脂肪細胞におけるACTHの生物活性と特異的結合能
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概要
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The earliest biological event in the action of ACTH upon its target tissue is thought to be an interaction of ACTH peptide with the cell membrane. This study was undertaken in order to clarify the relationship between ACTH's ability and its specific binding to fat cells. First of all, the lipolytic abilities of ACTH analogues were compared with ACTH (1-24) in order to determine what portion of the ACTH molecule is responsible for the lipolytic action of the hormone. Secondly, competitive displacement experiments were carried out in order to explain the action of the adrenergic blocking agent, phentolamine, on the lipolysis of ACTH.<BR>Isolated fat cells were incubated with several kinds of ACTH peptides in the presence or absence of adrenergic blocking agents at 37°C for 2 hrs. Then the lipolytic action of ACTH was determined by measuring the free fatty acid levels in the medium. These lipolytic activities were expressed as the ratios of the amount of free fatty acid released to that of 1μg/ml epinephrine. On the other hand, 80μg/0.2ml of fat cell ghosts were incubated at 4°C for 40 min. with 3-4×10<SUP>4</SUP> cpm of <SUP>125</SUP> I-labelled ACTH (1-24) and unlabelled ACTH (1-24) in the presence or absence of adrenergic blocking agents. After centrifugation, the radioactivities of the fat cell ghosts were measured.<BR>I. Dose dependent responses were found in the lipolysis stimulated with ACTH (1-24) at the concentration of 10<SUP>-10</SUP> -10<SUP>17</SUP>M. Then, lipolytic abilities of ACTH analogues were compared with ACTH (1-24). The relative lipolytic activity of ACTH (1-18) was 91.2 and 105.7% of ACTH (1-24) at the concentration of 10<SUP>-7</SUP> and 10<SUP>-6</SUP>M, respectively. Therefore, ACTH (1-18) showed almost the same equivalent lipolytic activity as that of ACTH (1-24). The relative lipolytic activity of ACTH (1-14) was 17.3 and 69.9% at the concentration of 10-7 and 10<SUP>-6</SUP>M, respectively. It was therefore concluded that ACTH (1-14) showed an apparent lipolytic activity. The relative lipolytic activity of ACTH (1-10) was 2.7 and 4.2 at the concentration of 10<SUP>-7</SUP> and 10<SUP>-6</SUP>M, respectively. These values do not differ significantly from the control value (basal lipolytic activity in the absence of ACTH), however, ACTH (1 10) stimulated lipolysis at the concentration of 10<SUP>-5</SUP>M. Therefore, it was ascertained that ACTH (1-10) had a slight lipolytic activity.<BR>When fat cell ghosts were incubated with <SUP>125</SUP> I-labelled ACTH (1-24), 18.0% of total radioactivities bound the fat cell ghosts. Displacement of <SUP>125</SUP> I-labelled ACTH (1-24) occurred in the presence of unlabelled ACTH (1-24). Displacement of binding was not seen at the concentration under 10<SUP>-7</SUP>M of ACTH. But the radioactivities of ACTH in the ghost cells showed a liner decrease according to an increase in the concentration of unlabelled ACTH (1-24) added. 6.3% of the bound radioactivity was not displaced over the concentration of 10<SUP>-4</SUP>M of ACTH. Displacement of labelled ACTH binding the fat cell ghosts did not occur with the addition of insulin and epinephrine. It seemed that ACTH has no common receptor with those of insulin and epinephrine.<BR>Second, influences of ACTH analogues on the binding of ACTH (1-24) with fat cell ghosts were examined. Unlabelled ACTH analogues caused also the displacement of labelled ACTH (1-24). Evaluation of binding experiments by the Scatchard method showed that not less than two receptor sites were present in adipocytes. The affinity constants were 1.0×10<SUP>-9</SUP>M (low affinity constant was 1.3×10<SUP>-7</SUP>M) for ACTH (1-24), 7.6×10<SUP>-10</SUP>M (1.7×10<SUP>-7</SUP>M) for ACTH (1-18) and 2.2×10<SUP>-9</SUP>M (9.4×10<SUP>-8</SUP>M) for ACTH (1-10). ACTH (1-18) showed the same extent of lipolytic activity and binding ability to the fat cells as ACTH (1-24).
- 一般社団法人 日本内分泌学会の論文