Existence and distribution of serotonin and its 5-HT2-like binding site in the brain : pituitary axis of juvenile and sexually recrudescing female rainbow trout (Oncorhynchus mykiss)
Agrawal, Smriti Mona
Master of Science
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In the first phase of this study, mammalian serotonin2-like (5-HT2-like) binding sites in juvenile rainbow trout (Oncorhynchus mykiss) hypothalamus, were examined by radioligand binding assay using the tritiated analog of a selective serotonin antagonist, ketanserin ([3H]ketanserin), as the radioligand. Specific [3H]ketanserin binding (Bsp) to juvenile hypothalamus membrane was tissue-dependent where Bsp increased linearly with tissue concentration. Therefore, 1 hypothalamus-equivalent per tube (1100 ± 115 cpm/mg protein) was subsequently used throughout the rest of the first phase. In association experiments (n=5), Bsp increased progressively with time to achieve equilibrium binding levels (1192+ 120 cpm/mg protein) which remained stable for at least 60 min thereafter; kobs, and k+i were 0.032 and 0.048 min-1 nM-1 respectively. This consistent, and relatively stable association of radioligand to the binding site indicates good stability of [3H]ketanserin binding to this binding site. In dissociation experiments, Bsp completely dissociated within 20 min following addition of excess ketanserin; k_i, and t1/2 were 0.0803 min-1 and 8.7min, respectively. This pattern of [3H]ketanserin binding to this binding site is consistent with the association and dissociation kinetics of radioligand binding to a receptor. Bsp was saturable (2500 ± 256 cpm/mg protein); Scatchardcalculated values for the equilibrium dissociation constant (KD) and capacity (BMAX) were 0.48nM, and 125 fmol/mg protein, respectively, indicating the presence of a finite population of high-affmity 5-HT2-like binding sites. Bsp was differentially displaced by various competitors, with a rank order of potency of ketanserin = mianserin > ritanserin > 5-HT = spiperone » methiothepin mesylate > metergoline = DOI > 2-methyl-5-HT > amethyl- 5-HT »»5-HIAA = reserpine. This rank order suggests that specific 5-HT2 agonists and antagonists displace specifically bound [3H]ketanserin more effectively compared to non-specific competitors, and in a manner comparable with the 5-HT2-like binding site of mammals. Collectively, these findings provide pharmacological evidence for the existence of a 5HT2-like receptor subtype in the trout hypothalamus.