ROLE OF GUANINE-NUCLEOTIDE-BINDING PROTEINS, G(I-ALPHA-3) AND G(S-ALPHA), IN DOPAMINE AND THYROTROPIN-RELEASING-HORMONE SIGNAL-TRANSDUCTION- EVIDENCE FOR COMPETITION AND COMMONALITY

It is clear that dopamine (DA) at high concentrations (>100 nmol/l) in hibits the release of prolactin (PRL). Paradoxically, this monoamine a t low concentrations (<10 nmol/l) has also been shown to augment PRL s ecretion. One possible explanation for these divergent effects is that DA binds receptors capable of interacting with multiple G protein sub types that recruit opposing intracellular signaling pathways within la ctotropes. To identify G proteins which couple DA receptor activation to PRL secretion, we have selectively immunoneutralized the activity o f G(i alpha 3) and G(s alpha) in primary cultures of rat pituitaries a nd subsequently tested the ability of these cultures to respond to hig h and low dose DA. Specifically, permeabilized pituitary cell cultures from random-cycling female rats were treated with control immunoglobu lins (IgGs; 50 mu g/ml) purified from preimmune serum (PII) or IgGs di rected against the C-terminal portion of G(i alpha 3) or G(s alpha). A fter immunoneutralization of these G proteins, cells were challenged w ith 10 or 1000 nmol DA/l and the relative amount of PRL released was a ssessed by reverse hemolytic plaque assay. Results were expressed as % of basal values and compared. Under control conditions (PII), 1000 nm ol DA/l inhibited (61.4 +/- 7.6% of basal values; mean +/- S.E.M.) whi le 10 nmol DA/l augmented (120.0 +/- 7.0%) PRL release in five separat e experiments. Treatment of cells with anti-G(i alpha 3) attenuated th e inhibitory effect of high dose DA (87.3 +/- 14.5%). However, elimina tion of G(i alpha 3) activity did not significantly alter the PRL stim ulatory effect of 10 nmol DA/l (121.0 +/- 5.2%). Interestingly, immuno neutralization of G(s alpha) resulted in a reciprocal shift in the act ivity of the lower dose of DA from stimulatory to inhibitory (69.7 +/- 7.3%) while combined treatment of anti-G(i alpha 3) and anti-G(s alph a) abrogated the responsiveness of pituitary cell cultures to either D A treatment (1000 nmol/l, 70.7 +/- 12.5% and 10 nmol/l, 87.5 +/- 21.4% ). These data reveal that ligand-activated DA receptors can interact w ith both G(i alpha 3) and G(s alpha). Elimination of the stimulatory c omponent (G(s alpha) favors the DA receptor activation of the inhibito ry pathway (G(s alpha)) suggesting a competition between negative and positive intracellular signaling mechanisms in normal lactotropes. In addition to DA treatment, we also challenged permeabilized pituitary c ells with 100 nmol thyrotropin-releasing hormone (TRH)/l as a positive control for secretory integrity. As anticipated, TRH stimulated PRL r elease to 188.0 +/- 31.0% of basal values under control conditions. Un expectedly, immunoneutralization of G(s alpha) completely blocked the ability of TRH to induce PRL release (101.8 +/- 12.0%). This neutraliz ing effect was specific to G(s alpha) in that blockade of G(i alpha 3) activity had no significant effect on TRH-stimulated PRL release (166 .2 +/- 13.1%). These data are the first to support a direct role of G( s alpha) in TRH signal transduction within PRL-secreting cells.