Lesioning of locus coeruleus projections by DSP-4 neurotoxin treatment: effect on amphetamine-induced hyperlocomotion and dopamine D2 receptor binding in rats.

DSP-4 is a neurotoxin highly selective for the noradrenergic nerve terminals of the locus coeruleus projections. Data on the effect of DSP-4 treatment on amphetamine-induced hyperlocomotion are contradictory. In this study, DSP-4 (50 mg/kg) caused reduction of noradrenaline levels by 70% in the cerebral cortex and by 79% in the cerebellum. This treatment resulted in upregulation of dopamine D2 receptors in the striatum as evidenced by [3H]-raclopride binding. In an open field test, DSP-4 reduced locomotor activity. D-Amphetamine (1.5 mg/kg) caused a similar increase in locomotor activity in control and DSP-4-pretreated animals not familiar to the apparatus. However, when the rats were habituated to the test apparatus, the effect of amphetamine on horizontal activity was significantly larger in the DSP-4-pretreated animals. These data suggest that supersensitivity of D2 receptors develops after locus coeruleus denervation, but that the enhanced efficacy of amphetamine in DSP-4-treated rats is masked by neophobia.

Mechanism of modulation of [3H]raclopride binding to dopaminergic receptors in rat striatal membranes by sodium ions.

The mechanism of modulation of [3H]raclopride binding to dopaminergic receptors in rat brain striatal membranes by sodium ions was studied by means of equilibrium and kinetic measurements. Among different mono- and divalent cations studied, only sodium and lithium ions significantly enhanced [3H]raclopride binding to rat striatal membranes, but the effect of lithium was considerably smaller if compared with that of sodium. The equilibrium binding studies revealed that the increase in Na+ concentration from 0.5 to 150 mM increased both the radioligand affinity and the number of binding sites. The meaning of these changes was established by kinetic studies, which yielded hyperbolic plots of [3H]raclopride binding rate constants over the radioligand concentration. These plots correspond to the two-step ligand binding reaction mechanism, involving fast binding equilibrium followed by a slow isomerization of the receptor-antagonist complex. Sodium ions did not influence the antagonist affinity for the receptor sites in the first step of the binding process, nor the rate of isomerization of the receptor-ligand complex, but slowed down the rate of deisomerization. This led to a change in the value of the receptor-ligand dissociation constant Kd determined under equilibrium conditions. The same change in deisomerization rate was also sufficient to alter the receptor density (Bmax), measured by the conventional ligand binding procedure.

Modulation of [3H]quinpirole binding to dopaminergic receptors by adenosine A2A receptors.

Alteration of ligand binding to dopamine D2 receptors through activation of adenosine A2A receptors in rat striatal membranes has been studied by means of kinetic analysis. The binding of dopaminergic agonist [3H]quinpirole to rat striatal membranes was characterized by the constants Kd = 1.50+/-0.09 nM and Bmax = 115+/-2 fmol/mg of protein. The kinetic analyses revealed that the binding had at least two consecutive and kinetically distinguishable steps, the fast equilibrium of complex formation between receptor and agonist (KA = 5.9+/-1.7 nM), followed by a slow isomerization equilibrium (Ki = 0.06). Activation of adenosine A2A receptors by CGS 21680 caused enhancement of the rate [3H]quinpirole binding, altering mainly the formation of the receptor-ligand complexes (KA) as well as the isomerization rate of this complexes (ki), while the deisomerization rate (k[-i]) and the apparent dissociation rate remained unchanged.

Kinetic evidence for isomerization of the dopamine receptor-raclopride complex.

The binding kinetics of the specific dopamine D2 antagonist [3H]raclopride to dopamine D2 receptors in rat neostriatum were studied. The pseudo-first-order rate constants of [3H]raclopride binding with these membranes revealed a hyperbolic dependence upon the antagonist concentration, indicating that the reaction had at least two consecutive and kinetically distinguishable steps. The first step was fast binding equilibrium, characterized by the dissociation constant KA = 12 +/- 3 nM. The following step corresponded to a slow isomerization of the receptor-antagonist complex, characterized by the isomerization equilibrium constant Ki = 0.11. The dissociation constant Kd = 1.3 nM, calculated from these kinetic data, was similar to Kd = 2.4 nM, determined from equilibrium binding isotherm for the radioligand. Implications of the complex reaction mechanism on dopamine D2 receptor assay by [3H]raclopride were discussed.

Optimization of synthesis of agarose-based lectin affinity sorbents.

Different amounts of wheat germ agglutinin were immobilized to agarose gel, previously activated by different amounts of the coupling agent divinyl sulfone. The effectiveness of these affinity sorbents was characterized specific binding of ovomucoid with the gel. These studies revealed the formation of clear optima in binding capacity of the affinity gel, depending on conditions of its synthesis. The ratio between the concentration of the coupling agent and immobilized lectin were found to be crucial for optimization of binding properties of the affinity sorbent.