Somatotropes and thyrotropes in the rat anterior pituitary gland cosecrete substance P: analysis by the sandwich cell immunoblot assay.

Substance P (SP) which is synthesized and secreted by anterior pituitary cells has been suggested to alter pituitary functions as a local modulator. We determined which cell type(s) of rat anterior pituitary gland secretes SP. A new technique, named the sandwich cell immunoblot assay (CIBA), was developed to identify two protein substances that are secreted simultaneously from the same cells. Monodispersed anterior pituitary cells were sandwiched with pairs of transfer membranes and incubated to absorb secretory substances on the membranes. Small reference points for identifying the location of cell blots were simultaneously put on the pairs of transfer membranes after the incubation. The validity of the sandwich CIBA was revealed by the following: (1) cell blots were detected at the same locations on the pairs of transfer membranes that were immunostained with the same antisera against the anterior pituitary hormones, and (2) the areas of these cell blots detected at the same locations on different membranes correlated well. When one of the pair of transfer membranes was immunostained for SP and the remaining immunostained for one of the anterior pituitary hormones, it was found that 78 and 27% of SP-immunoreactive cell blots showed also GH and TSH immunoreactivity, respectively. None of the SP-immunoreactive cell blots showed immunoreactivity for PRL, ACTH, LH or FSH. These results suggest that the sandwich CIBA is useful to identify two substances cosecreted from a cell and that a subpopulation of rat somatotropes or thyrotropes cosecretes SP.