Rat liver and kidney contain high densities of σ1 and σ2 receptors: characterization by ligand binding and photoaffinity labeling

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Abstract

Rat liver and kidney were investigated for the presence of sigma (σ) receptor subtypes by radioligand binding with three highly selective σ probes and by photoaffinity labeling using [H]azido-di-o-tolyguanidine ([3H]azido-DTG). [3(+)- Pentazocine, a highly selective σ1 probe, bound to sites in liver membranes with Kd = 7.5 nM and Bmax = 2929 fmol/mg protein. [3H](+)-Pentazocine binding sites in kidney had Kd = 23.3 nM and Bmax = 229fmol/mg protein. [3H1,3- Di-o-tolylguanidine ([3H]DTG) and [3H](+)-3-(3-hydroxyphenyl-N-(1-propyl)piperidine ([3H](+)-3-PPP) label both σ1 and σ2 receptors. Parameters for [3H]DTG in the liver were Kd = 17.9 nM and Bmax - 11 895 fmol.mg protein. Similar parameters were observed for [3H](+)-3-PPP, Kd = 51.9 nM and Bmax = 11 070 fmol/mg protein. [3H]DTG bound to rat kidney with Kd = 45.8 nM and Bmax = 1190 fmol/mg protein. The observation that either [3H]DTG or [3H](+)-3-PPP labeled a higher number of sites relative to [3H](+)-pentazocine suggested that liver and kidney contain both subtypes of σ receptor. This was confirmed by competition studies vs. [3H](+)-pentazocine and [3H]DTG (in the presence of dextrallorphan to mask σ1 sites). In both tissues, [3H](+)-pentazocine labeled sites with high affinity for haloperidol and enantioselectivity for (+)-benzomorphans over (−)-benzomorphans. [3H]DTG + dextrallorphan labeled sites in both tissues which also high affinity for haloperidol, but which had the characteristic σ2 property of low affinity for (+)-benzomorphans and enantioselectivity for (−)-benzomorphans over the corresponding (+)-isomer. Similar results were obtained with [3H](+)-3-PPP + dextrallorphan. Several novel aryl diamines, such as 1S, 2R-cis-N-[2-(3,4-dichlorophenylethyl]-N-methyl-2-(1-pyrrolidinyl)cyclohexylamine BD737) and N-2- (3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine (BD1008), bound to both sites with high affinity. Photoaffinity labeling with 10 nM [3H]azido-DTG resulted in specific labeling of polypeptides of 25 kDa and 21.5 kDa and 21.5 kDa. Dextrallorphan (100 nM or 500 nM) completely blocked labeling of the 25 kDa polypeptide, but had no effect on labeling of the lower molecular weight protein. (+)-10,11-Dihydro-5-methyl-5H-dibenzol[a,d]cyclohepten-5,10-imine((+)_-MK-801) had no effect on labeling of either polypeptide. These data are consistent with the notion that the 25 kDa and 21.5 proteins represent σ2 and σ2 receptors, respectively. Thus, rat liver and kidney contain high densities of σ1 and σ2 receptors, with σ2 sites comprising 75–80% of the total σ population. The high density of σ receptor subtypes may indicate important functions in hepatic and renal tissues.

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