Differential effects of forskolin on tyrosine hydroxylase gene transcription in identified brainstem catecholaminergic neuronal subtypes in organotypic culture.
The regulation of gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, was studied in brainstem noradrenergic nuclei, locus coeruleus (LC), A2 and A1, in vitro. Several novel experimental approaches employed in this study included: (i) the development of a slice-explant model in which these brainstem nuclei maintained a high survival of the noradrenergic neurons, an organotypic topology and the coexpression of two identifying markers in addition to TH, i.e. norepinephrine transporter (NET) and vesicular monoamine transporter 2 (VMAT2); (ii) quantitative analysis of TH transcription in these nuclei was made using a labelled intronic probe to measure TH heteronuclear RNA (hnRNA) and (iii) the use of tetrodotoxin in the media to eliminate spontaneous neural activity in these nuclei, thereby providing a basal state as the starting point for the study of TH transcription under various pharmacological perturbations. In the presence of TTX, the adenylcyclase stimulator, forskolin, produced a 155% increase in LC, a 130% increase in A1, and a 220% increase in A2 in TH hnRNA as compared to control nuclei. This effect of forskolin was abolished in the LC and A1 by the PKA inhibitor, H89 (5 microm), but not by the MAP kinase pathway (MEK) inhibitor, PD98059 (75 microm). In contrast, the robust increase in TH transcription produced by forskolin in A2 neurons, was completely inhibited by PD98059, and only partially inhibited by H89, showing that induced TH transcription is mediated by different kinase pathways in specific central noradrenergic neuronal subtypes.
Rusnak
Milan
M
Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.
Gainer
Harold
H
D000818:Animals; D000831:Animals, Newborn; D001640:Bicuculline / Q000494:pharmacology; D001933:Brain Stem / Q000166:cytology*; D002395:Catecholamines / Q000235:genetics / Q000378:metabolism*; D002452:Cell Count / Q000379:methods; D005576:Colforsin / Q000494:pharmacology*; D004347:Drug Interactions; D018756:GABA Antagonists / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects*; D007150:Immunohistochemistry / Q000379:methods; D017403:In Situ Hybridization / Q000379:methods; D066298:In Vitro Techniques; D007546:Isoquinolines / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D050484:Norepinephrine Plasma Membrane Transport Proteins; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013449:Sulfonamides / Q000494:pharmacology; D027981:Symporters / Q000378:metabolism; D013779:Tetrodotoxin / Q000494:pharmacology; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics / Q000378:metabolism*; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins
D002395:Catecholamines; D018756:GABA Antagonists; D007546:Isoquinolines; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D047428:Protein Kinase Inhibitors; C493454:Slc18a2 protein, rat; C493226:Slc6a2 protein, rat; D013449:Sulfonamides; D027981:Symporters; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins; D005576:Colforsin; D013779:Tetrodotoxin; D014446:Tyrosine 3-Monooxygenase; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D001640:Bicuculline