The Effects Of Hydrogen Sulfide/EDHF And Total Flavones Of Rhododendra On Bk_Ca Channels In Rat Middle Cerebral Artery Smooth Muscle Cells

Endothelium-derived hyperpolarizing factor(EDHF) is the third relaxing factors which produced by vascular endothelium following nitric oxide(NO) and prostacyclin(CO).The phenomena of EDHF have been found in different peripheral vessels and the substance of EDHF varies in different types or different size vessels. It has been confirmed that H2 S was the EDHF in rat cerebral artery. The underlying mechanisms of vasorelaxant effects by H2 S on cerebral vessels are still unclear. Total flavones of rhododendra can make vasodilation and protection against stroke.But the relationship between H2 S and vasodilation effects made by TFR remains unknown. By using patch clamps and other technologies,we try to investigate the vasodilation mechanisms of H2 S and TFR on rat middle cerebral artery and clarify the possible relationship between them.Purpose: 1. To observe the influences of exogenous H2 S on BKCa current and protein in rat middle cerebral artery smooth muscle cells. Explore the vasodilation mechanisms of H2 S on electrophysiological and molecular sides to prove that H2 S is an EDHF in cerebral artery. 2. To observe the influences of TFR on BKCa current in rat middle cerebral artery smooth muscle cells. Explore the protective mechanisms of TFR on electrophysiological side. 3. To explore the possible relationship between H2 S and influences of TFR on BKCa current in rat middle cerebral artery smooth muscle cells.Methods: 1. Isolating the MCA smooth muscle cells by enzymatic hydrolysis. 2.Recording the currents of BKCa channel in MCA smooth muscle cells by whole-cell patch clamp technology and grouping as follows:(1) Selecting the suitable smooth muscle cells and forming whole-cell condition,recording the influence of Na HS on BKCa currents in MCA smooth muscle cells with incremental concentration.(2) Recording the influence of TFR on BKCa currents in MCA smooth muscle cells with incremental concentration.(3) Exploring the relationship between endogenous H2 S and TFR on BKCa currents in MCA smooth muscle cells by the application of PPG to inhibit the produce of H2 S. 3. MCA smooth muscle cells were cultivated in primary culture and were identificated by immunohistochemistry. 4.Detecting the influences of Na HS on BKCa channel protein by western blot method.Results: 1. As patch clamp results showed, the currents of BKCa could be recorded and were inhibitated by IBTX with 100 nmol/L concentration in isolated smooth muscle cells. 2. As patch clamp results showed, Na HS(10、100、500nmol/L)could activate BKCa channel with concentration-dependent phenomenon and the activation could be blocked by the application of IBTX. 3. As patch clamp results showed, the current of BKCa could be increased by TFR(33.3、100、300 mg/L)with concentration-dependent phenomenon(P<0.001)and the activation could be blocked by the application of IBTX. 4. As patch clamp results showed, TFR could also activate the current of BKCa despite the usage of 100 mmol/L PPG(inhibitor of endogenous H2 S synthetase). 5. In immunohistochemistry identification experiment, many brown myofilament were showed in the intracytoplasm of cells under the microscope. It confirmed that the cultivated cells were smooth muscle cells.6. As western blot results showed,compared with contral group, the protein of BKCa channel of all groups treated with Na HS had no statistical differences(P>0.05).Conclusion: 1. The mechanisms of vasodilation induced by H2S(EDHF)in rat MCA related to the activation of smooth muscle cell BKCa currents and H2 S may not be able to change the expression of BKCa protein. 2. TFR could activate BKCa currents in MCA smooth muscle cells and this may be one of the reasons why TFR could make vasorelaxant effects.