The Role Of Calcium Desensitization Of Vascular Smooth Muscle In Vascular Hyporeactivity And The Regulation Of TNF-α Following Endotoxic Shock

There is vascular hyporeactivity in the prolonged period of various shock, including hemorrhagic shock and septic shock. Vascular hyporeactivity is that blood vessels are refractory to vasoconstrictors or vasodilatators, which plays an important role in the incidence,development and therapy of shock.Meanwhile, it is associated with the failure of boosting pressure leading to multiple organ dysfunction syndrome. The occurrence of vascular hyporeactivity after shock may be related to receptor desensitization, or the membrane hyperpolarization of vascular smooth muscle cell(VSMC). However, recovering the receptor sensitivity and improving the polarization state of the cell membrane only partially ameliorate the vascular reactivity. Our previous study showed that calcium desensitization existed in VSMC following hemorrhagic shock, and played important role in vascular hyporeactivity. Whether there is calcium desensitization and it also plays an important role in vascular hyporeactivity in endotoxic shock is not clear. The characteristic of the pathophysiology in endotoxic shock is systemic inflammatory response syndrome(SIRS). The continuing express of a great number of cytokines took part in the Pathophysiology of endotoxic shock.Previous study have demonstrated that sepsis-like shock can be induced by TNF-α.There is no report whether TNF-αtakes part in the regulation of calcium desensitization and the development of vascular hyporeactivity following endotoxic shock is not determined. To elucidate above issues we used endotoxic shock model of rabbits to investigate the role of calcium desensitization of vascular smooth muscle in vascular hyporeactivity, the regulatory effects and the possible mechanisms of TNF-αon calcium sensitivity following endotoxic shock.Methods:The superior mesenteric artery(SMA) from normal and LPS induced endotoxic shock rabbits was adopted to assay the vascular reactivity and calcium sensitivity via observing the contraction initiated by norepinephrine(NE) and Ca2+ under depolarizing conditions(120mmol/LK+)with isolated organ perfusion system.Meanwhile,the phosphorylation of MLC20 was measured by western blot. Rho kinase activity and plasma TNF-αlevels were measured by ELISA. The experiments were conducted in three parts. In the first part, we observed whether calcium desensitization was existed in the hyporesponsive blood vessels following endotoxic shock or not, and if calcium desensitization of blood vessel played an important role in vascular hyporeactivity by observing AVP and insulin, the calcium sensitivity regulating agents could regulate the vascular reactivity through regulating the calcium sensitivity. In the second part,in order to confirm the regulatory effect of TNF-αon the calcium sensitivity of blood vessels following endotoxic shock,we observed serum TNF-αlevels from rabbits at different time after 1mg/kg LPS intravenous injection and the regulatory effect of different concentration of TNF-αon calcium sensitivity of SMA. In the third part, in order to study the possible mechanisms of TNF-αon calcium sensitivity of blood vessels following endotoxic shock, we observed the role of Y-27632,a specific inhibitor of Rho-kinase, and PKC inhibitor Staurosporine on calcium sensitivity of SMA incubating with low concentration of TNF-αand effects of TNF-αon the activity of Rho kinase .Results:1. As compared with the normal control group , the vascular reactivity of SMA to NE and Ca2+ was increased in the early period at 30 min and 1 hour following 1mg/kg LPS intravenous injection, the cumulative dose-response curves of SMA to NE and Ca2+ were shifted to the left, the maximal contraction (Emax) of NE and Ca2+ was significantly increased (P<0.05 or p<0.01).But the vascular reactivity of SMA to NE and Ca2+ was decreased in the later period, the cumulative dose-response curves of SMA to NE and Ca2+ were shifted to the right, the maximal contraction (Emax) of NE and Ca2+ were significantly decreased (p<0.01).Meanwhile, the phosphorylation of MLC20 to SMA was increased in the early period at 30 min and 1 hour following 1mg/kg LPS intravenous injection, the phosphorylation of MLC20 to SMA was greatly decreased at 2 hour after 1mg/kg LPS intravenous injection(P<0.01),but was slightly increased in the later period at 4 hour and 6 hour following 1mg/kg LPS intravenous injection(P<0.05).All these results suggested that the calcium sensitivity was decreased following endotoxic shock.2. Calcium sensitivity regulating agent AVP(1×10-9mol/L) made the cumulative dose-response curve of SMA to NE and Ca2+ shift to the left, and increased the maximal contraction (Emax) of SMA to NE and Ca2+(p<0.05 or p<0.01);however, insulin(1×10-7mol/L) made the cumulative dose-response curve of SMA to NE and Ca2+ shift to the right and decreased the contractile response of NE and Ca2+(p<0.05 or p<0.01). These results suggested that calcium desensitization played an important role in the development of vascular hyporeactivity.3. As compared with the normal control group,serum TNF-αbegan to increase at 2 hours and reached the peak at 4 hour after LPS administration and almost decreased to the normal control level at 6 hour following LPS administration (P<0.05,P<0.01). Lower concentration of TNF-α(20ng/ml) incubated with SMA significantly increased the vascular reactivity of SMA to Ca2+, while higher concentration of TNF-α(200ng/ml) significantly decreased the vascular reactivity of SMA to Ca2+ (P<0.05,P<0.01).These results showed that TNF-αcan regulate the calcium sensitivity of vascular smooth muscle in endotoxic shock rabbits.4. Rho-kinase inhibitor Y-27632 could abolish TNF-α(20ng/ml) induced increase of vascular reactivity of SMA to Ca2+. PKC inhibitor Staurosporine had no effect on this effect of TNF-α(20ng/ml) (P>0.05). As compared with the normal control group, the Rho kinase activity of SMA was increased in the early period (at 30 min and 1 hour) following 1mg/kg LPS intravenous injection(P<0.05), but the Rho kinase activity of SMA was significantly decreased in the later period (at 6 hour) following 1mg/kg LPS intravenous injection(P<0.01). Meanwhile,lower concentration of TNF-α(20ng/ml) significantly increased the Rho kinase activity and phosphorylation of MLC20 of SMA(P<0.05,P<0.01),but higher concentration of TNF-α(200ng/ml) significantly decreased the Rho kinase activity and phosphorylation of MLC20(P<0.05,P<0.01), meanwhile Y-27632(10-5mol/L) abolished the TNF-α(20ng/ml) induced increase of Rho kinase activity and MLC20 phosphorylation(P<0.01).These results suggested the regulatory effect of TNF-αon the calcium sensitivity of SMA following endotoxic shock rabbits were closely related to Rho-kinase but not to PKC.Conclusion:1. Calcium desensitization existed in the vascular smooth muscle following endotoxic shock,which played an important role in vascular hyporeactivity endotoxic shock.2. TNF-αplayed an important role in the regulation of calcium sensitivity of vascular smooth muscle following endotoxic shock. Lower concentration of TNF-αsignificantly increased the vascular reactivity of SMA to Ca2+, while higher concentration of TNF-αsignificantly decreased the vascular reactivity of SMA to Ca2+.3. The regulatory effect of TNF-αon the calcium sensitivity of vascular smooth muscle following endotoxic shock rabbits was closely related to Rho-kinase.