The major neurological consequence of cocaine abuse is inhibition of dopamine reuptake. As a consequence, cocaine abuse has been found to disrupt plasma prolactin levels. Availability of a cell culture system for studying dopaminergic repression of the prolactin gene would help understand how cocaine disrupts physiological regulation of this gene. However, since GH rat pituitary cell lines, previously employed for many studies of prolactin gene regulation, lack dopamine receptors (D2R), little is yet known about how dopamine represses expression of the prolactin gene. We have recently shown that expression in GH3 cells of either of two forms of the D2R receptor, D2L or D2S, permits dopaminergic regulation of a co- transfected rat prolactin-CAT construct. The major aim of the proposed research is to use this system to elucidate the cellular pathway that mediates dopaminergic repression of the prolactin gene. We plan first to investigate whether a dopaminergic agonist, quinpirole, can regulate prolactin gene transcription in a GH3 cell line stably expressing transfected D2L cDNA. We will then employ promoter-CAT constructs containing fragments of the prolactin promoter, transiently transfected into this cell line, to localize and characterize a dopaminergic repressor element (DARE) in the prolactin promoter. Further investigation at the DNA level will depend upon whether these initial studies implicate the pituitary-specific transcription factor pit-1 in dopamine action. If so, dopamine-induced modifications of pit-1 will be investigated; if not, the gene-proximal dopamine mediator will be cloned. More gene-distal mediators (both small molecules and macromolecules) of dopaminergic repression will be investigated by employing: (1) transfection as above to determine whether a DARE co-localizes with a regulatory element for Ca2+ and/or cyclic AMP; (2) pharmacological studies of dopaminergic regulation of endogenous prolactin gene expression. Finally, we will use similar approaches to identify and characterize a DARE in the mouse fos gene promoter.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Research Project (R01)
Project #
5R01DA007859-03
Application #
2120293
Study Section
Drug Abuse Biomedical Research Review Committee (DABR)
Project Start
1992-03-13
Project End
1996-02-28
Budget Start
1994-03-15
Budget End
1995-02-28
Support Year
3
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Mount Sinai School of Medicine
Department
Physiology
Type
Schools of Medicine
DUNS #
City
New York
State
NY
Country
United States
Zip Code
10029
Gaiddon, C; Tian, J; Loeffler, J P et al. (1996) Constitutively active G(S) alpha-subunits stimulate Pit-1 promoter activity via a protein kinase A-mediated pathway acting through deoxyribonucleic acid binding sites both for Pit-1 and for adenosine 3',5'-monophosphate response element-binding protein. Endocrinology 137:1286-91
Coleman, D T; Chen, X; Sassaroli, M et al. (1996) Pituitary adenylate cyclase-activating polypeptide regulates prolactin promoter activity via a protein kinase A-mediated pathway that is independent of the transcriptional pathway employed by thyrotropin-releasing hormone. Endocrinology 137:1276-85
Gaiddon, C; Mercken, L; Bancroft, C et al. (1995) Transcriptional effects in GH3 cells of Gs alpha mutants associated with human pituitary tumors: stimulation of adenosine 3',5'-monophosphate response element-binding protein-mediated transcription and of prolactin and growth hormone promoter activity via Endocrinology 136:4331-8
Coleman, D T; Bancroft, C (1995) Nicotine acts directly on pituitary GH3 cells to inhibit prolactin promoter activity. J Neuroendocrinol 7:785-9
Tian, J; Ma, H W; Bancroft, C (1995) Constitutively active Gq-alpha stimulates prolactin promoter activity via a pathway involving Raf activity. Mol Cell Endocrinol 112:249-56
Tian, J; Chen, J; Bancroft, C (1994) Expression of constitutively active Gs alpha-subunits in GH3 pituitary cells stimulates prolactin promoter activity. J Biol Chem 269:33-6
Yan, G; Chen, X; Bancroft, C (1994) A constitutively active form of CREB can activate expression of the rat prolactin promoter in non-pituitary cells. Mol Cell Endocrinol 101:R25-30