Identification by the sequential cell immunoblot assay of a subpopulation of rat dopamine-unresponsive lactotrophs

The responsiveness to dopamine of PRL secretion from individual lactotrophs of female rats was investigated by the use of a newly developed sequential cell immunoblot assay. In this assay, PRL secretion from the same single lactotrophs that had been cultured on plastic coverslips was quantified before and after dopamine treatment by a combination of direct absorption of PRL secreted on protein-blotting transfer membranes, immunostaining, and microscopic image analysis. The assay was sensitive enough to detect 0.03 fg PRL/pixel and was specific for PRL. The range of PRL secretion from single pituitary cells in culture was 0.03-1.92 pg/cell.h. PRL secretion was increased with time of incubation and reached a maximum by 80-160 min. There was a significant correlation in PRL secretion from the same lactotrophs between the first and second 60-min incubations. When medium used in the second incubation contained no dopamine, amounts of PRL secreted during the second incubation period were 21-204% of those secreted during the first incubation period. Inclusion of 10(-8)-10(-6) M dopamine in the second incubation medium increased in a dose-dependent manner the proportion of lactotrophs whose PRL secretion was suppressed significantly compared with that during the first incubation period. However, PRL secretion from approximately 6% of the total lactotrophs was not suppressed even by dopamine at concentrations over 10(-6) M. The present study demonstrates that the sequential cell immunoblot assay is a useful means to quantify repeatedly hormone secretion from individual endocrine cells in culture. Furthermore, these results suggest that there is a subpopulation of rat PRL-secreting lactotrophs that are unresponsive to dopamine.