Molecular Mechanism Of ATP Release By Cerebral Artery Smooth Muscle Cells After Subarachnoid Hemorrhage

Objective(s):Subarachnoid hemorrhage is a serious common diseases,but the pathological mechanism of complex for its happened is not clear at present usually put the oxygenated hemoglobin in the blood(foxy-hemoglobin,OxyHb)is one of the important reasons causing vasospasm Rho kinase has been proved to be the G protein Rho downstream target of monomer,it can participate in continuous through phosphorylation and inhibit vasoconstriction RhoA/Rho kinase pathways play a role in the constriction of OxyHb,used the Rho OxyHb in the blood vessels of contraction mechanism of translocation ATP has significantly vasoconstriction,and studies have found that smooth muscle cells through autocrine secretion of ATP function,but did not clarify whether ATP can role in cerebral vasospasm after SAH caused by,so I want to confirm the change of ATP,under the condition of SAH to discuss whether it could be related to the CVS gap junction is one of the most common form of intercellular communication they are composed of membrane proteins,forming the passage between the ions and small molecules,these ions and small molecules connected to the adjacent cell cytoplasm.Studies have shown that in the classification of different groups,including the vertebrates,exists with certain invertebrate gap junction protein was named innexins homologous genes,clear detection by PSI-BLAST search,and named it pannexin-1(Panx1)a large number of studies have shown that the activation of Panx1 leads to extracellular ATP levels,ATP in the cell as intracellular metabolites by Panx1 channel is released under the condition of physiological and pathological physiology,cell ATP release;Extracellular ATP on the extracellular enzyme(mainly CD39 and CD73)before the rapid degradation of adenosine,autocrine and paracrine role the role of extracellular ATP(P2R)mediated by P2 cell surface receptors,including across the cell membrane cation channels P2X receptors(P2XR)G protein coupled receptor P2Y receptors can purines(P2YR)P2XR on behalf of the ATP gating homogeneous or ion channels,is composed of three subunits,is one of the most important sub P2X1 P2X2 P2X3P2X4 and P2X7,ATP is sensitive to P2X1 and P2X4,and ATP affects body function by activating P2X purinable receptors.This topic with ATP as the theoretical basis for important signaling molecule vasoconstriction,through exploring Subarachnoid Hemorrhage,Subarachnoid Hemorrhage,SAH)when the concentration of ATP and vasospasm inhibition of Rho and relations with ATP content and SAH pannexin-1 Rho and express the relation between the quantity change and pannexin-1 P2X1 after SAH and P2X4 express the change of the quantity,so as to prove the existence of SAH afterbrain arterial smooth muscle cells of the release of ATP in CVS mechanism.Methods:Arterial smooth muscle cells were cultured in an oxygenated hemoglobin(OxyHb)environment to simulate the SAH microenvironment.After pharmacological inhibition of Rho and pannexin-1(PANX1)in cerebral artery smooth muscle cells,the ATP concentration in the culture group was detected by chemluminescence method,and the expression levels of PANX1 and P2X of panligase protein released by OxyHb receptor protein Rho and ATP receptor P2X were detected by western blot.1.Simulated in vitro subarachnoid hemorrhage microenvironment:(1)culture of rat vascular smooth muscle cell lines;(2)in vitro induced subarachnoid hemorrhage model;(3)the concentration of ATP released into the cell medium was detected.2.Specific inhibitors inhibit related genes:(1)use specific inhibitors to inhibit Rho and pannexin-1(PANX1)genes in cerebral artery smooth muscle cells;(2)the concentration of ATP in the medium was detected by chemiluminescence to verify whether the increase of ATP promoted by OxyHb was realized by rho-panx1 pathway.3.Immunoprotein imprinting:(1)first detect the OxyHb receptor protein RhoA;(2)PANX1 of panligase protein releasing ATP was detected;(3)the expression levels of ATP receptors P2X1 and P2X4 were detected.4 statistical methods:k-s test was used to determine whether the sample data met the normal distribution,and Levene test was used to determine whether the sample data met the homogeneity of variance.Kruskal-wallis H test was used for the comparison of multiple groups that did not conform to the normal distribution.The results of ppaired comparison were expressed as Mean standard deviation(Mean SD)or median(quartile)by the Bonferroni correction method.The test level was=0.05,and the difference was statistically significant if P<0.05 on both sides.Results:1.OxyHb can induce the increase of ATP release in cerebral artery smooth muscle cells,and the relative concentration of ATP is the highest when OxyHb concentration is 10μM for 24 hours(t=16.076,P=0.000,t=16.142,P=0.000,t=16.142,P=0.000).2.Inhibition of Rho or PANX1 can reduce ATP release(tl=8.333,P1<0.001).T2＝27.913,P2<0.001).3.The expression of Rho PANX1 protein in cells cultured in OxyHb mixed medium increased.4.4.The protein expression of P2X1 P2X4 in cells cultured in OxyHb mixed medium increased,with the highest expression of P2X1 on the 14th day and the highest expression of P2X4 on the 7th day.Conclusion(s):1.Increased release of ATP in the brain after subarachnoid hemorrhage.2.There is a rho-pannexin-1 pathway in arterial smooth muscle cells that releases ATP extracellular.3.The expression of ATP receptor P2X is also up-regulated.4.Cerebral artery smooth muscle cells after SAH can promote the release of extracellular ATP and up-regulation of the receptor P2X through rho-panxl pathway,which may be one of the mechanisms causing cerebral hemodynamic disorder after SAH and provide a new idea for the clinical solution of sah-induced vasospasm.It provides a new idea for clinical treatment of vasospasm caused by SAH.