The D2 receptor: blocked transcription in GH3 cells and cellular pathways employed by D2A to regulate prolactin promoter activity.
Although the GH3 line of somatolactotropic rat pituitary cells has proven useful for many regulation studies, the absence of functional D2 receptors on these cells long prevented their use in studies of dopaminergic action. However, it is now possible to employ GH3 cells expressing recombinant D2 receptors for such investigations. We have investigated both the level at which expression of functional D2 receptors in GH3 cells is blocked, and the cellular pathways employed by the major pituitary D2 receptor isoform, D2A, to inhibit prolactin (PRL) gene transcription. In run-off transcription assays with nuclei from either parental GH3 cells or a GH3 cell line stably expressing a D2A expression vector, Pit-1 gene transcription was detectable in either cell line, but only the latter cell line yielded detectable D2 receptor transcription, implying that the block in D2 receptor expression by GH3 cells is transcriptional. Further investigations employed GH3 cells transiently co-transfected with a D2A expression vector plus a rat PRL promoter construct (-1957)PRL-CAT. Pertussis toxin blocked repression by quinpirole, a D2 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 D2A-mediated repression of the PRL promoter.(ABSTRACT TRUNCATED AT 250 WORDS)
Fischberg
D J
DJ
Department of Physiology and Biophysics, Mount Sinai School of Medicine, City University of New York, NY 10029, USA.
Bancroft
C
C
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002120:Calcium Channel Agonists / Q000494:pharmacology; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D004873:Ergolines / Q000494:pharmacology; D005786:Gene Expression Regulation; D037342:Pertussis Toxin; D010902:Pituitary Gland / Q000378:metabolism*; D011388:Prolactin / Q000235:genetics*; D011401:Promoter Regions, Genetic; D019257:Quinpirole; D051381:Rats; D017448:Receptors, Dopamine D2 / Q000235:genetics* / Q000502:physiology; D011994:Recombinant Proteins; D013134:Spiperone / Q000378:metabolism; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D002120:Calcium Channel Agonists; D004268:DNA-Binding Proteins; D004873:Ergolines; C494186:Pou1f1 protein, rat; D017448:Receptors, Dopamine D2; D011994:Recombinant Proteins; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; D010566:Virulence Factors, Bordetella; D019257:Quinpirole; D013134:Spiperone; D013973:Thyrotropin-Releasing Hormone; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D011388:Prolactin; D015500:Chloramphenicol O-Acetyltransferase; D037342:Pertussis Toxin; D002118:Calcium