Fish Physiology and Biochemistry vol. 11 no. 1-6 pp 139-144 (1993) Kugler Publications, Amsterdam/New York

Catecholamine synthesis in the rainbow trout (Oncorhynchus mykiss) brain: modulation of tyrosine hydroxylase activity C. Saligaut, S. Bennani and T. Bailhache Laboratoirede Physiologie des Rigulations, URA CNRS 256 - Campus de Beaulieu, 35042 Rennes Cedex, France.

Keywords: rainbow trout, dopamine,norepinephrine, hypothalamus, pituitary, telencephalon, preoptic area, tyrosine hydroxylase, estradiol, 2-hydroxyestradiol

Abstract Levels of catecholamines and tyrosine hydroxylase (TH) activity were measured in brain homogenates from female rainbow trout. In triploid fish or in diploid fish in ovarian recrudescence, the patterns of catecholamine content expressed as a function of in vitro TH activity vary in different areas of the brain. Km for the pterin cofactor is lower in the telencephalon than in the hypothalamus. Dopamine (DA) and 2-hydroxyestradiol (20HE 2) inhibit TH activity (by competitive and non-competitive interaction respectively). The K i for DA were different in the telencephalon and the hypothalamus and this could explain the different patterns of catecholamine levels and TH activity for these two structures. 20HE 2 inhibits TH activity in vitro; a catechol moiety is required since estradiol (E2 ) is not inhibitory. However, the exact mechanism of inhibition remains unclear. The rapid effect of 20HE2 cannot explain the previously reported activation of catecholamine synthesis by E2 in vivo.

Resume Les taux de cat6cholamines c6r6brales et l'activit6 in vitro de la tyrosine hydroxylase (TH) ont et6 determines chez la truite arc en ciel femelle. Chez des animaux triploides ou diploides en recrudescence ovarienne, les taux de cat6cholamines sont corrl1es differemment a l'activit6 TH selon la structure cerebrale considered. Le telenc6phale pr6sente un Km apparent pour la pterine plus faible que l'hypothalamus. La dopamine (DA) et le 2-hydroxyestradiol (20HE 2) inhibent l'activite de la TH selon des m6canismes respectivement competitifs et non comp6titifs. Les diff6rents Ki pour la DA obtenus au niveau du tlenc6phale et de l'hypothalamus pourraient expliquer les diff6rentes relations entre taux de cat6cholamines et activity TH obtenues pour ces deux structures. Le 20HE 2 inhibe egalement l'activite TH in vitro: la presence d'un radical catechol s'avere n6cessaire (I'oestradiol (E2 ) n'est pas actif par lui-meme), mais le m6canisme de l'inhibition reste a determiner. Les effets rapides du 20HE2 ne peuvent expliquer l'activation de la synthese cat6cholaminergique par l'E 2 in vivo demontr6e anterieurement.

Correspondence to: C. Saligaut

140 Introduction Biological interactions between estrogens and catecholamines have been recently reported in the rainbow trout: before ovulation a decrease of hypothalamic and hypophysial dopamine turn-over can be correlated to a decrease in blood estradiol levels (Saligaut et al. 1992a). Estradiol (E2) replacement of ovariectomized trout at the beginning of vitellogenesis increases both norepinephrine (NE) and dopamine (DA) levels in the hypothalamus, but not in other brain areas, suggesting structure specific modulations of catecholamine synthesis (Saligaut et al. 1992b). In mammals, tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of catecholamines (Levitt et al. 1965) and requires a pterin cofactor (Udenfriend et al. 1965). Long-term regulation of TH partly involves changes in the enzyme gene expression (Blum et al. 1987), whereas rapid modulation of TH appears to be accomplished by direct inhibition of enzymatic activity; for instance, catechol estrogens and catecholamines compete with the endogenous pterin cofactor (competitive ligation) (Lloyd and Weisz 1978; Lloyd and Ebersole 1980; Foreman and Porter 1980; Panek et al. 1987). In the present study, we have determined whether some rapid modulations of TH activity could explain structure-specific effects of estradiol on catecholamine synthesis in trout (Saligaut et al. 1992b). We focused our experiments on the effects of catecholamines, E2, or E2 derivatives (20HE 2) at the pterin site level (determination of TH kinetic parameters in vitro).

Materials and methods All the experiments were performed on the female rainbow trout (Oncorhynchusmykiss), kept in aerated and recirculated fresh water at 13°C + 1, under a natural photoperiod. In vitro TH activity This was determined by assaying 3-4 dihydroxyphenylalanine (DOPA) formed from the substrate

L-tyrosine (Nagatsu et al. 1979). Incubation (13 min.) was at 30°C (the optimal temperature for in vitro TH activity in trout (data not shown)) in a standard incubation medium (150 1l final volume; final concentrations in brackets): 1 M acetate buffer pH 6 (66 mM), 6 mM L-tyrosine in 0.01 M HCI (60 AM-1 mM), 10 mM 6-methyl-5,6,7,8-tetrahydropterin (6MPH 4 , 80-660 /M; Sigma) in 1.5 M mercaptoethanol, 10-50 al of brain homogenate in 0.25 M sucrose, catalase (2,600 U; Boehringer Mannheim, Germany), 12 mM ferrous ammonium sulfate (830 AM), 0.15 M ascorbic acid (10 mM) and water. The reaction was stopped with 600 l of cold 0.5 M perchloric acid containing amethyl DOPA (Sigma) as an internal standard in an ice bath. Catechol compounds were extracted by aluminium oxide at pH 8.0, eluted with 0.1 N HCI and assayed by HPLC (Saligaut et al. 1992a). Preliminary experiments demonstrated that TH activity was proportional to the amount of tissue assayed (100-900 g protein) and remained linear for periods up to 15 min. of incubation (data not shown). The incubation of standard L-DOPA with brain homogenates under optimal conditions did not result in a loss of L-DOPA, demonstrating the absence of DOPA decarboxylase activity (Ginns et al. 1988). Potential TH activity This was measured using saturating concentrations of L-tyrosine (1 mM) and pterin cofactor (600 AM) in the incubation medium for the hypothalamus, telencephalon, pituitary and preoptic area of triploid fish (weighing about 800g; Cornec fishfarm, France) or diploid fish in ovarian recrudescence (weighing about 1,500g; gonadosomatic index < 1%). Total catecholamine levels (DA + NE) were assayed by HPLC (Saligaut et al. 1992b) and compared with TH activity. TH kinetic parameters Vma, Km and Ki were determined from double reciprocal plots (Lineweaver-Burk and Dixon plots) obtained by incubating telencephalic or hypothalamic homogenates (from fish in ovarian recrudescence) with subsaturating concentrations of 6MPH 4 and adding various concentrations of


Catecholamine synthesis in the rainbow trout (Oncorhynchus mykiss) brain: modulation of tyrosine hydroxylase activity.

Levels of catecholamines and tyrosine hydroxylase (TH) activity were measured in brain homogenates from female rainbow trout. In triploid fish or in d...
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