肺血管平滑肌细胞钾通道分布不均--性及其病理生理调控

Potassium channel plays an important role in the physiologic and pathophysiologic regulation of pulmonary vascular smooth muscle tension and in its hypoxic reaction. In this project, we used whole cell patch clamp, molecular biology and other research techniques and applied specific agonists and antagonists to investigate the heterogenic distribution of different subtypes of potassium channel in normal as well as chroni hypoxic pulmonary vascular branches and vascular cells. We also investigated the relevant intracellular signaling mechanisms. The effects of long term application of NO on the potassium was also observed. These researches are useful for understanding the pathogenesis of hypoxic pulmonary hypertensin and can provide theoretic bases for clinical therapy. The purpose of this project was, with the use of the technology of whole cell patch clamp and under the condition of hypoxia,to investigate: (1)the reactions of different potassium channels to hypoxia; (2)the regulatory effects of various intracellular secong messengers, such as cAMP, cGMP and PKC, on voltage-gated potassium channel; (3)the regulatory effects of endothelin-1 (ET-1) on voltage-gated potassium channel in pulmonary artery smooth muscle cells from hypoxia-exposed rats; (4)the ion mechanisms of long term application of NO precursor L-arginin for the treatment of chroni hypoxic pulmonary hypertension; (5)the effects of cro on hypoxic pulmonary hypertension and its mechanism; and (6)the effects of lev, the levogyrate cro, on pulmonary artery endothelial cells and smooth muscle cells on normal and hypoxic culture conditions.Results1.Many spindle shaped cells were obtained via acute enzyme seperation metnod and identified to be PASMC with ?-actin stain. The outward current recorded with whole cell patch clamp technique was proved to be potassium current while the K+ in the pipette was replaced with Cs+. The peak potassium current at +50 mV was 225.5 ( 3.6 pA/pF. The current of votalge-gated channel was stable and did not change with time. At the beginning of whole cell patch clamp, the tow are almost overlapped (Fig.1). 2. Chronic hypoxia decreased the resting membrane potential of PASMCs but had no effect on the resting membrane capacitance. Chronic hypoxia significantly inhibited the potassium ion current of voltage-gated potassium channelbut increased the potassium ion current of Ca2+-sensitive potassium channel (Fig2 and Fig3)..3. The voltage-gated K + current in PASMCs from normal rats was inhibitd by PKC and was reversed by inhibitor of PKC. GFX. On the other hand, PKC had no effect on the voltage-gated K+ currents in PASMCs from rats expsed to chronic hypoxia. These results suggest that PKC has different effect on voltage-gated K+ current under different conditions.(Fig4,5,6).4. The voltage-gated K+ current was significantly inhibited by cAMP in normal rats from 150.0 ( 9.4 pA/pF to 133.0 ( 1.8 pA/pF at +50 mV (n+6, P<0.05). This effect was reversed by H-89, cAMP+PKA inhibitor, and the voltage-gated K+ current measured was 102.8 ( 11.6 pA/pF and 103.8 ( 10.4 pA/pF,respectively (n=6, P>0.05). The voltage-gated K+ current in hypoxia group was not affected by cAMP and the current at 50 mV before and after the application of cAMP was 72.0 ( 2.3 pA/pF and 79.9 ( 3.3 pA/pF, respectively (n=6, P<0.05).(Fig7,8,9)5. cGMP exhibited inhibitory effects of voltage-gated potassium channel of PASMC from both normal and chronic hypoxic rats and this inhibition effect was reversed by H-8, an inghibitor of PKG, suggesting that the inhibitory effect of cGMP is dependent on the signaling pathway of protein kinase G Fig10,11,12,13)..6. Immunohistochemistry technique was used to investigate the expression of ET-1 and its receptor ETAand ETB along the axis of intra-pulmonary arteries. The results showed that along the axis of pulmonary arteries the expression of ET-1 increased gradually from the proximal end to the distal end. The positive staining was mainly located in the inner and medial membrain of pulmonary arteries. Chronic hypoxia led to an inc

钾通道在调控肺血管平滑肌生理、病理生理张力和氧反应笥中起重要作用。本课题拟用膜片钳技术、分子生物学技术及其它技术，结合特异的激动／拮抗剂，阐明不同亚型钾通道在正常／慢性缺氧肺血管段和血管平滑肌细胞分布不均一性，其细胞内调控机制以及长期应用ＮＯ对其影响，有助于进一步明确缺氧肺动脉高压发病机理，并为临床治疗提供理论基础。