| Background and objectiveDecreased systemic Vascular reactivity or no reactions are defined as vascular hyporesponsiveness, Its main clinical manifestations are vasoconstriction dysfunction and refractory hypotension. Numerous clinical studies have found that perioperative obstructive jaundice (OJ) patients often have decreased peripheral vascular resistance and low blood pressure, and furthermore the blood vessels are not sensitive to endogenous and exogenous substances. Its exact mechanism remains unclear. Receptor desensitization, internal environment disorders, vascular smooth muscle cells (VSMCs) membrane hyperpolarization are considered as potential mechanisms. Vascular tone is mainly regulated by vascular smooth muscle, and potassium channels are essential in the regulation of vascular smooth muscle contraction and relaxation. The potassium channels in vascular smooth muscle cells are divided into four kinds:Kv (voltage-dependent potassium channel), KATP(ATP-sensitive potassium channels), Kir (inward rectifier potassium channel), Kca (calcium-activated potassium channels). The role of KATP and BKCa are very important. In this study, we want to use animal experiments and molecular biological experiments to prove the structure and function of potassium channels in obstructive jaundice rats vascular smooth muscle cells change or not. To explore the links between the changes and obstructive jaundice vascular hyporesponsiveness.Contents and methodsFirst, male Sprague-Dawley rats were selected to establish bile duct ligation (BDL) model, anesthetized each group rats to detect blood biochemical indices. Preparation of thoracic aortic rings of rats to measure each group rats artery contraction and relaxation reactivity to different concentrations of the drugs(NE & SNP). Changes in vascular tone were recorded and analyzed after treated with different type potassium channel blockers. Then thoracic aortic smooth muscle cells were obtained by enzymatic digestion, and using whole-cell patch-clamp technique to recording each group rats vascular smooth muscle cells KATP and BKCa currents,then recording currents after treated with blockers like charybdotoxin and glibenclamide. We analyze and get the specificity channel currents. We extracte total RNA and proteins from fresh thoracic aorta tissue of two groups rats to detect the amount of each channel protein and mRNA. We use HE staining and immunofluorescence staining techniques to observe pathology and molecular changes in the arterial tissue of BDL and Sham rats.Main resultsWe found that the vascular contractile response of BDL rats to vasopressor like NE are reduced, while the vascular reaction of BDL rats to antihypertensive drugs like SNP are increased. After using different types of potassium channel blocker, We found that the vascularl reactivity of BDL rats are obviously restored compared with the control group rats. Especially blocking BKCa、KATP lead to BDL rat thoracic aorta contractile response to NE significant recovery compared with control and sham group rats thoracic aorta, while blocking Kv, Kir channel that is not obvious. Using whole-cell patch-clamp recordings in primary cultured rat isolated thoracic aortic VSMCs confirmed BKca current and KATP currents significantly increased in the BDL rats thoracic aortic smooth muscle cells. After further experiments, we found BDL rats thoracic aorta BKca channels and KATP channel regulatory subunit BKCa-β1, SUR-2B protein levels and mRNA levels were upregulated, and their respective constitutive subunits BKCa-α and Kir6.1 have no changes. We found that BDL rats compared with Sham rats have no significant pathological changes after the thoracic aorta HE staining. We also confirmed the vascular smooth muscle BKCa-β1 and SUR-2B upregulation after specific immunofluorescence staining.ConclusionIn this study, We found that obstructive jaundice caused widespread vascular hyporesponsiveness. The results from the determination of vascular tone indicates that the mechanism may be the excessive activation of potassium channels in vascular smooth muscle cells, especially BKca and KATP.Western blot and RT-PCR results are further to prove molecular mechanisms underlying abnormal changes may be two regulatory subunits of BKca and KATP are upregulated in gene and protein levels. |
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