Molecular cloning, characterization and expression of two tryptophan hydroxylase (TPH-1 and TPH-2) genes in the hypothalamus of Atlantic croaker: down-regulation after chronic exposure to hypoxia.
Recently we discovered that hypoxia causes marked impairment of reproductive neuroendocrine function in Atlantic croaker, a marine teleost, which is due to a decline in hypothalamic serotonergic activity. As a first step in understanding the molecular responses of the hypothalamic serotonergic system to hypoxia, we cloned and characterized the genes for the enzymes regulating the rate-limiting step in serotonin biosynthesis, tryptophan hydroxylase (TPH-1 and TPH-2) in the croaker brain. The full-length croaker TPH-1 and TPH-2 cDNAs contain open reading frames encoding proteins with 479 and 487 amino acids, respectively, which are highly homologous to the TPH-1 (76-93%) and TPH-2 (64-92%) proteins of other vertebrates. Croaker TPH-1 and TPH-2 mRNA expression was detected throughout the brain but was greatest in the hypothalamic region. Both Northern blot analysis and real-time PCR showed that TPH-1 (transcript size approximately 2.1 kb) and TPH-2 ( approximately 1.9 kb) mRNA levels were significantly decreased in the hypothalami of croaker exposed for 2 weeks to hypoxic conditions compared with those in fish exposed to normoxic conditions. Immunohistochemistry of hypothalamic neurons with TPH antibodies showed reduced expression of TPHs in hypoxia-exposed fish compared with normoxic fish. Western blot analysis confirmed that hypoxia caused a marked decline in hypothalamic TPH protein levels, which was associated with decreases in hypothalamic TPH enzyme activity and 5-hydroxytryptophan levels. These results suggest that TPH is a major site of hypoxia-induced down-regulation of serotonergic function in croaker brains. Moreover, they provide the first evidence that hypoxia decreases the expression of TPH transcripts in vertebrate brains.
Rahman
M S
MS
University of Texas at Austin, Marine Science Institute, 750 Channel View Drive, Port Aransas, TX 78373, USA. rahman@mail.utexas.edu
Thomas
P
P
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D015536:Down-Regulation / Q000502:physiology*; D005393:Fish Diseases / Q000201:enzymology*; D007031:Hypothalamus / Q000166:cytology / Q000201:enzymology*; D000860:Hypoxia / Q000201:enzymology / Q000662:veterinary*; D051379:Mice; D009474:Neurons / Q000201:enzymology; D010473:Perciformes; D010802:Phylogeny; D011863:Radioimmunoassay / Q000379:methods; D051381:Rats; D012701:Serotonin / Q000378:metabolism; D014365:Tryptophan Hydroxylase / Q000145:classification / Q000235:genetics* / Q000378:metabolism
D012701:Serotonin; D014365:Tryptophan Hydroxylase