ヒト副腎皮質腫瘍に於けるACTHreceptorについて
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The responsiveness of human adrenocortical tumors to ACTH was investigated by measuring the specific binding of <SUP>125</SUP>I-ACTH to a crude extract of membrane from human adrenocortical tumors and changes of adenylate cyclase activity in the membranes after the in vitro administration of ACTH. <BR>Porcine ACTH was iodinated by the lactoperoxidase method and purified by ab-sorption to Quso G-32 and Bio-Gel P-10 column chromatography. The crude extracts of the membranes were prepared from bovine adrenals and human adrenocortical tumors : 6 primary aldosteronism, 3 Cushing's syndrome and 1 virilizing adrenal carcinoma. <BR>Bovine adrenal cortices and human adrenocortical tumors were homogenized in a 10mM Tris-HC1 buffer (pH 7.8) containing 0.25M sucrose and 1mM MgC1<SUB>2</SUB>. The homogenate was centrifuged at 600g for 10 min. at 4°C. The supernatant was centrifuged at 20,000g for 20 min. at 4°C, and then the pellet was washed several times with 10mM Tris-HC1 buffer (pH 7.8). The washed pellet was suspended in the same buffer and stored at - 80°C until use. For the binding study of <SUP>125</SUP>I-ACTH, the crude extract of the membrane was incubated in a 10mM Tris-HC1 buffer (pH 7.8) containing 0.3% human serum albumin with cold ACTH and <SUP>125</SUP>I-ACTH for 30 min. in an ice bath. At the end of the incubation, the reaction mixture was centrifuged at 3,000 rpm for 50 min. at 4°C. Radioactivity of the pellet and the supernatant was counted in a well typed scintillation counter. Adenylate cyclase activity in the membrane was determined by radioimmunoassay of cyclic AMP formed from unlabelled ATP in an ATP regenerating system containing 5mg creatine phosphate and 0.1mg/ml creatine kinase. Steroidogenesis of human adrenocortical tumors was determined as follows : the slices were incubated in a Krebs-Ringer bicarbonate buffer (pH 7.4) for 30 min. at 37°C under an atmosphere of 95% 0<SUB>2</SUB> : CO<SUB>2</SUB>, and the amount of steroid secreted in the medium was measured by radioimmunoassay. <BR>The crude extracts of membranes from bovine adrenals bound <SUP>125</SUP>I-ACTH rapidly. And they were displaced by porcine ACTH, ACTH<SUB>1-24</SUB>, ACTF1<SUB>1-18</SUB>, but not by ACTH<SUB>1-10</SUB>. These bindings were temperature dependent and inhibited by divalent cations, Ca<SUP>2+</SUP> and Mg<SUP>2+</SUP>. Dissociation constant (Kd) was 1.2 × 10<SUP>-7</SUP>M. Adenylate cyclase activity was stimulated by ACTH<SUB>1-24</SUB>. <BR>The crude extracts of membranes from 6 patients with primary aldosteronism showed specific binding to porcine ACTH and their Kd ranged from 3.1 × 10<SUP>-7</SUP> -10 × 10<SUP>-7</SUP>M. Adenylate cyclase in their crude extracts of membranes were stimulated to 2-5 times above the basal level by 4 × 10<SUP>-6</SUP>M ACTH<SUB>1-24</SUB>. Steroidogenesis of both aldosterone and cortisol was increased 2-4 times over the basal levels after the addition of 1 × 10<SUP>-5</SUP>M ACTH<SUB>1-24</SUB>. In Cushing's syndrome, the crude extracts of membranes bound specifically the porcine ACTH and their Kd ranged from 12 × 10<SUP>-7</SUP> -51 × 10<SUP>-7</SUP>M. Adenylate cyclase activities of their membranes were 1.4 -3 times higher than the basal levels in the presence of 0.4 - 1.0 × 10<SUP>-5</SUP>M ACTH<SUB>1-24</SUB>. Steroidogenesis of cortisol in 2 out of 3 patients with Cushing's syndrome did not increase after the addition of 1× 10<SUP>-5</SUP>M ACTH<SUB>1-24</SUB>. The other one was not investigated. In virilizing adrenal carcinoma, specific binding to porcine ACTH was not observed, and adenylate cyclase activity was not stimulated by ACTH<SUB>1-24</SUB>. Steroidogenesis of both cortisol and DEHA did not increase after the addition of ACTH<SUB>1-24</SUB>. In all human adrenocortical tumors, adenylate cyclase activities were stimulated by NaF. <BR>The results indicate
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