Improved sensitivity to adrenocorticotrophin after purification and pre-incubation of isolated rat adrenal cells

Abstract

A highly sensitive bioassay for ACTH was developed, in which isolated [rat] adrenal cells, purified previously by passage through a 5% BSA [bovine serum albumin] layer were pre-incubated for 1 h. The detection limit for hACTH1-39 in this system is 0.19 .+-. 0.10 (SD) fmol/ml. As a result of the purification step the ED50 for h[human]ACTH1-39 decreased from 14.1 .+-. 4.8 to 6.9 .+-. 1.2 fmol/ml. Pre-incubation of the purified cells decreased this value significantly to 4.0 .+-. 1.5 fmol/ml. For ACTH1-24 the increase of sensitivity after purification was even higher, the ED50 changing from 7.1 .+-. 4.2 to 1.6 .+-. 0.2 fmol/ml. After pre-incubation of the purified cells this value amounted to 0.8 .+-. 0.3 fmol/ml. After purification and pre-incubation of the adrenal cells the changes observed for the ED50 of hACTH1-32 and of a highly purified human hypophyseal extract paralleled exactly the change observed for hACTH1-39. The improved sensitivity of the purified cell suspension might in fact represent a decrease in ACTH degradation. Both purification and pre-incubation could lead to removal or inactivation of enzymes which attach the polypeptide hormone. This hypothesis is supported by experiments in which the cell suspension has been contaminated with an adrenal homogenated prior to the purification and pre-incubation steps. Pre-incubation of purified isolated adrenal cells has increased the sensitivity and that the amino acid sequence 33-39 is not involved in protecting the molecule from proteolytic attack.