Specific binding of [3H]ketanserin to hypothalamus membranes of juvenile rainbow trout, Oncorhynchus mykiss.
This study examines the existence and pharmacological specificity of [3H]ketanserin binding in hypothalamus of juvenile rainbow trout. Hypothalamic membranes were incubated with [3H]ketanserin (selective 5HT2 antagonist) under several experimental conditions; reactions were terminated by filtration and bound radioactivity was counted by liquid scintillation spectroscopy. Tissue dilution experiments revealed that specific [3H]ketanserin binding (B(sp)) was tissue dependent; 1 hypothalamus equivalent per tube (1100 +/- 115 cpm/mg protein) was subsequently used throughout the rest of this study. In association experiments, B(sp) increased progressively with time, achieved equilibrium binding levels (1192 +/- 120 cpm/mg protein) within 80 min, and remained stable for at least 60 min thereafter; k(obs), and k(+1) were 0.032 and 0.048 min(-1) x nM(-1), respectively. In dissociation experiments, B(sp) completely dissociated within 20 min following addition of excess ketanserin; k(-1) and t1/2 were 0.0803 min(-1) and 8.7 min, respectively. B(sp) was saturable (2500 +/- 256 cpm/mg protein); Scatchard-calculated values for the equilibrium dissociation constant (K(D)) and capacity (Bmax) were 0.48 nM, and 125 fmol/mg protein, respectively. B(sp) was differentially displaced by structurally related competitors, with a rank order of potency of ketanserin = mianserin > ritanserin > serotonin (5HT) = spiperone >> methiothepin mesylate > metergoline = DOI ((+/-)-2-5-dimethoxy-4-iodoamphetamine hyrobromide) > 2-methyl-5HT > alpha-methyl-5HT >>>> 5HIAA (5-hydroxyindole acetic acid) = reserpine. These findings provide pharmacological evidence for the presence of a 5HT2-like receptor subtype in the trout hypothalamus.