Age-related alterations in prolactin secretion by individual cells as assessed by the reverse hemolytic plaque assay

The objectives of this study were to determine: 1) if lactotropes from old rats, compared to those from young rats, secrete a greater amount of prolactin (PRL) per cell, 2) if the percentage of pituitary cells secreting PRL changes with age; and 3) how estradiol (E₂), dopamine (DA), or thyrotropin-releasing hormone (TRH), or the combination of these factors influences both of these parameters in old rats. To meet these objectives we used the reverse hemolytic plaque assay (RHPA), because this method allows us to determine both the percentage of pituitary cells secreting prolactin during the experimental period and the amount of hormone released by each secreting pituitary cell. These parameters were measured in young (2-3 mo old) or old (17-19 mo old) female Sprague-Dawley rats. Animals were ovariectomized (OVX) for 10 days or OVX for 1 wk and then treated with E₂ for 3 days. Rats were killed, anterior pituitaries were removed, and cells were enzymatically dispersed and prepared for use in the RHPA. Pituitary cells were treated in vitro with vehicle, DA, or TRH for 1 or 2 h. In terms of percentage of pituitary cells secreting PRL, old OVX and E₂-treated rats exhibited a greater percentage of secretory cells than young at both 1 and 2 h of incubation. Administration of E₂ increased the percentage of cells secreting PRL in both young and old rats. DA reduced the percentage of cells secreting PRL at the highest dose tested (10^-5 M) regardless of age or E₂ status following incubation for 1 h. Under TRH-stimulated conditions, old rats had a greater percentage of cells secreting PRL than did young rats under TRH-stimulated conditions. TRH increased the percentage of cells secreting PRL in young OVX rats and in old animals regardless of E₂ status, but had no effect in young animals treated with E₂ following 1 or 2 h of incubation. In terms of the amount of PRL secreted per cell (i.e. the area of plaques formed), old OVX and E₂-treated rats secreted less PRL per cell than did young animals following 1 or 2 h or incubation. Administration of E₂ increased the basal secretion of PRL in both young and old rats. In addition, old rats were less responsive to the inhibitory effects of DA and the stimulatory effects of TRH. Thus, incubation with DA for 1 h reduced the secretion of PRL per cell at the highest dose tested (10^-5 M) in young OVX rats, but had no effect in old OVX rats. Similarly, DA reduced the secretion of PRL at the higher doses (10^-6 and 10^-5 M) in young E₂-treated rats, but only at the highest dose (10^-5 M) in old E₂-treated animals. TRH increased the secretion of PRL per cell at all doses tested (10^-9 to 10^-7 M) in young OVX rats, but only at the higher doses (10^-8 and 10^-7 M) in old OVX rats. Likewise, TRH increased the secretion of PRL at the highest doses (10^-8 and 10^-7 M) in young E₂-treated rats, but had no effect in old E₂-treated animals. We have demonstrated that alterations in the regulation of PRL in terms of both the percentage of pituitary cells secreting PRL and the amount of PRL secreted per cell occur in old rats. It is suggested from these findings that an increase in the percentage of cells secreting PRL and decrease in the responsiveness of pituitary cells from older rats play a major role in the age-related elevation in plasma concentrations of PRL that has been reported to occur in old animals.