Multiple pathways of sigma(1) receptor ligand uptakes into primary cultured neuronal cells

Although many antipsychotics have affinities for a receptors, the transport ation pathway of exogenous cr, receptor ligands to intracellular type-1 sig ma receptors are not fully understood. In this study, sigma (1) receptor li gand uptakes were studied using primary cultured neuronal cells. [H-3](+)-p entazocine and [H-3](R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperaz in-1-yl]methyl-2-pyrrolidinone L-tartrate (MS-377), used as a selective sig ma (1) receptor ligands, were taken up in a time-, energy- and temperature- dependent manner, suggesting that active transport mechanisms were involved in their uptakes. cr, receptor ligands taken up into primary cultured neur onal cells were not restricted to agonists, but also concerned antagonists. The uptakes of these ligands were mainly Na+-independent. Kinetic analysis of [H-3](+)-pentazocine and [H-3]MS-377 uptake showed K-m values (muM) of 0.27 and 0.32, and V-max values (pmol/mg protein/min) of 17.4 and 9.4, resp ectively. Although both ligands were incorporated, the pharmacological prop erties of these two ligands were different. Uptake of [H-3](+)-pentazocine was inhibited in the range 0.4-7.1 muM by all the cr, receptor ligands used , including N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine m onohydrochloride (NE-100), a selective a, receptor ligand. In contrast, the inhibition of [H-3]MS-377 uptake was potently inhibited by haloperidol, ch aracterized by supersensitivity (IC50, approximately 2 nM) and was inhibite d by NE-100 with low sensitivity (IC50), 4.5 muM). Moreover, kinetic analys is revealed that NE-100 inhibited [H-3]MS-377 uptake in a noncompetitive ma nner, suggesting that NE-100 acted at a site different from the uptake site s of [H-3]MS-377. These findings suggest that there are at least two uptake pathways for sigma (1) receptor ligands in primary cultured neuronal cells (i.e. a haloperidol-sensitive pathway and another, unclear, pathway). In a ddition, pretreatment of cells with a calmodulin antagonist, N-(6-aminohexy l)-5-chloro-1-naphthalene sulfonamide (W-7), a myosin light chain kinase in hibitor, 1-(5-chloronaphthalene-1-sulfonyl)homopiperazine (ML-9), or micros omal Ca2+-ATPase inhibitors resulted in a reduction of the amount of or rec eptor ligand uptake. These findings suggest that the Ca2+ pump on the endop lasmic reticulum and/or calmodulin-related events might be involved in the regulation of the uptake of sigma receptor ligands into primary neuronal ce lls. (C) 2001 Elsevier Science BN. All rights reserved.