Dj. Fischberg et C. Bancroft, THE D-2 RECEPTOR - BLOCKED TRANSCRIPTION IN GH(3) CELLS AND CELLULAR PATHWAYS EMPLOYED BY D-2A TO REGULATE PROLACTIN PROMOTER ACTIVITY, Molecular and cellular endocrinology, 111(2), 1995, pp. 129-137
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.