Etiology of the differences in corticotropin-releasing hormone-induced adrenocorticotropin secretion of black and white women.

After i.v. oCRH, plasma immunoreactive ACTH (ACTH-IR) is significantly greater in blacks than in whites; however, there is no corresponding increase in cortisol secretion. To test the hypothesis that there are black-white differences in adrenal responsiveness to ACTH that underlie this phenomenon, weight-, age-, and education-matched black (n = 10) and white (n = 10) women were i.v. infused with 5 differing doses of ACTH1-24 (0, 0.003, 0.01, 0.1, and 1 microgram/kg) with measured plasma cortisol and DHEA. To test the alternative hypothesis that greater post-CRH plasma ACTH-IR in blacks is caused by qualitative differences in circulating ACTH-immunoreactive peptides, we collected pre- and post-CRH plasma from 5 black and 5 white women and measured ACTH-IR after sample fractionation, using high-pressure liquid chromatography. There were no racial differences in adrenal responsiveness to differing doses of ACTH1-24 and no differences in the distribution of the forms of ACTH-IR before CRH. After CRH, whites had predominant ACTH-IR peaks at the retention times of ACTH1-39 and ACTH1-39-sulfoxide, whereas blacks had prominent peaks at several additional retention times. The post-CRH ratio of intact to total ACTH was significantly lower in blacks than in whites (0.27 +/- 0.17 vs. 0.71 +/- 0.17, P < 0.003). We conclude that there are qualitative differences in post-CRH circulating ACTH-IR in blacks and whites, leading to a greater immunoreactive to bioactive ACTH ratio in blacks. Such differences in the circulating forms of ACTH can account for greater CRH-stimulated ACTH-IR in blacks.

Processing of pro-opiomelanocortin in GH3 cells: inhibition by prohormone convertase 2 (PC2) antisense mRNA.

The processing of pro-opiomelanocortin (POMC) was examined in GH3 cells, a rat sommatomammotrope cell line, by transiently-transfecting the cells with mouse POMC cDNA. The peptide products were extracted, chromatographed on HPLC and identified by specific radioimmunoassay. POMC was processed to generate ACTH-related peptides, beta-endorphin and Lys-gamma 3- MSH, with complete disappearance of the POMC precursor. The ACTH-related molecules were identified as ACTH1-14, ACTH1-15, ACTH1-17, as well as ACTH1-39. GH3 cells which were not transfected with POMC cDNA did not contain endogenous POMC-related peptides. RT-PCR demonstrated that GH3 cells contain prohormone convertase 2 (PC2) mRNA but no PC1 mRNA. To determine if PC2 was the enzyme responsible for POMC processing in this cell line, GH3 cells were stably-transfected with PC2 antisense cDNA. A cell line was obtained which showed an absence of PC2 protein compared to control untransfected GH3 cells, indicating successful hybridization of PC2 antisense mRNA to the endogenous PC2 mRNA. When this cell line was then transiently-transfected with POMC cDNA, POMC was not processed. The results from these experiments suggest that PC2 alone can correctly process POMC to biologically active smaller peptides in vivo. Additionally, the GH3 cell line with and without incorporation of PC2 antisense cDNA can be used as a model system to study the role of PC2 in the post-translational processing of other prohormones and proproteins in vivo.

Pyroglutamyl peptidase-II ("thyroliberinase") activity in human serum: influence of weight and thyroid status.

The tripeptide hormone, TRH, is metabolized by three enzymes, the most specific of which is pyroglutamyl peptide hydrolase-II (also termed thyroliberinase), a metalloenzyme present in serum and brain. Because pyroglutamyl peptidase-II activity in rat serum is regulated by thyroid hormone levels, we tested the hypothesis that this activity is similarly altered in humans. We studied serum pyroglutamyl peptidase-II activity in 6 patients with hyperthyroidism, 18 patients with hypothyroidism, and 31 euthyroid, normal weight volunteers. Because TRH [or its metabolite cyclo(His-Pro)] is believed to be an important hormone regulating appetite and metabolism, we also evaluated pyroglutamyl peptidase-II activity in 27 euthyroid patients with obesity. Serum pyroglutamyl peptidase-II activity was elevated in patients with hypothyroidism (mean +/- SEM, 33.9 +/- 3.7 nmol/mL.h) compared to that in euthyroid, normal weight volunteers (24.5 +/- 2.8 nmol/mL.h; P < 0.05), but not that in patients with hyperthyroidism (28.3 +/- 4.1 nmol/mL.h; P = NS). Euthyroid obese patients had the highest pyroglutamyl peptidase-II activity (43.6 +/- 2.8 nmol/mL.h; P < 0.0001 vs. normal weight volunteers). Pyroglutamyl peptidase-II activity was positively correlated with body mass index (r2 = 0.30; P < 0.0001). After correction for body mass index, there were no difference in pyroglutamyl peptidase-II activity in hypothyroid, hyperthyroid, and euthyroid individuals. We conclude that serum pyroglutamyl peptidase-II activity is regulated by, or regulates, body weight.