Use of cell fusion techniques to probe the mechanism of catecholamine-induced desensitization of adenylate cyclase in frog erythrocytes.
Use of cell fusion techniques to probe the mechanism of catecholamine-induced desensitization of adenylate cyclase in frog erythrocytes.
复制标题
利用细胞融合技术探讨儿茶酚胺诱导青蛙红细胞腺苷酸环化酶脱敏的机制。
DOI：
10.1016/0304-4165(80)90231-7
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发表时间：
1980
期刊：
影响因子：
--
通讯作者：
Lefkowitz,RJ
中科院分区：
文献类型：
--
作者：
Pike,LJ;Lefkowitz,RJ
儿茶酚胺敏感的腺苷酸环化酶系统似乎至少由三种组分组成：β-肾上腺素能受体（R组分）、腺苷酸环化酶的催化单位（C组分）和核苷酸调节蛋白（N组分），负责介导鸟嘌呤核苷酸对系统的影响。用细胞融合技术研究了这三种成分在蛙红细胞同源脱敏过程中的作用。用二环己基碳二亚胺（DCCD）抑制一群蛙红细胞中的β受体功能，而用苯基乙二醛抑制另一群蛙红细胞中的N和C组分。利用仙台病毒融合两种修饰细胞，构建了含有来自每种细胞类型的组分的异源β-肾上腺素能受体-腺苷酸环化酶系统。当来自先前对儿茶酚胺脱敏的细胞的β受体与来自新鲜红细胞的N-C组分偶联时，所得的杂交体表现出对异丙肾上腺素的致密化反应。相比之下，当新鲜细胞的β-肾上腺素能受体与来自脱敏红细胞的N-C组分偶联时，杂交细胞对异丙肾上腺素的反应性没有明显降低。在对照实验中证明了这种再敏感是加入新鲜β-肾上腺素能受体的结果。蛙红细胞同时对儿茶酚胺和前列腺素E_1脱敏，并用DCCD修饰，使β-肾上腺素能受体失活，但不使前列腺素受体失活。当通过细胞融合向这些双重脱敏的红细胞提供新鲜的β-肾上腺素能受体时，只有β-肾上腺素能反应恢复到对照水平。在杂交体中，对前列腺素的反应保持脱敏，表明观察到的儿茶酚胺刺激的腺苷酸环化酶活性的再敏化是特异性的，并且是由于添加了新鲜的β-肾上腺素能受体。这些数据表明，在青蛙红细胞，同源脱敏主要是受体相关的改变的结果。
The catecholamine-sensitive adenylate cyclase system appears to be comprised of at least three components; the beta-adrenergic receptor (R component), the catalytic unit of adenylate cyclase (C component) and a nucleotide regulatory protein (N component), responsible for mediating the effects of guanine nucleotides on the system. Cell fusion techniques were used to investigate the role of these three components in the process of homologous desensitization in the frog erythrocyte. Dicyclohexylcarbodiimide (DCCD) was used to inhibit β-receptor function in one population of frog erythrocytes, whilst phenyl glyoxal was employed to inactivate the N and C components in a second population of frog erythrocytes. Using Sendai virus to fuse the two types of modified cell, heterologous β-adrenergic receptor-adenylate cyclase systems were constructed which contained components from each cell type. When beta receptors from cells previously desensitized to catecholamines were coupled to N-C components derived from fresh erythrocytes, the resulting hybrid exhibited a densitized response to isoproterenol. By contrast, when β-adrenergic receptors from fresh cells were coupled to N-C components derived from desensitized erythrocytes, no decreased responsiveness to isoproterenol was apparent in the hybrid. That this resensitization was the result of the addition of fresh β-adrenergic receptors was demonstrated in a control experiment. Frog erythrocytes were desensitized simultaneously to catecholamines and prostaglandin E1and modified with DCCD which inactivates the β-adrenergic receptor but not the prostaglandin receptor. When fresh β-adrenergic receptors were supplied by cell fusion to these doubly desensitized erythrocytes, only the β-adrenergic response was restored to control levels. The response to prostaglandin remained desensitized in the hybrids, indicating that the observed resensitization of catecholamine-stimulated adenylate cyclase activity was specific and was due to the addition of fresh β-adrenergic receptors. These data suggest that in the frog erythrocyte, homologous desensitization is primarily the result of receptor-related alterations.