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本实验室先前的研究已证实,普罗托品(protopine,Pro)舒张家兔主动脉的作用,可能与其增加血管平滑肌细胞内cAMP和cGMP水平有关。为了深入探讨Pro的扩血管作用机制,实验采用等张收缩记录大鼠离体血管条张力,利用Fura-2/ AM负载的大鼠胸主动脉培养细胞直接测定细胞内游离Ca2+浓度([Ca2+]i),并应用同位素γ-32P-ATP催化活性法测定蛋白激酶C (PKC)活性等方法,分别观察了Pro的相关效应。结果表明,Pro(30和100μmol/L)明显降低去甲肾上腺素(NA)和高钾所致的动脉条收缩幅度,使二者的量效曲线呈非平行右移,最大反应压低;pD21值分别为3.7±0.25和3.97±0.15;Pro(50和100 μmol/L)对静息状态下的[Ca2+]没有任何影响,但对NA和高钾引起的[Ca2+]i升高均有明显抑制作用;Pro(30和100 μmol/L);对未经NA处理血管条的胞浆和胞膜PKC活性均无明显影响:但在NA预处理的血管条,Pro使NA所升高的胞浆内PKC的活性趋于降低,而明显升高胞膜PKC的活性,对PKC的总活性无明显影响。结果提示,在有NA存在的情况下,Pro似能促使PKC从胞浆向细胞膜转移,其扩血管效应似为其降Ca2+作用、升高cAMP和cGMP的作用及其对PKC影响等几方面的综合结果。
Previous studies in our laboratory have confirmed that the effect of protopine (Pro) on aortic relaxation in rabbits may be related to increasing cAMP and cGMP levels in vascular smooth muscle cells. In order to further explore the vasodilator mechanism of Pro, the isometric contraction of isolated rat vascular strips was used to measure the intracellular Ca2 + concentration (Ca2 +) in Fura-2 / AM loaded rat thoracic aorta cells. i), and the activity of protein kinase C (PKC) was measured by isotope gamma-32P-ATP catalytic activity assay. The related effects of Pro were observed. The results showed that Pro (30 and 100 μmol / L) significantly reduced the contractions of arterial strips caused by norepinephrine (NA) and hyperkalemia, which caused a non-parallel shift of the dose- (3.7 ± 0.25 and 3.97 ± 0.15, respectively); Pro (50 and 100 μmol / L) had no effect on resting [Ca2 +] but significant inhibition of [Ca2 +] i induced by NA and potassium Pro (30 and 100 μmol / L) had no significant effect on the cytosolic and membrane PKC activities of NA-treated vascular strips. However, in NA preconditioned strips, Pro increased the NA PKC activity tends to decrease, while significantly increased cell membrane PKC activity, the total activity of PKC no significant effect. The results suggest that in the presence of NA, Pro seems to promote the PKC translocation from the cytoplasm to the cell membrane, and its vasodilator effect is similar to its role in lowering Ca2 +, increasing cAMP and cGMP and its effect on PKC Comprehensive results.