We evaluated the effects of the lipophilic nonpeptide corticotropin-releasing hormone (CRH) type 1 receptor antagonist antalarmin on the behavioral, neuroendocrine, and autonomic components of the stress response in adult male rhesus macaques. After oral administration, significant antalarmin concentrations were detected in the systemic circulation and the cerebrospinal fluid by a mass spectrometry-gas chromatography assay developed specifically for this purpose. Pharmacokinetic and dose-response studies suggested that an oral dose of 20 mg/kg was optimal for behavioral and endocrine effects. We then administered this dose in a double-blind, placebo-controlled fashion to monkeys exposed to an intense social stressor: namely, placement of two unfamiliar males in adjacent cages separated only by a transparent Plexiglas screen. Antalarmin significantly inhibited a repertoire of behaviors associated with anxiety and fear such as body tremors, grimacing, teeth gnashing, urination, and defecation. In contrast, antalarmin increased exploratory and sexual behaviors that are normally suppressed during stress. Moreover, antalarmin significantly diminished the increases in cerebrospinal fluid CRH as well as the pituitary-adrenal, sympathetic, and adrenal medullary responses to stress. We conclude that CRH plays a broad role in the physiological responses to psychological stress in primates and that a CRH type 1 receptor antagonist may be of therapeutic value in human psychiatric, reproductive, and cardiovascular disorders associated with CRH system hyperactivity.

Background: Alternative splicing of the transcripts of the human glucocorticoid receptor gene results in two mutually exclusive products, the dassic, ligand-binding glucocorticoid receptor (hGRa), and a dominant negative non-ligand-binding isoform, hGR(3. Materials and Methods: We examined the existence of and quantified both hGRa and hGR,B isoforms in a panel of human tissues, as well as in intact and fractionated HeLa cells, using specific quantitative Western blots and/or immunocytochemistry. We studied the potential interactions of hGR,B with heat shock protein (hsp) 90 and/or hGRa using crossimmunoadsorption/precipitation procedures followed by Western blots. Results: For the first time, we demonstrated the natural existence of the hGRI3 protein, which was widely expressed in human tissues. The ratio of immunoreactive hGRa to hGR(3 varied from 0.2 to 1.0 among different tissues, and was approximately 0.2 in HeLa cells. In the latter, both isoforms were distributed in the cytoplasm and nucleus in the absence of the hormonal ligand, and translocated into the nucleus after addition of dexamethasone. The cytosolic and nudear hGRa-to-hGR/3 ratio remained the same before and after dexamethasone exposure, suggesting that upon activation the two isoforms translocated into the nucleus in equal proportions. hGRa-and hGR(3-specific antibodies cross-adsorbed and precipitated cytosolic and nuclear glucocorticoid hGRa and hGR,B, respectively, as well as hsp9o, suggesting that hGRa and hGRP are in complex with hsp9O and/or each other. Conclusions: The hGRi3 protein is widely expressed throughout the human body and present mostly in the cytoplasm of human cells, in complex with hsp9o and other proteins. In the presence of glucocorticoid, hGR/3 probably heterodimerizes with ligand-bound hGRa and translocates into the nudeus to act as a dominant negative inhibitor of the classic receptor.