Phase-dependent resetting of the adrenal clock by ACTH in vitro

The adrenal cortex has a molecular clock that generates circadian rhythms in glucocorticoids, yet how the clock is synchronized to the external environment is unknown. Using mPER2::Luciferase (mPER2Luc) knockin mice, in which luciferase is rhythmically expressed under the control of the mouse Per2 clock gene, we hypothesized that ACTH transmits entrainment signals to the adrenal. Adrenal explants were administered ACTH at different phases of the mPER2Luc rhythm. Treatment with ACTH 1–39 produced a phase delay that was phase-dependent, with a maximum at circadian time (CT)18; ACTH did not alter the period or amplitude of the rhythm. Forskolin produced a parallel response, suggesting that the phase delay was cAMP-mediated. The response to ACTH was concentration-dependent and peptide-specific. Pulse administration (60 min) of ACTH 1–39 also produced phase delays restricted to late CTs. In contrast to ACTH 1–39, other ACTH fragments, including α-melanocyte-stimulating hormone, which do not activate the melanocortin 2 (MC2/ACTH) receptor, had no effect. The finding that ACTH in vitro phase delays the adrenal mPER2luc rhythm in a monophasic fashion argues for ACTH as a key resetter, but not the sole entrainer, of the adrenal clock.

Advantages of IRMA over RIA in the Measurement of ACTH

A technically simple and rapid two-site immunoradiometric assay (IRMA) for human ACTH, based on monoclonal antibodies (MAbs), was compared with a clinically validated ACTH radioimmunoassay (RIA). Both methods measure ACTH 1–39 in unextracted plasma and cross-react <0·5% with ACTH fragments. ACTH levels were assessed in 103 patient samples: for concentrations in the range 5·3–1000 ng/L, results by the two methods were significantly correlated ( r = 0.82, n = 86, P < 0·001). The IRMA was more sensitive and had a wider working range than the RIA (detection limits 5·3 ng/L (IRMA) vs 11 ng/L (RIA); CV < 10% between 19 and 1000 ng/L (IRMA) and CV < 15% between 30 and 400 ng/L (RIA)). In two patients for whom discrepant results were obtained, measurement of ACTH by bioassay and ACTH precursors by direct IRMA demonstrated the greater accuracy of the ACTH IRMA result. The improved performance of the IRMA combined with its many practical advantages compared to RIA, make it ideal for use in detailed clinical and physiological studies which have previously been hampered by the poor reliability of ACTH measurement.

Immunoradiometric assay of corticotropin with use of avidin-biotin separation.

We have developed a "sandwich"-type immunoradiometric assay for corticotropin (ACTH), with a detection limit of 2 ng/L. Two antibodies are used: a mouse monoclonal antibody directed against ACTH[1-17] and labeled with 125I; and a purified polyclonal goat antibody directed against ACTH[34-39] and conjugated to biotin. We could separate 125I-labeled antibody bound to ACTH from 125I-labeled antibody not bound to ACTH by using an avidin-biotin bridge, with avidin bound to a polystyrene ball. This assay reacts with ACTH[1-39] but shows no reaction with ACTH fragments [1-24], [1-17], or [34-39], or with melanotropin, endorphins, or lipotropin. This assay is sensitive enough to detect ACTH in plasma of all normal adults. Concentrations measured in 94 adults between 0800 and 1000 hours were normally distributed on a log scale, with a mean of 19.5 ng/L and a 95% range of 7.1 to 53.8 ng/L. Dexamethasone given at 2300 hours to 14 adults suppressed ACTH to less than 4 ng/L in 13 of the subjects and to 8 ng/L in the 14th. Metyrapone given to 13 adults at 2300 hours increased ACTH to 245.3 ng/L (95% range, 90.1 to 667.7 ng/L). This assay accurately classified patients with disorders of the adrenal system.