[ 3 H]‐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, [ 3 H]‐thioperamide, to rat cerebral cortical membranes. 2 The binding of [ 3 H]‐thioperamide, to rat cerebral cortical membranes reached equilibrium after incubation with [ 3 H]‐thioperamide after 8–10 h at 4°C. Equilibrium was maintained for up to 18 h of incubation. Addition of 1 μ m ( R )‐α‐methylhistamine rapidly dissociated [ 3 H]‐thioperamide from its binding sites. From these kinetic experiments a dissociation constant of 0.3 nM was obtained for [ 3 H]‐thioperamide. 3 Saturation experiments with [ 3 H]‐thioperamide using 1 μ m ( R )‐α‐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 [ 3 H]‐thioperamide to rat cerebral cortical membranes. 4 Saturation experiments with [ 3 H]‐thioperamide using 0.3 μ m iodophenpropit to define nonspecific binding were best analysed according to a two site model. For the high affinity [ 3 H]‐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 [ 3 H]‐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, [ 3 H]‐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 [ 3 H]‐thioperamide, 50–60% of the total binding was potently displaced by the H 3 agonists histamine, ( R )‐α‐methylhistamine, ( S )‐α‐methylhistamine, imetit and immepip. Displacement of the binding of 0.3 nM [ 3 H]‐thioperamide binding exhibited clear stereoselectivity for the R and S isomers of α‐methylhistamine. 7 Binding of 0.3 nM [ 3 H]‐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 [ 3 H]‐thioperamide with affinities comparable to the low affinity site found with 0.3 nM [ 3 H]‐thioperamide. 8 Ondansetron and haloperidol did not displace binding of 5 nM [ 3 H]‐thioperamide at concentrations at which the former are known to bind to 5‐HT 3 or σ receptors, respectively. On the other hand, nonselective cytochrome P 450 inhibitors displaced the binding of 5 nM [ 3 H]‐thioperamide from both rat cerebral cortical membranes and rat liver microsomes. 9 It is concluded that the histamine H 3 antagonist, [ 3 H]‐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 [ 3 H]‐thioperamide a low affinity, high density, non‐H3 receptor binding site(s) in rat brain. The latter is probably due to binding to cytochrome P 450 isoenzymes.