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High-affinity binding of [3H]cimetidine to a heme-containing protein in rat brain.

ABSTRACT: [(3)H]Cimetidine (3HCIM) specifically binds to an unidentified site in the rat brain. Because recently described ligands for this site have pharmacological activity, 3HCIM binding was characterized. 3HCIM binding was saturable, heat-labile, and distinct from the histamine H(2) receptor. To test the hypothesis that 3HCIM binds to a cytochrome P450 (P450), the effects of nonselective and isoform-selective P450 inhibitors were studied. The heme inhibitor KCN and the nonselective P450 inhibitor metyrapone both produced complete, concentration-dependent inhibition of 3HCIM binding (K(i) = 1.3 mM and 11.9 muM, respectively). Binding was largely unaffected by inhibitors of CYP1A2, 2B6, 2C8, 2C9, 2D6, 2E1, and 19A1 but was eliminated by inhibitors of CYP2C19 (tranylcypromine) and CYP3A4 (ketoconazole). Synthesis and testing of CC11 [4(5)-(benzylthiomethyl)-1H-imidazole] and CC12 [4(5)-((4-iodobenzyl)-thiomethyl)-1H-imidazole] confirmed both drugs to be high-affinity inhibitors of 3HCIM binding. On recombinant human P450s, CC12 was a potent inhibitor of CYP2B6 (IC(50) = 11.7 nM), CYP2C19 (51.4 nM), and CYP19A1 (140.7 nM) and had a range of activities (100-494 nM) on nine other isoforms. Although the 3HCIM binding site pharmacologically resembles some P450s, eight recombinant human P450s and three recombinant rat P450s did not exhibit 3HCIM binding. Inhibition by KCN and metyrapone suggests that 3HCIM binds to a heme-containing brain protein (possibly a P450). However, results with selective P450 inhibitors, recombinant P450 isoforms, and a P450 antibody did not identify a 3HCIM-binding P450 isoform. Finally, CC12 is a new, potent inhibitor of CYP2B6 and CYP2C19 that may be a valuable tool for P450 research.

SUBMITTER: Stadel R

PROVIDER: S-EPMC2586985 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

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High-affinity binding of [3H]cimetidine to a heme-containing protein in rat brain.

Stadel Rebecca R Yang Jun J Nalwalk Julia W JW Phillips James G JG Hough Lindsay B LB

Drug metabolism and disposition: the biological fate of chemicals 20071219 3

[(3)H]Cimetidine (3HCIM) specifically binds to an unidentified site in the rat brain. Because recently described ligands for this site have pharmacological activity, 3HCIM binding was characterized. 3HCIM binding was saturable, heat-labile, and distinct from the histamine H(2) receptor. To test the hypothesis that 3HCIM binds to a cytochrome P450 (P450), the effects of nonselective and isoform-selective P450 inhibitors were studied. The heme inhibitor KCN and the nonselective P450 inhibitor mety ...[more]

PMID: 18094038

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Project description:Binding of D,L-(E)-2-amino-4-[(3)H]-propyl-5-phosphono-3-pentenoic acid ([(3)H]-CGP39653), a selective antagonist at the glutamate site of the NMDA receptor, is modulated by glycine in rat brain tissue. We have further investigated this phenomenon in rodent and human brain by means of receptor binding and quantitative autoradiography techniques. In rat cerebral cortical membranes the glycine antagonist 3-[2-(Phenylaminocarbonyl)ethenyl]-4,6-dichloro-indole-2-carboxylic acid sodium salt (GV150526A) did not change basal [(3)H]-CGP39653 binding, but competitively reversed the high affinity component of [(3)H]-CGP39653 binding inhibition by glycine, with a pK(B) value of 8.38, in line with its affinity for the glycine site (pK(i)=8.49 vs. [(3)H]-glycine). Glycine (10 microM) significantly decreased [(3)H]-CGP39653 affinity for the NMDA receptor (with no change in the B(max)), whereas enhanced L-glutamate affinity (P<0.05, paired-samples Student's t-test). In rat brain sections the addition of GV150526A (30 microM) to the incubation medium increased [(3)H]-CGP39653 binding to 208% of control (average between areas), indicating the presence of endogenous glycine. The enhancement presented significant regional differences (P<0.05, two-way ANOVA), with striatum higher than cerebral cortex (282 and 187% of control, respectively; P<0.05, Fisher's LSD). On the contrary, there was not any significant variation in affinity values of [(3)H]-CGP39653, L-glutamate, glycine and GV150526A in striatal and cortical membranes. These results confirmed the existence of regionally distinct NMDA receptors subtypes with different glycine/glutamate allosteric modulation. Whole brain autoradiography revealed an uneven distribution of [(3)H]-CGP39653 binding sites in human brain. High levels of binding were determined in hippocampus and in cingulate, frontoparietal and insular cortex. Intermediate to low levels of binding were found in diencephalic nuclei and basal ganglia. [(3)H]-CGP39653 binding was increased to 216% of control (mean between areas) by 30 microM GV150526A. The enhancement, however, did not present significant regional differences. These results introduce GV150526A as a useful tool to identify NMDA receptor subtypes by means of receptor autoradiography; moreover, they demonstrate that the allosteric inhibition of [(3)H]-CGP39653 binding by glycine parallels an increase in receptor affinity to the endogenous ligand L-glutamate. Finally, this study provides the first detailed anatomical description of the regional distribution of [(3)H]-CGP39653 binding sites in human brain.

Dataset Information

High-affinity binding of [3H]cimetidine to a heme-containing protein in rat brain.

Publications

High-affinity binding of [3H]cimetidine to a heme-containing protein in rat brain.

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