Effect of dopamine withdrawal on activation of adenylate cyclase and phospholipase C in enriched lactotrophs.

In order to define the roles of cAMP and inositol phosphates (IPx) in the mechanisms governing dopamine (DA)-regulated PRL release, we have carried out studies with enriched lactotrophs from dispersed anterior pituitaries of estrogen-treated rats. Changes in the intracellular levels of cAMP as well as IPx were determined in response to the acute addition or removal of DA. The withdrawal of DA from the incubation medium in cells cultured with DA (500 nM) for 24 h resulted in a rapid and significant increase in cAMP concentration from 1.29 +/- 0.098 to 3.89 +/- 0.199 pmol/dish. On the other hand, the administration of DA for 10 min to cells cultured without it resulted in a significant decrease in intracellular cAMP (from 3.04 +/- 0.208 to 1.62 +/- 0.057 pmol/dish). Similarly, PRL released into the medium was stimulated (95.1 +/- 9%) or inhibited (46.9 +/- 6%) after DA withdrawal or addition, respectively. Cells incubated 24 h with [3H]inositol and DA (500 nM) responded to DA withdrawal with a significant increase in the concentration of [3H]IPx (5148 +/- 199 vs. 8,376 +/- 164 cpm/dish), whereas acute DA administration had no effect on the level of [3H]IPx. The administration of 8-Br-cAMP (0.5 and 2.5 mM) and forskolin (10 microM) for 10 min to cells tonically cultured in the presence of DA had no effect on the intracellular concentration of [3H]IPx, although they decreased the relative proportion of [3H]IP3 fraction from 22.1% to 11.6%, 13.6%, and 9.6%, respectively. The administration of TRH (100 nM), either under tonic DA inhibition or 10 min after the transient removal of DA inhibition, resulted in a similar stimulation of IPx formation (from 5,625 +/- 155 to 21,830 +/- 100 and 24,870 +/- 80 cpm/dish, respectively). However, the release of PRL induced by TRH was potentiated 6-fold (38.2 +/- 2.17 vs. 227 +/- 41 ng/dish) by the transient removal of DA. These findings support the conclusions that: 1) DA receptors in lactotrophs are negatively coupled to adenylate cyclase as seen during the addition or removal of DA. 2) DA receptors are negatively coupled to phospholipase C; however activation is only seen upon the removal of DA. 3) The effect of DA withdrawal on the formation of IPx is not secondary to the activation of adenylate cyclase. 4) The potentiation of the PRL response to TRH after DA withdrawal does not involve increased production of IPx.