Effect of Adrenalectomy and Cortisol on the <i>in vivo</i> Rate of Incorporation of Phenylalanine-¹⁴C into Rat Corticotropin

Abstract

A method has been developed for studying the rate of in vivo ACTH synthesis by measuring the incorporation of phenylalanine-¹⁴C into ACTH in the rat. At 1 h after the intra-peritoneal injection of labeled amino acid the anterior pituitary was obtained and ACTH was extracted with hot glacial acetic acid, precipitated with ether, and adsorbed onto oxidized cellulose. The ACTH fraction was then eluted with 0.1 N hydrochloric acid. This fraction was shown to contain radioactive ACTH and did not contain free phenylalanine-¹⁴C. Changes in the rate of ACTH synthesis were estimated by changes in specific activity of the ACTH following a 1 h exposure of the rat to isotope. At 7, 14, and 24 days following adrenalectomy the rate of pituitary ACTH labeling doubled, while at 40 days, there was a three- to fivefold increase over the control rate. Administration of cortisol to the adrenalectomized but not to the intact rat decreased the rate of pituitary ACTH labeling. Nearly total inhibition of ACTH synthesis was caused by cortisol (0.5 mg/100 g body wt.) within 2 h. It was found that changes in the rate of incorporation of labeled amino acids into ACTH were not a reflection of general changes in the rate of tissue protein synthesis.

Effects of Hypothalamic Extract and Corticosterone on Incorporation of ¹⁴C-Phenylalanine into Newly Synthesized ACTH-Like Protein <i>in vitr</i><i>o</i>

“…The elevation of ACTH synthesis after adrenalectomy supports what others (12,15) have found by techniques of extraction and isolation of radioactively labeled rat ACTH by oxycellulose adsorption. Holub (6) also reported the suppression of ACTH synthesis by cortisone acetate.…”

“…These findings support the work of Wool et al (15), who used crude bovine ACTH as a marker corti cotropin for radioactively tagged ACTH, and Stackhouse et al (12), who reported rat ACTH by bioassay in their oxycellulose eluates. Neither of those reports examined this procedure as to its efficacy for extracting only rat pitu itary ACTH and particularly in terms of the starting material.…”

“…Holub (6) also reported the suppression of ACTH synthesis by cortisone acetate. Stackhouse et aL (12), on the other hand, found that cortisol acetate injection failed to affect the in vivo ACTH labeling in intact rats but these injections did suppress the increase in rat ACTH synthesis induced by adrenalectomy. Others (1,13) showed that prednisolone did not affect the in vitro incorporation of l4C-labeled amino acid into rat ACTH.…”

“…Perhaps improvement in the technique, such as measurement of the amount of ACTH in every radioactive sample and expressing the radioactive counts as specific activi ty (12) would improve the sensitivity and enable one to obtain a good dose response with HSME extract.…”

“…We subjected rats to bilateral adrenalectomy and cortisone injections to determine if that method o f ACTH isolation produced the same results as those published by others (1,6,12,15). In addition, we manipulated the in vitro addition of corticosterone and HSME extract.…”