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

1 The purpose of the present study was to characterize the binding of the histamine H-3 receptor antagonist, [H-3]-thioperamide, to rat cerebral cortical membranes. 2 The binding of [H-3]-thioperamide to rat cerebral cortical membranes reached equilibrium after incubation with [H-3]-thioperamide after 8-10 h at 4 degrees C. Equilibrium was maintained for up to 18 h of incubation. Addition of 1 mu M (R)-alpha-methylhistamine rapidly dissociated [H-3]-thioperamide from its binding sites. From these kinetic experiments a dissociation constant of 0.3 nM was obtained for [H-3]-thioperamide. 3 Saturation experiments with [H-3]-thioperamide using 1 mu M (R)-alpha-methylhistamine to define nonspecific binding were best analysed according to a single site model. A dissociation constant (K-D) of 0.80 +/- 0.06 nM (n=3) and a maximal number of binding sites (B-max) of 73 +/- 20 fmol mg(-1) protein (n=3) were obtained for the binding of [H-3]-thioperamide to rat cerebral cortical membranes. 4 Saturation experiments with [H-3]-thioperamide using 0.3 mu M iodophenpropit to define nonspecific binding were best analysed according to a two site model. For the high affinity [H-3]-thioperamide site a K-D value of 1.1 +/- 0.3 nM (n=3) and B-max value of 162 +/- 108 fmol mg(-1) protein (n=3) were obtained whereas K-D and B-max values for the low affinity site were 96 +/- 19 nM and 4346 +/- 3092 fmol mg(-1) protein (n=3), respectively. 5 Using 5 nM [H-3]-thioperamide, the binding was hardly displaced by H-3 agonists within concentration-ranges expected to bind to the histamine H-3 receptor. Under these conditions, [H-3]-thioperamide binding was fully displaced by various H-3-antagonists, yet most H-3 antagonists showed K-i values different from those expected for the histamine H-3 receptor. 6 Using 0.3 nM [H-3]-thioperamide, 50-60% of the total binding was potently displaced by the H-3 agonists histamine, (R)-alpha-methylhistamine, (S)-alpha-methylhistamine, imetit and immepip. Displacement of the binding of 0.3 nM [H-3]-thioperamide binding exhibited clear stereoselectivity for the R and S isomers of alpha-methylhistamine. 7 Binding of 0.3 nM [H-3]-thioperamide was completely displaced by several H-3 antagonists (thioperamide, iodophenpropit, iodoproxyfan, and burimamide) and biphasic displacement curves were obtained; the K-i values for the high affinity site corresponded well with the expected values for the H-3 receptor. Antagonists fully displaced the binding of 5 nM [H-3]-thioperamide with affinities comparable to the low affinity site found with 0.3 nM [H-3]-thioperamide. 8 Ondansetron and haloperidol did not displace binding of 5 nM [H-3]-thioperamide at concentrations at which the former are known to bind to 5-HT3 or a receptors, respectively. On the other hand, nonselective cytochrome P-450 inhibitors displaced the binding of 5 nM [H-3]-thioperamide from both rat cerebral cortical membranes and rat liver microsomes. 9 It is concluded that the histamine H-3 antagonist, [H-3]-thioperamide, can be used as a radioligand to study the histamine H-3 receptor in rat brain, provided that subnanomolar concentrations are used in displacement studies. Moreover, the specific binding should be defined with an H-3 agonist, since most H-3 antagonists share with [H-3]-thioperamide a low affinity, high density, non-H-3 receptor binding site(s) in rat brain. The latter is probably due to binding to cytochrome P-450 isoenzymes.