The D 2 receptor: blocked transcription in GH 3 cells and cellular pathways employed by D 2A to regulate prolactin promoter activity

Abstract

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 2 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 Ca 2+, 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 Ca 2+ channel antagonist were independent, suggested that regulation of intracellular Ca 2+ 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.