Non-competitive interaction between raclopride and spiperone on human D-receptors in intact Chinese hamster ovary cells.

We recently investigated the binding properties of the antagonists [(3)H]-raclopride and [(3)H]-spiperone to intact Chinese hamster ovary cells expressing recombinant human D(2long)-dopamine receptors (CHO-D(2L) cells). Compared with saturation binding with [(3)H]-raclopride, raclopride reduced [(3)H]-spiperone binding with to low potency in competition binding experiments. The present findings illustrate the ability of spiperone to inhibit [(3)H]-raclopride binding non-competitively. While raclopride only decreases the apparent K(D) of [(3)H]-raclopride in saturation binding experiments, spiperone only decreases the number of sites to which [(3)H]-raclopride binds with high affinity. Also, while the IC(50) of raclopride depends on the concentration of [(3)H]-raclopride in competition experiments, this is not the case for spiperone. Kinetic studies reveal that the binding of raclopride at its high affinity sites does not affect the association of subsequently added [(3)H]-spiperone nor the rebinding of freshly dissociated [(3)H]-spiperone to the same or surrounding receptors. Yet, spiperone does not affect the dissociation rate of [(3)H]-raclopride and raclopride does not affect the (genuine) dissociation rate of [(3)H]-spiperone. The easiest way to interpret the present findings in molecular terms is to assume that D(2L)-receptors or their dimeric complexes possess two distinct binding sites: one with high affinity/accessibility for [(3)H]-raclopride and the other one with high affinity/accessibility for [(3)H]-spiperone. The ability of bound spiperone to inhibit high affinity raclopride binding while the reverse is not the case suggests for the occurrence of non-reciprocal allosteric interactions. These new findings could point at the occurrence of allosteric interactions between different classes of D(2)-receptor&nbsp;antagonists.</p

Antagonist-radioligand binding to D2L-receptors in intact cells.

D(2)-dopamine receptors mediate most of the physiological actions of dopamine and are important recognition sites for antipsychotic drugs. Earlier binding studies were predominantly done with broken cell preparations with the tritiated D(2)-receptor antagonists [(3)H]-raclopride, a hydrophilic benzamide, and [(3)H]-spiperone, a highly hydrophobic butyrophenone. Here we compared [(3)H]-raclopride and [(3)H]-spiperone binding properties in intact Chinese Hamster Ovary cells stably expressing recombinant human D(2L)-receptors. Specific binding of both radioligands occurred to a comparable number of sites. In contrast to the rapid dissociation of [(3)H]-raclopride in both medium only and in the presence of an excess of unlabelled ligand [(3)H]-spiperone dissociation was only observed in the latter condition, and it was still slower than in broken cell preparations. However, this could not explain the pronounced difference in the potency of some unlabelled ligands to compete with both radioligands. To integrate these new findings, a model is proposed in which raclopride approaches the receptor from the aqueous phase, while spiperone approaches the receptor by lateral diffusion within the&nbsp;membrane.</p

Angiotensin IV displays only low affinity for native insulin-regulated aminopeptidase (IRAP).

&lt;p&gt;Radioligand binding studies revealed that Ang IV binds to insulin-regulated aminopeptidase (IRAP)/&amp;#39;AT(4) receptors&amp;#39; with high affinity. Yet, as these experiments were routinely carried out in the presence of chelators, only the catalytic zinc-depleted apo-form of IRAP was labelled. While the chelators remove the catalytic zinc from IRAP and protect Ang IV from proteolytic degradation, the aminopeptidase N selective inhibitor &amp;#39;7B&amp;#39; only exerts the latter effect. By using 7B along with the new stable Ang IV-analog [(3) H]AL-11, we here show that the native enzyme is only a low-affinity target for Ang IV.&lt;/p&gt;</p