A sensitive and simple in vitro assay for corticotropin-releasing substances utilizing ACTH release from cultured anterior pituitary cells.

Graded doses of rat hypothalamic extract (HE) were added to dishes containing dispersed, pooled rat adenohypophyseal cells cultured for several days. ACTH secretion into the medium gave a linear log-dose response curve over a 100-fold range between 0.01 and 1 mg of NIH-HE (0.0125-1.25 rat hypothalamus). Forty percent of the maximal ACTH secretion in response to a given dose of HE occurred within 3 min. No decrease in intracellular ACTH occurred at any time or with any dose of HE, indicating that secretion was always balanced by production. ACTH secretion stopped as soon as HE-containing medium was replaced by medium without HE. The same cultured cells could be satisfactorily used in repetitive assays performed on the same or different days.