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 proven useful for many regulation studies, the absence of functional D-2 receptors on these cells long prevented their use in studies of dopaminergic action. However, it is now possible to employ GH(3) cells expressing recombinant D-2 receptors for such investigations. We have investigated both the level at which expression of functional D-2 receptors in GH(3) cells is blocked, and the cellular pathways employed by the major pituitary D-2 receptor isoform, D-2A, to inhibit prolactin (PRL) gene transcription. In run-off transcription assays with nuclei from 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 either cell line, but only the latter cell line yielded detectable D-2 receptor transcription, implying that the block in D-2A receptor expression by GH(3) cells is transcriptional. Further investigations employed GH(3) cells transiently co-transfected with a D-2A expression vector plus a rat PRL promoter construct (-1957)PRL-CAT. Pertussis toxin blocked repression by quinpirole, a D-2 agonist, of PRL-CAT activity, demonstrating that this action is mediated by a pertussis toxin-sensitive G protein. The observations that neither of two agents expected to raise intracellular Ca2+, Bay K8644 or thyrotropin-releasing hormone, prevented quinpirole repression of PRL-CAT activity, and that the repressive effects on this construct of quinpirole and the Ca2+ channel antagonist were independent, suggested that regulation of intracellular Ca2+ levels does not play a major role in D-2A-mediated repression of the PRL promoter. By contrast, cellular over-expression of the cAMP mediator protein kinase A completely inhibited quinpirole repression of PRL-CAT activity, suggesting a role for this kinase in D-2A-mediated repression of PRL gene transcription.