THE D-2 RECEPTOR - BLOCKED TRANSCRIPTION IN GH(3) CELLS AND CELLULAR PATHWAYS EMPLOYED BY D-2A TO REGULATE PROLACTIN PROMOTER ACTIVITY

Although the GH(3) line of somatolactotropic rat pituitary cells has p roven useful for many regulation studies, the absence of functional D- 2 receptors on these cells long prevented their use in studies of dopa minergic action. However, it is now possible to employ GH(3) cells exp ressing recombinant D-2 receptors for such investigations. We have inv estigated both the level at which expression of functional D-2 recepto rs in GH(3) cells is blocked, and the cellular pathways employed by th e major pituitary D-2 receptor isoform, D-2A, to inhibit prolactin (PR L) gene transcription. In run-off transcription assays with nuclei fro m either parental GH(3) cells or a GH(3) cell line stably expressing a D-2A expression vector, Pit-1 gene transcription was detectable in ei ther cell line, but only the latter cell line yielded detectable D-2 r eceptor transcription, implying that the block in D-2A receptor expres sion by GH(3) cells is transcriptional. Further investigations employe d GH(3) cells transiently co-transfected with a D-2A expression vector plus a rat PRL promoter construct (-1957)PRL-CAT. Pertussis toxin blo cked repression by quinpirole, a D-2 agonist, of PRL-CAT activity, dem onstrating that this action is mediated by a pertussis toxin-sensitive G protein. The observations that neither of two agents expected to ra ise intracellular Ca2+, Bay K8644 or thyrotropin-releasing hormone, pr evented quinpirole repression of PRL-CAT activity, and that the repres sive effects on this construct of quinpirole and the Ca2+ channel anta gonist were independent, suggested that regulation of intracellular Ca 2+ levels does not play a major role in D-2A-mediated repression of th e PRL promoter. By contrast, cellular over-expression of the cAMP medi ator protein kinase A completely inhibited quinpirole repression of PR L-CAT activity, suggesting a role for this kinase in D-2A-mediated rep ression of PRL gene transcription.