Perfused Rat Isolated Anterior Pituitary Cell Column as Bioassay for Factor(s) Controlling Release of Adrenocorticotropin: Validation of a Technique

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Abstract
A suspension of isolated rat anterior pituitary cells, with viability of 90% was mixed with 0.5 g preswollen Bio-Gel P2 and packed into a column. ACTH in the fractions collected from the perfused column was measured by immunoassay and bioassay with well correlated results (r = 0.84,P = 0.01). Cells were stimulated with 2–4-min pulses or crude stalk median eminence extract (SME) and responses were recorded as total ACTH released in excess of background. A log doseresponse curve was obtained over the range 0.0025–0.1 SME/ml (one SME = extract of 3–4 mg tissue/ml), giving a highly significant regression of response on dose (P < 0.01) with an index of precision of 0.164 ± 0.007 (mean ± SEM, n = 8). LH, as measured by immunoassay, was also released in response to SME. No significant ACTH release was seen with a cortex extract. ACTH elease was stimulated by 56 mM potassium ions; SMEstimulated ACTH release was dependent on calcium and magnesium ions and was inhibited by the presence of corticosterone (0.2 μg/ml) in the perfusate. Arginine vasopressin, lysine vasopressin, arginine vasotocin (AVT), and oxytocin were the only substances found to release ACTH, but not LH, in a dose-related manner, but responses were significantly nonparallel with those of SME. The system is easy to run and has been shown to be sensitive, precise, accurate, and specific for corticotropin-releasing factor in vitro.

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