Measurement of the secretion of a small peptide at the single cell level by the cell immunoblot assay: thyroidectomy increases the number of substance P-secreting anterior pituitary cells.

The present study developed a technique for quantification of a small peptide secreted by single endocrine cells and investigated the cellular basis for the effects of endocrine environments on their secretion by using this technique. To quantify substance P (SP) secreted by monodispersed rat anterior pituitary cells and to estimate the relative abundance of SP-secreting cells, the cell immunoblot assay was improved by several modifications, including the use of incubation chambers and addition of immunostaining procedures of fixation by paraformaldehyde and elimination of nonspecifically stained blots. Single pituitary cells formed SP-specific blots on protein-blotting transfer membranes that constituted a floor portion of the incubation chamber. A combination of microscopic image analysis and SP standard curve made it possible to quantify the amount of SP stained as blots. SP secretion from single pituitary cells showed a large variation ranging from 0.14-144 fg/cell.h. The mean SP secretion and the number of SP-secreting cells were increased in an incubation time-dependent manner, with plateau levels of 20.3 fg/cell and 30.2 in a chamber area of 8 x 8 mm, respectively, at 3 h. Thyroidectomy significantly increased the number of SP-secreting cells and the total amount of SP secretion but decreased the mean SP secretion from single pituitary cells. This decrease in the mean SP secretion in the thyroidectomized rats was found to be due to a relative abundance of cells that secrete small amounts of SP. The present study demonstrates that the cell immunoblot assay is useful for quantifying peptide secretion at the single cell level and suggests that thyroidectomy-induced increase in SP secretion from anterior pituitary cells is not due to an increase in SP secretion per cell but due to an increase in the number of SP-secreting cells.