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 pituita ry cells has been suggested to alter pituitary functions as a local mo dulator. We determined which cell type(s) of rat anterior pituitary gl and secretes SP. A new technique, named the sandwich cell immunoblot a ssay (CIBA), was developed to identify two protein substances that are secreted simultaneously from the same cells. Monodispersed anterior p ituitary cells were sandwiched with pairs of transfer membranes and in cubated to absorb secretory substances on the membranes. Small referen ce points for identifying the location of cell blots were simultaneous ly put on the pairs of transfer membranes after the incubation. The va lidity of the sandwich CIBA was revealed by the following: (1) cell bl ots were detected at the same locations on the pairs of transfer membr anes that were immunostained with the same antisera against the anteri or 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 re maining immunostained for one of the anterior pituitary hormones, it w as found that 78 and 27% of SP-immunoreactive cell blots showed also G H and TSH immunoreactivity, respectively. None of the SP-immunoreactiv e cell blots showed immunoreactivity for PRL, ACTH, LH or FSH. These r esults suggest that the sandwich CIBA is useful to identify two substa nces cosecreted from a cell and that a subpopulation of rat somatotrop es or thyrotropes cosecretes SP.