Effects Of Ischemic Preconditioning And Pharmacological Preconditioning On Vascular Reactivity And Calcium Sensitivity Following Hemorrhagic Shock And Its Relationship To Rho-kinase In Rats

After severe trauma or shock, including hemorrhagic, endotoxic, and septic shock, vascular reactivity to vasoconstrictors and vasodilators is greatly reduced. This reduced vascular reactivity plays an important role in the incidence, development, and outcome of the shock and interferes with the therapy for shock. Therefore, it's very important to study the therapy for vascular hyporeactivity following shock. A considerable number of experimental studies have indicated that ischemic preconditioning (IPC) could improve the tolerance to an ischemic injury in various organs, including the heart, kidney, liver, stomach, intestine, brain, skeletal muscle and lung. Several studies showed that Large-conductance calcium-activated K+ channel(Bkca) was involved in the cadioprotective effect of ischemic preconditioning, and BKca opener, NS1619 pretreatment can induce the protection in heart. Whether IPC and NS1619 preconditioning protect against shock-induced vascular hyporeactivity and their mechanism are not clear. Previous studies in our lab showed that vascular hyporeactivity may be related to the calcium desensitization of the contractile apparatus (a decrease of force/Ca2+ ratio) in vascular smooth muscle cell (VSMC), and calcium desensitization played an important role in vascular hyporeactivity. Rho-kinase was involved in the regulation of calcium sensitivity of vascular smooth muscle. Rho-kinase agonist Ang-Ⅱsignificantly increased the vascular reactivity and calcium sensitivity following hemorrhagic shock, which suggested that Rho-kinase maybe a regulatory target to restore shock-induced vascular hyporeactivity. Whether IPC and NS1619 preconditioning improve vascular reactivity and calcium sensitivity of blood vessel by activating Rho-kinase and its mechanism are not clear. To elucidate these problems we used hemorrhagic shock model of rats to investigate the effects of IPC and NS1619 preconditioning on vascular reactivity and calcium sensitivity following hemorrhagic shock and its relationship to Rho-kinase. Methods:The experiments were conducted in two parts. In these two parts, we observed the effect of IPC and NS1619 preconditioning on the vascular reactivity and calcium sensitivity following hemorrhagic shock and its relationship to Rho-kinase in rats respectively. First, we observed the effect of different IPC methods and different doses of NS1619 preconditioning on the survival rate and survival time, and determined the optimal IPC method and optimal dose of NS1619 preconditioning. The response of mean arterial pressure (MAP) and contractile response of SMA to norepinephrine (NE, 3μg/kg) (reflected by the changes of blood vessel diameter) in vivo and the effects of IPC on them were observed after hemorrhagic shock.With isolated superior mesenteric artery (SMA), the vascular reactivity and calcium sensitivity were determined via observing the contraction initiated by NE and Ca2+ under depolarizing conditions (120mmol/L K+) with isolated organ perfusion system. The effect of IPC and NS1619 preconditioning on the vascular reactivity and calcium sensitivity following hemorrhagic shock were observed. Rho-kinase activity was expressed as the ratio of phosphorylation levels of MPYT (pMPYT) to those of MYPT, which was measured by with western blot analysis. RhoA activity in the two parts was measured respectively with the method of pulldown and G-LISA. The changes of Rho-kinase and RhoA activities of SMA at late shock and the effects of IPC and NS1619 preconditioning on them were observed. Meanwhile, the effects of Rho-kinase and RhoA antagonists on vascular reactivity and calcium sensitivity, and the effects of RhoA antagonists on Rho-kinase activity of SMA from IPC and NS1619 preconditioning rats (3h after shock) were also studied. The activities of p115-RhoGEF, PDZ-RhoGEF and LARG were measured by the RhoGEF activity assay kit. The effect of IPC and NS1619 preconditioning on the activities of p115-RhoGEF, PDZ-RhoGEF and LARG of SMA at late shock (3h after shock)were observed, and the relationship among the changes of Rho-kinase and RhoA activity and those of RhoGEF activity were analyzed.Results:1. IPC and NS1619 preconditioning significantly increased the survival time and survival rate of hemorrhagic shock rat.2. In vivo, the pressor response of NE was increased and contractile response of SMA to NE were significantly increased at early shock (P<0.01), but they were significantly decreased at late shock (P<0.01). IPC significantly increased the pressor response of NE and the contractile response of SMA to NE as compared with shock control group at late shock(P<0.01).3. As compared with sham operated group, the vascular reactivity of SMA was increased significantly at early shock (10min after shock). Its cumulative dose-response curve to NE was significantly shifted to the left, and the maximal contractions (Emax) of SMA to NE was significantly increased (P<0.05 or P<0.01). But as shock prolonged, the vascular reactivity and the calcium sensitivity of SMA was decreased, the cumulative dose-response curves of SMA to NE and Ca2+ at late shock(3h after shock) shifted to the right, Emax of NE and Ca2+ were significantly decreased(P<0.05 or P<0.01). IPC and NS1619 preconditioning significantly increased the contractile response of SMA to NE and Ca2+ as compared with shock control group. The Emax of SMA to NE and Ca2+ in the IPC group and the NS1619 preconditioning group were significantly higher than that in the shock control group (p<0.01).4. At late shock (3h after shock), the activity of Rho-kinase and RhoA were significantly decreased as compared with sham operated group (p<0.05), IPC and NS1619 preconditioning significantly increased the activities of Rho-kinase and RhoA as compared with shock control group(p<0.05). Y-27632 and C3 Transferase, Rho-kinase and RhoA antagonist, significantly abolished the protect effect of IPC and NS1619 preconditioning on vascular reactivity and calcium sensitivity following hemorrhagic shock. The cumulative dose-response curves of SMA to NE and Ca2+ significantly shifted to the right, and Emax of NE and Ca2+ were significantly decreased (p<0.05 or p<0.01). Meanwhile, RhoA antagonist C3 Transferase signicantly dereased IPC-induced and NS1619 preconditionging-induced increase of the activity of Rho-kinase of SMA at late shock (3h after shock) (p<0.05).5. As compared with sham operated group, PDZ-RhoGEF activity was significantly decreased at late shock (3h after shock) (P<0.05), but the activity of p115-RhoGEF and LARG did not change (P>0.05). IPC and NS1619 preconditioning significantly increased shock-induced dereased PDZ-RhoGEF activity (P<0.05). It was positively correlated with the changes of Rho-kinase and RhoA activities induced by IPC and NS1619 preconditioning. Conclusions:1. IPC and NS1619 preconditioning can improve shock-induced reduced vascular reactivity and calcium sensitivity, improve the survival time and survival rate of hemorrhagec shock rats.2. RhoA/Rho-kinase plays important roles in the protective effect of IPC and NS1619 preconditioning on the shock-induced vascular hyporeactivity. IPC and NS1619 preconditioning may activate RhoA and Rho-kinase.3. Among three RhoGEFs, PDZ-RhoGEF is the only one involved in the protective effect of IPC on the shock-induced vascular hyporeactivity as the upstream regulator of RhoA/Rho-kinase. PDZ-RhoGEF may be a new regulating target to restore the vascular hyporeactivity following hemorrhagic shock.