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We have reported that both an extract of rat hypothalamus-stalk-median eminence (HE) and 5-hydroxytryptamine (5-HT) will stimulate the release of ACTH biological activity from acutely, non-enzymically dispersed rat pars intermedia (PI) cells. We have also found that the ACTH activity within the PI cells is composed of 4 chromatographically separable entities, but only a small proportion of the total ACTH activity co-elutes with ACTH^1-39. In the present study we subjected the media, after incubation of PI cells with either HE or 5-HT, to sequential chromatography on Sephadex G-50 F and G-100 SF. We found the same 4 ACTH-like moieties as in the PI cell extracts. Only the release of the 2 earliest eluting components, presumably of much larger molecular dimensions than ACTH^1-39, was enhanced by either HE or 5-HT. There was no augmented release of the ACTH component having molecular dimensions similar to ACTH¹"³⁹. Thus, in our in vitro system, the ACTH biological activity released from PI cells is not the size of ACTH^1-39, but is composed of at least 2 much larger molecules which possess ACTH (as well as some MSH) biological activity.

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Enzymatically dispersed cells of the rat pars nervosa – pars intermedia (NI) were observed by scanning electron microscopy after 1, 2 and 4 days in culture. The cells attached to the plate slowly, requiring about 3–4 days for the majority to adhere. The epithelial cells became attached singly and in clumps and branched chains, often over a bed of fibroblast-like cells. After the first day in culture, few surface features were evident on the NI cells, but by day 2, the surfaces showed a small number of blebs. In 4-day cultures, the cells revealed extensive areas with blebs and microvilli, and a few structures resembling cilia were observed. The adrenocorticotrophic hormone content of the cells after 4 days in culture was similar to that found in fresh tissue.