SUMMARY A reproducible method for isolating intact cells from the adrenal glands of a small number of rats (four to six) is described. The incubation conditions and fluorometric corticosteroid assay were modified to enable a large number of incubations to be carried out with each batch of cells. Linear regression analysis of log-dose—response curves for adrenocorticotrophin (ACTH) analogues yields potency ratios with 95% confidence limits which are usually less than ± 20%. The ED50 of the assay is about 50 pg/ml for natural ACTH and 7 pg/ml for corticotrophin-(1–24)-tetracosapeptide. Insulin and prostaglandin E2 had no effect on the steroidogenic response to ACTH while cyclic AMP at high doses was steroidogenic but gave a much steeper log-dose—response curve than ACTH. α- and β-melanocytestimulating hormone (MSH) were both agonists but only at concentrations 1 × 106 times higher than those of ACTH. Cells isolated from rabbit adrenals were similar to those from the rat in their responsiveness to ACTH but were unresponsive to amounts of MSH sufficient to stimulate rat adrenal cells.

An isolated rat adrenal cell bioassay was used to measure blood concentrations in rats after infusion of synthetic human ACTH, corticotrophin-(1-24)-tetracosapeptide or [D-Ser1, Lys17, Lys18]corticotrophin-(1-18)-octadecapeptide amide. Lower blood levels were found with the 1-24 peptide than with human ACTH and the highest levels were found with the 1-18 peptide. These results suggest that the 1-24 peptide which is almost equipotent with natural ACTH in vivo may be more potent at the receptor and corroborate findings to this effect obtained with isolated adrenal cells. The high potency and prolonged action of the 1-18 analogue in vivo are also explained by these results. Low arterial blood concentrations of the 1-24 peptide and human ACTH were found during infusion, suggesting that substantial inactivation must be occurring in a single passage through the lungs. The effects of renal ligature on blood concentrations indicated that the kidney is involved in handling the 1-18 peptide and that human ACTH is also cleared by this organ. After infusion the fall in blood concentrations was biphasic. It is suggested that the rapid phase is due to clearance of peptides in the circulation which results in a fall to lower blood concentrations which are sustained by slow release of peptide from binding sites which act as a depot.

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