Effect of proteolytic enzyme inhibitors on steroidogenic action of corticotropin and its synthetic analogues.

Abstract

The in vitro effect of Kunitz pancreatic trypsin inhibitor (KPTI) on the release of corticosterone from rabbit adrenals in the presence of three different preparations of corticotropins has been investigated. These preparations include highly purified porcine corticotropin, Pcorticotropin and Gly^a-*^ corticotropin. The effect of KPTI was found to be more marked in sustaining the action of corticotropin with shorter peptide chain than that with longer peptide chain. KPTI per se did not show any stimulatory effects on steroidogenesis. It may be concluded that KPTI protects shorter chain corticotropin against digestion by proteolytic enzymes. The above mentioned effect of KPTI was not demonstrated, however, with soybean trypsin inhibitor. (Endocrinology 90: 1652, 1972) T T HAS BEEN known that corticotropins are •*• inactivated by proteolytic enzymes. White and Gross (1) reported that plasmin inactivated corticotropins in blood. Other proteolytic enzymes, such as trypsin, pepsin and liver cathepsin have been also recognized as corticotropin inactivators (1). However, little is known as to whether corticotropins could be inactivated in the adrenal, during exerting their hormone action in adrenal. In the present report we employed proteolytic enzyme inhibitors to evaluate the possible influence of tissue proteolytic activities on the maintenance of steroidogenic action of corticotropins including synthetic analogues with shorter chain peptides. Our data suggest that the action of synthetic corticotropin analogue with 18 amino acids is markedly augmented by protecting against the degradation of the peptide. Materials and Methods A highly purified porcine corticotropin (HP corticotropin) with a potency of 120 U/mg was purchased from Calbiochem; synthetic P* corticotropin from Organon-Daiichi; Kunitz pancreatic trypsin inhibitor and soybean trypsin inhibitor from Worthington Biochemical Corp.; bovine serum albumin (Fr. V) from Armour Pharm. Co.; synthetic Gly-a-2 Received November 17, 1971, corticotropin was a gift from Dr. Otsuka of Shionogi Laboratory, Osaka. Adrenals were obtained from male rabbits weighing 2.0-2.5 kg. They were washed exhaustively by Krebs-Ringer bicarbonate buffer, pH 7.4 (KRB) and quadricepted after removal of thin fibrous capsules. Each adrenal preparation weighed 30-50 mg. Two pieces of quadricepted tissue from a pair of adrenal glands were placed in each incubation vessel containing 5 ml KRB with glucose (2 mg/ml) and bovine serum albumin (5 mg/ml) (G-KRB) in the presence or absence of trypsin inhibitors. The vessels were preincubated for 60 min in an atmosphere of 95% O2—5% CO2 at 37 C with shaking at 100 cycles/min. After preincubation, corticotropin preparations were added and incubation was continued for another 120 min under the same condition. The steroidogenic action was followed by measuring the amount of corticosterone released into the incubation medium. 0.5 ml aliquots of medium were removed at 0, 20, 40, 60 and 120 min for the assay of corticosterone which was performed by the method of Guillemin (2). Results Effect of KPTI on steroidogenic action of corticotropins The effect of three different corticotropin preparations on corticosterone production in rabbit adrenals were tested. As shown in Fig. 1A, HP corticotropin (0.04 y-g/ml), synthetic J3 NOTES AND COMMENTS 1653 FIG. 1. The effect of HP corticotropin, P' corticotropin and Gly-a-2 corticotropin on the corticosterone production in rabbit adrenals in the presence or absence of proteolytic enzyme inhibitors (KPTI or STI). Quadricepted adrenals were preincubated for 60 min with or without proteolytic enzyme inhibitors, each preparation of corticotropins was then added to incubation vessels as indicated with an arrow and incubation was continued for 120 min. Each value is the mean ± SE of four or five experiments. 12.0