We propose to investigate the molecular basis of the impaired ability of hypothalamic dopaminergic neurons of the aged brain to synthesize L-dihydroxyphenylalanine (DOPA), to release dopamine, and to respond to prolactin stimulation. We shall investigate the hypothesis that this impaired ability is a consequence of a deficiency of exposed prolactin receptors (PR) exists in the plasma membranes of aged dopaminergic neurons compared to young dopaminergic neurons and that such a deficiency is a result of altered fludity of the lipid membranes of the neurons. To do so, the number of exposed PR in the aging hypothalamus will be evaluated using radiolabelled monoclonal antibodies to prolactin, and the effects of alterations of lipid membrane fluidity on the available number of PR will be investigated. Lipid membrane fluidity will be changed by modifying the cholesterol-to-phospholipid ratio of the membranes. The consequences of such modifications on the responses of aging neurons to prolactin stimulation will be investigated by examining the intracellular events involved in the synthesis of dopamine, the in vitro synthesis (in organ culture) of tyrosine hydroxylase (TH), the transformation of TH from an inactive form through phosphorylation to an active form, the specific activity of TH per mole of the enzyme, the synthesis of DOPA, and the release of dopamine into hypophysial portal blood. Monoclonal antibodies against TH and against PR as well as affinity-purified, monospecific polyclonal antibodies against prolactin will be utilized in these stuides. These proteins will be used in blocking experiments as well as experiments in which isolation and/or quantification of TH and PR are undertaken. The activity of TH in vivo will be assessed by quantifying the rate of synthesis of DOPA in the median eminence. The rate of release of dopamine by hypothalamic dopaminergic neurons will be quantified by determining the rate of release of dopamine into hypophysial portal blood. Using these various approaches, we propose to ascertain the extent to which down-regulation of PR in the hypothalamus of the aging brain is involved in the reduced capacity of the aged brain to synthesize DOPA and release dopamine.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AG004344-05
Application #
3480053
Study Section
Reproductive Biology Study Section (REB)
Project Start
1984-01-01
Project End
1992-01-31
Budget Start
1988-02-01
Budget End
1989-01-31
Support Year
5
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Type
Schools of Medicine
DUNS #
City
Dallas
State
TX
Country
United States
Zip Code
75390
Porter, J C; Ijames, C F; Wang, T C et al. (1995) Purification and identification of pituitary cytotropic factor. Proc Natl Acad Sci U S A 92:5351-5
Kedzierski, W; Aguila-Mansilla, N; Kozlowski, G P et al. (1994) Expression of tyrosine hydroxylase gene in cultured hypothalamic cells: roles of protein kinase A and C. J Neurochem 62:431-7
Aguila-Mansilla, N; Kedzierski, W; Porter, J C (1993) Localization of a defect in hypothalamic dopaminergic neurons of the aged brain that results in impaired PKA-dependent activation of tyrosine hydroxylase. J Pharmacol Exp Ther 267:171-5
Aguila-Mansilla, N; Jorquera, B A; Kedzierski, W et al. (1991) Effect of cerebroventricular anterior pituitary grafts on in situ expression of tyrosine hydroxylase in dopaminergic neurons of the aged animal. Endocrinology 128:1303-9
Kedzierski, W; Porter, J C (1991) A novel non-enzymatic procedure for removing DNA template from RNA transcription mixtures. Biotechniques 10:210-4
Aguila-Mansilla, N; Kedzierski, W; Porter, J C (1991) Testicular inhibition of tyrosine hydroxylase expression in tuberoinfundibular and nigrostriatal dopaminergic neurons. Endocrinology 129:877-82
Porter, J C; Aguila-Mansilla, N; Ramin, S M et al. (1991) Tyrosine hydroxylase expression in hypothalamic cells: analysis of the roles of adenosine 3',5'-monophosphate- and Ca2+/calmodulin-dependent protein kinases in the action of pituitary cytotropic factor. Endocrinology 129:2477-85
Kedzierski, W; Porter, J C (1990) Quantitative study of tyrosine hydroxylase mRNA in catecholaminergic neurons and adrenals during development and aging. Brain Res Mol Brain Res 7:45-51
Porter, J C; Kedzierski, W; Aguila-Mansilla, N et al. (1990) The tuberoinfundibular dopaminergic neurons of the brain: hormonal regulation. Adv Exp Med Biol 274:1-23
Wang, P S; Gonzalez, H A; Reymond, M J et al. (1989) Mass and in situ molar activity of tyrosine hydroxylase in the median eminence. Effect of thyroidectomy and thyroid hormone replacement. Neuroendocrinology 49:659-63

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