The stimulatory and inhibitory effects of dopamine on prolactin secretion involve different G-proteins

The reverse hemolytic plaque assay (RHPA) was used in this study to further characterize the mechanism whereby low concentrations of dopamine (DA) stimulate PRL secretion in vitro. Female Sprague-Dawley rats were used as a source of anterior pituitary cells for the RHPA. Pituitary cells were infused into Cunningham chambers along with a suspension of protein-A-coated ovine red blood cells. Excess cells were rinsed from the chambers leaving a monolayer of cells attached to the glass. The cells were then incubated with solutions containing PRL antiserum (1:40) and various concentrations of DA. After 4 h, a solution containing guinea pig complement (1:60) was infused into the chambers. Thirty minutes later, the cells were fixed and plaques (zones of hemolysis) surrounding PRL-producing cells (lactotrophs) were measured and used as an index of the amount of PRL secreted. Control cells that received no DA had a mean plaque area of 8,000 microns 2 and two distinct subpopulations of plaque sizes. This biphasic population of cells consisted of a small and a large plaque producing population. The mean plaque area surrounding lactotrophs was significantly (P less than 0.05) decreased if 1 microM or 10 microM DA was present (4,500 microns 2 and 3,500 microns 2, respectively). These cells which received inhibitory concentrations of DA demonstrated a monophasic distribution of plaque-forming cells. On the other hand, mean plaque area was significantly (P less than 0.05) increased if 0.1 nM or 1 nM DA was presented to the cells (15,000 microns 2 and 14,500 microns 2, respectively). These cells receiving stimulatory doses of DA exhibited a multiphasic distribution of plaque-forming cells. The possibility that the two physiological opposing actions of DA on PRL secretion might be mediated by different GTP binding proteins was also examined using cholera toxin (CTX) and pertussis toxin (PTX). Anterior pituitary cells were pretreated with either CTX (50 micrograms/ml) or PTX (5 micrograms/ml) for 1 h before initiation of the RHPA. In the RHPA, cells received no DA, a stimulatory dose of DA (0.1 nM), or a inhibitory dose of DA (10 microM). The effects of toxin pretreatment on mean plaque area of DA-treated cells was determined. PTX pretreatment significantly attenuated the inhibitory effects of DA while having no effect on the stimulatory effects of DA on PRL secretion. CTX significantly (P less than 0.05) potentiated the stimulatory effects of DA on PRL secretion and had no effect on inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)