Püschel G P, Miura H, Neuschäfer-Rube F, Jungermann K
Institut für und Molekulare Zellbiologie, Georg-August-Universität, Göttingen, Germany.
Eur J Biochem. 1993 Oct 1;217(1):305-11. doi: 10.1111/j.1432-1033.1993.tb18247.x.
In perfused rat livers, infusion of prostaglandin F2 alpha (PGF2 alpha) or noradrenaline increased glucose and lactate output and reduced flow. Glucagon increased glucose output and decreased lactate output without influence on flow. Infusion of phorbol 13-myristate 14-acetate (PMA) for 20 min prior to these stimuli strongly inhibited the metabolic and hemodynamic effects of noradrenaline, reduced the metabolic actions of PGF2 alpha but did not alter the effects of glucagon. In isolated rat hepatocytes PGF2 alpha, noradrenaline and glucagon activated glycogen phosphorylase but only PGF2 alpha and noradrenaline increased intracellular inositol 1,4,5-trisphosphate (InsP3). The noradrenaline- or PGF2 alpha-elicited activation of glycogen phosphorylase and increase in InsP3 were largely reduced after preincubation of the cells for 10 min with PMA, whereas the glucagon-mediated enzyme activation was not affected. In contrast to PMA, the phorbol ester 4 alpha-phorbol 13,14-didecanoate, which does not activate protein kinase C, did not attenuate the PGF2 alpha- and noradrenaline-elicited stimulation of glucose output, glycogen phosphorylase and InsP3 formation. Stimulation of InsP3 formation by AlF4-, which activates phospholipase C independently of the receptor, was not attenuated by prior incubation with PMA. Plasma membranes purified from isolated hepatocytes had both a high-capacity, low-affinity and a low-capacity, high-affinity binding site for PGF2 alpha. The Kd of the high-capacity, low-affinity binding site was close to the concentration of PGF2 alpha that increased glycogen phosphorylase activity half-maximally. Binding to the high-capacity, low-affinity binding site was enhanced by guanosine 5'-O-(3-thio)triphosphate (GTP[S]). This high-capacity, low-affinity site might thus represent the receptor. The Bmax and Kd of the high-capacity site, as well as the enhancement by GTP[S] of PGF2 alpha binding to this site, remained unaffected by PMA treatment. It is concluded that, in hepatocytes, activation of protein kinase C by PMA interrupted the InsP3-mediated signal pathway from PGF2 alpha via a PGF2 alpha receptor and phospholipase C to glycogen phosphorylase at a point distal of the receptor prior to phospholipase C.
在灌注的大鼠肝脏中，输注前列腺素F2α（PGF2α）或去甲肾上腺素可增加葡萄糖和乳酸的输出，并减少血流量。胰高血糖素增加葡萄糖输出，减少乳酸输出，但对血流量无影响。在这些刺激之前20分钟输注佛波醇13 - 肉豆蔻酸酯14 - 乙酸酯（PMA）可强烈抑制去甲肾上腺素的代谢和血流动力学效应，降低PGF2α的代谢作用，但不改变胰高血糖素的作用。在分离的大鼠肝细胞中，PGF2α、去甲肾上腺素和胰高血糖素可激活糖原磷酸化酶，但只有PGF2α和去甲肾上腺素可增加细胞内肌醇1,4,5 - 三磷酸（InsP3）。细胞用PMA预孵育10分钟后，去甲肾上腺素或PGF2α引起的糖原磷酸化酶激活和InsP3增加在很大程度上受到抑制，而胰高血糖素介导的酶激活不受影响。与PMA不同，不激活蛋白激酶C的佛波醇酯4α - 佛波醇13,14 - 二癸酸酯不会减弱PGF2α和去甲肾上腺素引起的葡萄糖输出、糖原磷酸化酶和InsP3形成的刺激。AlF4 - 激活磷脂酶C而不依赖于受体，其对InsP3形成的刺激不会因预先用PMA孵育而减弱。从分离的肝细胞中纯化的质膜对PGF2α有一个高容量、低亲和力和一个低容量、高亲和力的结合位点。高容量、低亲和力结合位点的Kd接近使糖原磷酸化酶活性增加到最大一半时的PGF2α浓度。鸟苷5'-O -（3 - 硫代）三磷酸（GTP[S]）可增强与高容量、低亲和力结合位点的结合。因此，这个高容量、低亲和力位点可能代表受体。高容量位点的Bmax和Kd，以及GTP[S]对PGF2α与该位点结合的增强作用，不受PMA处理的影响。结论是，在肝细胞中，PMA激活蛋白激酶C在磷脂酶C之前的受体远端一点中断了从PGF2α经PGF2α受体和磷脂酶C到糖原磷酸化酶的InsP3介导的信号通路。
