Use of the Reverse Hemolytic Plaque Assay to Study the Regulation of Anterior Lobe Adrenocorticotropin (ACTH) Secretion by ACTH-Releasing Factor, Arginine Vasopressin, Angiotensin II, and Glucocorticoids*

Abstract

A reverse hemolytic plaque assay (RHPA) for ACTH was developed to study the responses of anterior lobe corticotropes to secretagogues-CRF, arginine vasopressin (AVP), angiotensin II (A-II), or to a 6- to 24-h pretreatment with corticosterone. Tests showed that optimal plaque formation was obtained after 3-4 h with 1:100-1:200 anti ACTH-(25-39) and 1:20-1:50 complement. Under optimal basal conditions, 6.6% of pituitary cells from normal male rats formed plaques. The addition of 50-90 .mu.g/ml ACTH to the anti-ACTH for 48 h before its use in the RHPA resulted in a decrease in percentage of ACTH plaques to levels but not different from those obtained with preimmune serum, or when complement was omitted from the assay (0.9-1.3%). There was a gradual increase in percentage of ACTH plaques to 9.8% of the population after exposure to increasing doses of CRF (0.1-10 nM). These same high percentages of ACTH plaques could also be obtained by the addition of 1 nM AVP or A-II with the lowest doses of CRF (100-500 pM). Exposure to 1 nM AVP alone resulted in no significant increases in percentages above basal values. Average plaque areas were increased to maximal levels of three to four .times. basal with increasing doses of CRF. Finally, when cells were pretreated with 100 nM corticosterone for up to 24 h, the percentage of plaques formed under basal conditions was reduced by 60% (2.6%) and it did not increase after exposure to 1 nM CRF. The data from the RHPA correlate well with previous studies of corticotropes. Since ACTH cells normally represent 10% of the anterior lobe cell population, the RHPA shows that a subset of corticotropes (6.6%) is actively secreting under basal conditions tested in this study. The remaining 3% can be stimulated to form plaques by high doses of CRF (> 1 nM) or low doses of CRF (100 pM) and 1 nM AVP or A-II. Glucocorticoids reduce the percentage of ACTH plaques formed under basal or stimulated conditions and allow no CRF-stimulated increase in plaque area. This correlates with recent reports that show glucocorticoids inhibit proopiomelanocortin messenger RNA synthesis and lower the number of pituitary CRF receptors.