[3H]-thioperamide as a radioligand for the histamine H3 receptor in rat cerebral cortex.

1. The purpose of the present study was to characterize the binding of the histamine H3 receptor antagonist, [3H]-thioperamide, to rat cerebral cortical membranes. 2. The binding of [3H]-thioperamide to rat cerebral cortical membranes reached equilibrium after incubation with [3H]-thioperamide after 8-10 h at 4 degrees C. Equilibrium was maintained for up to 18 h of incubation. Addition of 1 microM (R)-alpha-methylhistamine rapidly dissociated [3H]-thioperamide from its binding sites. From these kinetic experiments a dissociation constant of 0.3 nM was obtained for [3H]-thioperamide. 3. Saturation experiments with [3H]-thioperamide using 1 microM (R)-alpha-methylhistamine to define nonspecific binding were best analysed according to a single site model. A dissociation constant (KD) of 0.80 +/- 0.06 nM (n = 3) and a maximal number of binding sites (Bmax) of 73 +/- 20 fmol mg-1 protein (n = 3) were obtained for the binding of [3H]-thioperamide to rat cerebral cortical membranes. 4. Saturation experiments with [3H]-thioperamide using 0.3 microM iodophenpropit to define nonspecific binding were best analysed according to a two site model. For the high affinity [3H]-thioperamide site a KD value of 1.1 +/- 0.3 nM (n = 3) and Bmax value of 162 +/- 108 fmol mg-1 protein (n = 3) were obtained whereas KD and Bmax values for the low affinity site were 96 +/- 19 nM and 4346 +/- 3092 fmol mg-1 protein (n = 3), respectively. 5. Using 5 nM [3H]-thioperamide, the binding was hardly displaced by H3 agonists within concentration-ranges expected to bind to the histamine H3 receptor. Under these conditions, [3H]-thioperamide binding was fully displaced by various H3-antagonists, yet most H3 antagonists showed Ki values different from those expected for the histamine H3 receptor. 6. Using 0.3 nM [3H]-thioperamide, 50-60% of the total binding was potently displaced by the H3 agonists histamine, (R)-alpha-methylhistamine, (S)-alpha-methylhistamine, imetit and immepip. Displacement of the binding of 0.3 nM [3H]-thioperamide binding exhibited clear stereoselectivity for the R and S isomers of alpha-methylhistamine. 7. Binding of 0.3 nM [3H]-thioperamide was completely displaced by several H3 antagonists (thioperamide, iodophenpropit, iodoproxyfan, and burimamide) and biphasic displacement curves were obtained; the Ki values for the high affinity site corresponded well with the expected values for the H3 receptor. Antagonists fully displaced the binding of 5 nM [3H]-thioperamide with affinities comparable to the low affinity site found with 0.3 nM [3H]-thioperamide. 8. Ondansetron and haloperidol did not displace binding of 5 nM [3H]-thioperamide at concentrations at which the former are known to bind to 5-HT3 or sigma receptors, respectively. On the other hand, nonselective cytochrome P450 inhibitors displaced the binding of 5 nM [3H]-thioperamide from both rat cerebral cortical membranes and rat liver microsomes. 9. It is concluded that the histamine H3 antagonist, [3H]-thioperamide, can be used as a radioligand to study the histamine H3 receptor in rat brain, provided that subnanomolar concentrations are used in displacement studies. Moreover, the specific binding should be defined with an H3 agonist, since most H3 antagonists share with [3H]-thioperamide a low affinity, high density, non-H3 receptor binding site(s) in rat brain. The latter is probably due to binding to cytochrome P450 isoenzymes.