| Vasohyporesponsiveness (VHR) is one of the most important causes in the occurrence of circulatory failure and death in severe trauma and shock in clinic, but its mechanism remains uncertain. Several reports showed that hemorrhagic shock could not only upregulate protein tyrosine kinase (PTK) activity, but also the inhibition of protein tyrosine phosphorylation induced by PTK inhibitor genistein (GST) could restore vascular responsiveness of resistant artery in hemorrhagic and endotoxic shock, which means there maybe some relationship between protein tyrosine phosphorylation mediated by PTK and vasohyporesponsiveness. But till now, it is still not known which target protein(s) is (are) involved in the development of vasohyporesponsiveness in shock. Our previous work showed that large conductance calcium-activated potassium channel (BKCa) in superior mesenteric artery (SMA) was excessively activated following hemorrhagic shock in rat. It plays an important role in the occurrence of vasohyporesponsiveness. Studies showed that there are some tyrosine sites in αsubunit, which are associated with the modulation of BKCa channel activity. It was suggested that BKCa channel αsubunit maybe the downstream signal target protein of hemorrhagic shock-induced protein tyrosine phosphorylation. In this study, a hemorrhagic shock model was established in rat and the immunoprecipitation (IP) and Western blotting (WB), cell-attached patch clamp and classical vascular tension measurement were adopted to explore: (1) the effects of hemorrhagic shock on BKCa channel α subunit tyrosine phosphorylation and its modulation; (2) the role of BKCa channel α subunit tyrosine phosphorylation in the activation of BKCa channel and the development of vasohyporesponsiveness in hemorrhagic shock; (3) the role of BKCa channel αsubunit tyrosine phosphorylation in the vasohyporesponsiveness induced by nitric oxide (NO) and endothelin-1 (ET-1) and their signal transduction. It is aimed to elucidate the molecular mechanisms of vasohyporesponsiveness induced by hemorrhagic shock and provide the experimental basis for selecting the new molecular taget to treat vascular responsiveness. The main results and conclusions are as the followings:1. IP and WB results showed that hemorrhagic shock (30 mmHg, 2h and 4h) could up-regulate BKCa channel α subunit tyrosine phosphorylation in superior mesenteric artery (SMA) in a time-dependent manner, and it was demonstrated that PTK and protein tyrosine phosphatase (PTP) were involved in the modulation of BKCa channel α subunit tyrosine phosphorylation in primary cultured smooth muscle cell of SMA. 2. Hemorrhagic shock could induce BKCa channel activation in cell-attached configuration, which was characterized with the decrease of mean close time (Tmc) and slow close time constant (τcs), along with the increase of channel open probability (Po)and the half-activated potential (V?). PTK inhibitor GST could lengthen Tmc and τcs and decreased the Po and V?, while some kinds of PTK activator and PTP inhibitor Na3VO4 could restore the effects of GST, which means that BKCa channel α subunit tyrosine phosphorylation mediated by PTK and PTP was associated with the activation of BKCa channel in hemorrhagic shock rat. The results of experiment in vitro showed that GST could restore vasohyporesponsiveness of SMA from hemorrhagic shock rats, while Na3VO4 could cause a further decrease of vasoresponsiveness to NE, which was partly inhibited by 0.1mM TEA (1mM or less of TEA is considered to be able to specifically block BKCa channel), the results show that BKCa channel tyrosine phosphorylation was associated with the development of vasohyporesponsiveness following hemorrhagic shock. 3. A vasohyporesponsiveness model induced by L-arginine and ET-1 in vitro were adopted to explore the role of BKCa channel tyrosine phosphorylation in NO and ET-1 induced vasohypresponsiveness and their signal transduction. The results showed that BKCa channel inhibitor, low concentration of TEA (0.1mM), could partly res... |
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