , and reached its peak at 2h ,and then decreased at 3h and 4h after shock. 18α-GA can abolish this tendency induced by Ach. This result suggested that MEGJs play important roles in endothelium-dependent vasodilator reactivity following hemorrhagic shock.2. Connexins' type that involved in the regulation of vascular reactivity following hemorrhagic shockThe mRNA and protein expression of Cx37 and Cx40 gradually decreased at 0h , 0.5h , 1h, 2h after shock, gradually increased at 3h , 4h. While the expression of Cx43 decreased at 0h after shock,then gradually increased at 0.5h,1h and 2h after shock and then decreased at 3h,4h after shock.Cx40AODN improved the vascular response of SMA to myricetin and Ach in non hypoxia group,1h and 3h hypoxia groups,but Cx43AODN decreased their reactivity.These results showed that Cx40 and Cx43 took part in the regulation of vascular reactivity after hemorrhagic shock.Cx40 has a suppressing effect on endothelium-dependent vasoconstrictor and vasodilator reactivity, while Cx43 has an enhancing effect on endothelium-dependent vasoconstrictor and vasodilator reactivity.3. The possible mechanism of MEGJs regulating the endothelium-dependent vascular contractile reactivity following hemorrhagic shock(1) Effect of MEGJs on calcium sensitivity of SMA after hypoxia under the action of myricetinAs compared with control group,the calcium sensitivity of SMA significantly increased following 1h hypoxia and decreased following 3h hypoxia.Cx40AODN significantly increased the calcium sensitivity of hypoxia treated SMA,but Cx43AODN decreased them.These results indicated that Cx40 and Cx43 had negative and positive effects on calcium sensitivity respectively.(2) Effect of MEGJs on the MLCP activity of SMA after hypoxia under the action of myricetinAs compared with control group, the MLCP activity of SMA decreased after 1h hypoxia and increased after 3h hypoxia.Cx40AODN can decrease the increased MLCP activity induced by myricetin and Cx43AODN can increase it.It suggested that Cx40 and Cx43 had positive and negative effect on MLCP activity following hemorrhagic shock.(3) Effect of MEGJs on MLC20 phosphorylation of SMA after hypoxia under the action of myricetinAs compared with control group, the MLC20 phosphorylation of SMA after hypoxia significantly decreased.Myricetin increased the phosphorylation of MLC20 in all groups. Cx40AODN increased the phosphorylation of MLC20 induced by myricetin and Cx43AODN decreased it in normal control group and 1h hypoxia group. In 3h hypoxia group,Cx40AODN increased the phosphorylation of MLC20 induced by myricetin, but Cx43AODN had no effect on the phosphorylation of MLC20.(4) Effect of MEGJs on [Ca2+]i of VSMC under the action of myricetin[Ca2+]i of VSMC gradually increased at 30min and 2h after hypoxia. Myricetin increased [Ca2+]i of VSMC.Cx40AODN increased the lift of [Ca2+]i of VSMC induced by myricetin and Cx43AODN decreased it.(5) Effect of MEGJs on MLCK activity of SMA after hypoxia under the action of myricetinAs compared with control group, the MLCK activity of SMA increased in 1h hypoxia and decreased in 3h hypoxia.Myricetin,Cx40AODN and Cx43AODN had no effect on the MLCK activity.The results demonstrated that Cx40 can decrease the [Ca2+]i of VSMC while Cx43 can increase it.4. The possible mechanism of MEGJs regulating the endothelium-dependent vasodilator reactivity following hemorrhagic shock(1) Effect of MEGJs on MLCP activity of SMA after hypoxia under the action of Ach Ach can increase the MLCP activity of SMA in each group.Cx40AODN and Cx43AODN had no effect on the MLCP activity induced by Ach. These results suggested that Cx40 and Cx43 have no obivious effects on the MLCP activity of vessels after Ach and hypoxia treatment.(2) Effect of MEGJs on [Ca2+]i of VSMC after hypoxia under the action of Ach Ach can decrease [Ca2+]i of VSMC.Cx40AODN and Cx43AODN had no effect on [Ca2+]i of VSMC induced by Ach.The results suggested that Cx40 and Cx43 had no obivious impact on [Ca2+]i after Ach treatment.(3) Effect of MEGJs on MLCK activity of SMA after hypoxia under the action of Ach Ach can decrease the MLCK activity. Cx40AODN and Cx43AODN had no effect on the decreased MLCK activity induced by Ach. These results suggested that Cx40 and Cx43 had no obivious impact on the MLCK activity of hypoxia treated SMA.Conclusions:1. Vascular reactivity appears biphasic change following hemorrhagic shock. MEGJ plays important roles in regulating endothelium-dependent vasoconstrictor and vasodilator reactivity following hemorrhagic shock.2. Cx40 can depress endothelium-dependent vasoscontrictor and vasodilator reactivity. Cx43 can enhance endothelium-dependent vasoconstrictor and vasodilator reactivity.3. The possible mechanisms that MEGJ regulates endothelium-dependent vaso- constrictor reactivity following hemorrhagic shock may be mainly through Cx40 and Cx43 regulating the activity of MLCP , the phosphorylation level of MLC20 and vascular calcium sensitivity.4. MEGJ regulating endothelium-dependent vasodilator reactivity following hemo- rrhagic shock may not be related to MLCP/MLCK and MLC20 signal pathway.There may be other mechanism involved it.