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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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