Multiple pathways of σ1 receptor ligand uptakes into primary cultured neuronal cells

Abstract Although many antipsychotics have affinities for σ receptors, the transportation pathway of exogenous σ 1 receptor ligands to intracellular type-1 σ receptors are not fully understood. In this study, σ 1 receptor ligand uptakes were studied using primary cultured neuronal cells. [ 3 H](+)-pentazocine and [ 3 H]( R )-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperazin-1-yl]methyl-2-pyrrolidinone l -tartrate (MS-377), used as a selective σ 1 receptor ligands, were taken up in a time-, energy- and temperature-dependent manner, suggesting that active transport mechanisms were involved in their uptakes. σ 1 receptor ligands taken up into primary cultured neuronal cells were not restricted to agonists, but also concerned antagonists. The uptakes of these ligands were mainly Na + -independent. Kinetic analysis of [ 3 H](+)-pentazocine and [ 3 H]MS-377 uptake showed K m values (μM) of 0.27 and 0.32, and V max values (pmol/mg protein/min) of 17.4 and 9.4, respectively. Although both ligands were incorporated, the pharmacological properties of these two ligands were different. Uptake of [ 3 H](+)-pentazocine was inhibited in the range 0.4–7.1 μM by all the σ 1 receptor ligands used, including N , N -dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine monohydrochloride (NE-100), a selective σ 1 receptor ligand. In contrast, the inhibition of [ 3 H]MS-377 uptake was potently inhibited by haloperidol, characterized by supersensitivity (IC 50 , approximately 2 nM) and was inhibited by NE-100 with low sensitivity (IC 50 , 4.5 μM). Moreover, kinetic analysis revealed that NE-100 inhibited [ 3 H]MS-377 uptake in a noncompetitive manner, suggesting that NE-100 acted at a site different from the uptake sites of [ 3 H]MS-377. These findings suggest that there are at least two uptake pathways for σ 1 receptor ligands in primary cultured neuronal cells (i.e. a haloperidol-sensitive pathway and another, unclear, pathway). In addition, pretreatment of cells with a calmodulin antagonist, N -(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), a myosin light chain kinase inhibitor, 1-(5-chloronaphthalene-1-sulfonyl)homopiperazine (ML-9), or microsomal Ca 2+ -ATPase inhibitors resulted in a reduction of the amount of σ receptor ligand uptake. These findings suggest that the Ca 2+ pump on the endoplasmic reticulum and/or calmodulin-related events might be involved in the regulation of the uptake of σ receptor ligands into primary neuronal cells.