The Role Of Calcium Desensitization Of Vascular Smooth Muscle In Vascular Hyporeactivity Following Hemorrhagic Shock And Its Regulatory Molecules

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 receptor desensitization, including adrenergic, endothelin receptors'desensitization, or the membrane hyperpolarization of vascular smooth muscle cell(VSMC), initiated by the over production of NO, functional disorder of the K⁺ and Ca²⁺ channels of VSMC. However, antagonizing NO, ameliorating the function of the K⁺ and Ca²⁺ channels, recovering the receptor sensitivity of NE, Ang-â…¡and endothelin, and improving the polarization state of the cell membrane cannot restore the vascular reactivity completely. Our previous study showed that calcium overload and calcium desensitization (the decrease of force/Ca²⁺ ratio) coexisted in the myocardium cell following severe trauma or shock, which resulted in less reactivity of the myocardium cell to the traditional cardiotonics, with the increased intracellular calcium concentration([Ca²⁺]_i). In addition, calcium overload was also found in VSMC following shock, but the relationship between vascular hyporeactivity and calcium desensitization is still unknown. We supposed that calcium desensitization may exist in the vascular smooth muscle cell and play an important role in the regulation of vascular hyporeactivity following hemorrhagic shock. Objective: To observe the role of calcium desensitization of vascular smooth muscle in vascular hyporeactivity, the regulatory effects and the possible mechanisms of Rho-kinase, PKC and PKG on calcium sensitivity following hemorrhagic shock(HS) in rats. Methods: The superior mesenteric artery(SMA) from hemorrhagic shock model of rat was adopted to assay the vascular reactivity and calcium sensitivity via observing the contraction initiated by norepinephrine(NE) and Ca²⁺ under depolarizing conditions(120 mmol/L K⁺) with isolated organ perfusion system. The experiments were conducted in three parts. In the first part, we observed whether or not calcium desensitization was existed in the hyporesponsive blood vessels, and if calcium desensitization of blood vessel played an important role in vascular hyporeactivity by observing whether Ang-â…¡and insulin, the calcium sensitivity regulating agents, and fasudil, a relatively selective antagonist of Rho-kinase could regulate the vascular reactivity through regulating the calcium sensitivity. In the second part, using Rho-kinase agonist Ang-â…¡and antagonist fasudil, PKC agonist PMA and antagonist staurosporine, PKG agonist 8Br-cGMP and antagonist KT-5823 as tool agents, we studied the regulatory effect of Rho-kinase, PKC and PKG on the calcium sensitivity of SMA following hemorrhagic shock. In the third part, we studied the possible mechanisms of Rho-kinase, PKC and PKG on calcium sensitivity of VSMC by observing the role of Calyculin A, a MLCP antagonist in the regulation effects of Rho-kinase, PKC and PKG on calcium sensitivity. Results: (1)As compared with the normal control, the cumulative dose-response curve of SMA to NE and Ca2+ in shock group were shifted to the right, the maximal contraction(Emax) and pD2(-log[EC50]) of NE and Ca2+ were decreased significantly (P<0.05, P<0.01). It was suggested that the calcium sensitivity was decreased following hemorrhagic shock. (2)Ang-â…¡made the cumulative dose-response curve of NE and Ca2+ shift to the left, and increased the contractile response of NE and Ca2+(P<0.05, P<0.01); On the contrary, insulin made the cumulative dose-response curve of NE and Ca2+ shift to the right and decreased the contractile response of NE and Ca2+(P<0.05); Pretreatment with fasudil abolished the improvement effect of Ang-â…¡on vascular contraction initiated by NE, and reduced the calcium sensitivity. These results along with the first results suggested that calcium desensitization played an important role in the incidence of vascular reactivity. (3)Ang-â…¡, PMA and KT-5823 improved the calcium sensitivity of SMA and made the cumulative dose-response curve of SMA to Ca2+ shift to the left, their Emax was 0.630 g/mg, 0.595 g/mg and 0.624 g/mg, respectively, which were all higher than shock control(0.377 g/mg)(P<0.05, P<0.01); fasudil, staurosporine and 8Br-cGMP decreased the calcium sensitivity of SMA and made the cumulative dose-response curve of Ca2+ shift to the right, their Emax of Ca2+ was 0.242 g/mg, 0.230 g/mg and 0.256 g/mg, respectively, which were all lower than shock control(0.377 g/mg)(P<0.05, P<0.01). These results suggested that Rho-kinase, PKC and PKG had important regulatory effects on calcium sensitivity of vascular smooth muscle following hemorrhagic shock. (4)Calyculin A pretreatment further enhanced Ang-â…¡and PMA induced increase of calcium sensitivityand made the cumulative dose-response curve of Ca2+ shift to the left, their Emax was 0.902 g/mg and 0.998 g/mg, higher than Ang-â…¡group(0.630 g/mg) and PMA group(0.595 g/mg) respectively(P<0.05, P<0.01), yet Calyculin A inhibited 8Br-cGMP induced decrease of calcium sensitivity and made the cumulative dose-response curve of Ca2+ shift to the left. Its Emax was 0.377 g/mg, higher than the 8Br-cGMP group(0.256 g/mg)(P<0.05). These results suggested the regulatory effects of Rho-kinase, PKC and PKG on calcium sensitivity were closely related to MLCP. Conclusion: (1)Calcium desensitization existed in the vascular smooth muscle following hemorrhagic shock and played an important role in vascular hyporeactivity, which was one of the important mechanisms of the occurrence of vascular hyporeactivity. (2)Rho-kinase, PKC and PKG played important roles in the regulation of calcium sensitivity of vascular smooth muscle in hemorrhagic shock. (3)The mechanisms of Rho-kinase, PKC and PKG regulating the calcium sensitivity of vascular smooth muscle were possibly through MLCP.