In vitro binding characteristics of [3H]spiperone to the pituitary of the goldfish (Carassius auratus)

Homogenates of the pituitary of the goldfish (Carassius auratus) were incubated with 3H]spiperone under various experimental paradigms to evaluate the binding characteristics of the goldfish pituitary dopamine receptor. Binding was tissue specific as binding of 3H]spiperone to goldfish pituitary was greater than other tissue types examined, and the magnitude of binding was found to be dependent on pituitary (protein) content; also, specific binding was heat labile. Association was rapid and binding was reversible (dissociable) by addition of excess competing ligand (domperidone, a specific dopamine D2 receptor antagonist); the half-life (t1/2) of dissociation was 9.2 min and the estimated dissociation rate constant (k-1) was 7.56 x 10(-2) min-1); as well, the association rate was temperature dependent. Binding was saturable; saturation analysis using 3H]spiperone indicated a single class of binding sites with an estimated dissociation constant (Kd) and capacity of 7.39 +/- 1.23 x 10(-6) M and 31.56 +/- 2.72 x 10(-9) mol/mg protein, respectively. 3H]Spiperone binding was displaceable; displacement analysis using unlabeled domperidone indicated a single class of binding sites with estimated Kd and capacity of 2.94 +/- 0.54 x 10(-6) M and 19.47 +/- 3.12 x 10(-9) mol/mg protein, respectively. Binding was specifically inhibited by various dopamine antagonists and agonists. The density of binding sites differed significantly between regions of the goldfish pituitary; the number of binding sites in the pars distalis and neurointermediate lobes was estimated as 38.89 +/- 2.07 x 10(-9) mol/mg protein vs 109.45 +/- 25.33 x 10(-9) mol/mg protein, respectively; while the Kd's estimated as 3.73 +/- 0.248 x 10(-6) M vs 4.1 +/- 1.21 x 10(-6) M, respectively, were not significantly different. These data agree with previous in vivo and in vitro findings of the biological actions of dopamine agonists and antagonists in modifying gonadotropic hormone release in the goldfish and represent the first demonstration of the existence and binding characteristics of a dopamine/neuroleptic receptor in the pituitary of a nonmammalian vertebrate.