CHANGES IN HORMONE RESPONSIVENESS AND CYCLIC-AMP METABOLISM IN RAT HEPATOCYTES DURING PRIMARY CULTURE AND EFFECTS OF SUPPLEMENTING THE MEDIUM WITH INSULIN AND DEXAMETHASONE
CHANGES IN HORMONE RESPONSIVENESS AND CYCLIC-AMP METABOLISM IN RAT HEPATOCYTES DURING PRIMARY CULTURE AND EFFECTS OF SUPPLEMENTING THE MEDIUM WITH INSULIN AND DEXAMETHASONE
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DOI：
10.1111/j.1432-1033.1984.tb07904.x
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发表时间：
1984-01-01
期刊：
影响因子：
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通讯作者：
SONNE, O
中科院分区：
文献类型：
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作者：
CHRISTOFFERSEN, T;REFSNES, M;SONNE, O
大鼠肝细胞的原代单层培养物用于研究激素对cAMP系统的长期和急性作用。当肝细胞裂解物在不同的时间进行分析后的细胞，3个主要的变化，cAMP的代谢和调节观察：胰高血糖素敏感的腺苷酸环化酶活性逐渐下降的文化。相反，在正常成年雄性大鼠肝脏和新鲜分离的肝细胞中，儿茶酚胺敏感活性非常低，在接种细胞后显示出强烈而快速的增加。同时，有一个早期的海拔（峰值。6 h），随后高Km和低Km cAMP磷酸二酯酶活性均降低。这些酶的变化可能解释了这样的发现，即在完整培养的细胞中，接种后2-24 h内cAMP对胰高血糖素的反应减弱，随后培养48 h内对胰高血糖素和肾上腺素能药物的反应增加。培养基中补充地塞米松和/或胰岛素影响肝细胞中cAMP的形成和分解。在铺板时加入胰岛素适度增加了48 h时测定的腺苷酸环化酶活性，而地塞米松无显著影响。在地塞米松的存在下，胰岛素对裂解物中的腺苷酸环化酶活性，特别是胰高血糖素反应性产生更强且剂量依赖性（1 pM-1 μ M）的提高。胰岛素加地塞米松抵消了体外发生的胰高血糖素敏感性腺苷酸环化酶活性的丧失。cAMP磷酸二酯酶活性的动力学图显示3个亲和区的基板。具有高和中等底物亲和力的2个（Km apprxeq. 10 μ M）在地塞米松处理的细胞中减少。胰岛素部分阻止了地塞米松的这种作用。响应于胰高血糖素或β-葡萄糖在完整细胞中的cAMP积累在用地塞米松预处理的培养物中，肾上腺素能药物强烈增加。显然，胰岛素和糖皮质激素通过对cAMP系统施加长期影响来调节胰高血糖素和肾上腺素对肝细胞的作用。
Primary monolayer cultures of rat hepatocytes were used for studies of long-term and acute effects of hormones on the cAMP system. When hepatocyte lysates were assayed at various times after plating of the cells, 3 major changes in the metabolism of cAMP and its regulation were observed: Glucagon-sensitive adenylate cyclase activity gradually declined in culture. In contrast, catecholamine-sensitive activity, being very low in normal adult male rat liver and freshly isolated hepatocytes, showed a strong and rapid increase after seeding of the cells. Concomitantly, there was an early elevation (peak .apprxeq. 6 h) and a subsequent decrease in activity of both high-Km and low-Km cAMP phosphodiesterase. These enzymic changes probably explained the finding that in intact cultured cells the cAMP response to glucagon was diminished for 2-24 h after seeding, followed by an increase in the responsiveness to glucagon as well as to adrenergic agents up to 48 h of culture. Supplementation of the culture media with dexamethasone and/or insulin influenced the formation and breakdown of cAMP in the hepatocytes. Insulin added at the time of plating moderately increased the adenylate cyclase activity assayed at 48 h, while dexamethasone had no significant effect. In the presence of dexamethasone, insulin exerted a stronger and dose-dependent (1 pM-1 .mu.M) elevation of the adenylate cyclase activity in the lysates, particularly of the glucagon responsiveness. Insulin plus dexamethasone counteracted the loss of glucagon-sensitive adenylate cyclase activity occurring in vitro. Kinetic plots of the cAMP phosphodiesterase activity showed 3 affinity regions for the substrate. The 2 with high and intermediate substrate affinity (Km .apprxeq. 10 .mu.M) were decreased in the dexamethasone-treated cells. Insulin partly prevented this effect of dexamethasone. Accumulation of cAMP in intact cells in response to glucagon or .beta.-adrenergic agents was strongly increased in cultures pretreated with dexamethasone. Apparently, insulin and glucocorticoids modulate the effects of glucagon and epinephrine on hepatocytes by exerting long-term influences on the cAMP system.