Sigma Receptor-Mediated Alterations in Cellular Morphology and Viability
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The Journal of Neuroscience, January 1995. 75(l): 117-134 Cytotoxic E ffects of Sigma Ligands: Sigma Receptor-mediated Alterations in Cellular Morphology and Viability Bertold J. Vilner, Brian Ft. de Costa, and Wayne D. Bowen Unit on Receptor Biochemistry and Pharmacology, Laboratory of Medicinal Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892 The morphological effects of several neuroleptics as well as effect on the activity of sigma compounds. Increasing the other novel and prototypic sigma ligands were examined by pH from the normal range of 7.2-7.4 to pH 8.3-8.5 shifted addition to cultures of C6 glioma cells. Sigma ligands caused the dose curves (30, 100,300 NM) for all sigma compounds loss of processes, assumption of spherical shape, and ces- to the left. Under these conditions, DTG, (+)-3-PPP, and ben- sation of cell division. The time course and magnitude of zomorphans produced effects in 24 hr or less. Decreasing this effect were dependent on the concentration of sigma the medium pH to 6.5-6.7 markedly reduced the activity of ligand. Continued exposure to sigma compounds ultimately all sigma ligands, producing significant protection from cy- resulted in cell death. However, the morphological effect was totoxic effects. Importantly, compounds that lacked sigma reversible when sigma ligand was removed shortly after binding affinity showed neither synergism with 30 PM BD737 rounding. The potency of compounds to produce these ef- nor an increase in activity at higher pH. These results confirm fects generally correlated with binding affinity at sigma re- the sigma receptor specificity of this effect. Sigma ligands ceptors of C6 glioma cell membranes labeled with [3H](+)- had similar effects on other cells of neuronal and non-neu- pentarocine. At a concentration of 100 PM, haloperidol, ronal origin, including SK-N-SH and SH-SY5Y neuroblasto- reduced haloperidol, fluphenazine, perphenazine, trifluo- mas, NCB-20 hybridoma, NG 108- 15 neuroblastoma-glioma perazine, BD737, LR172, BD1006, and SH344 produced sig- hybrid, COS-7 (kidney), MRS-5 (lung), and PC12 pheochrom- nificant effects in 3-6 hr of exposure. Other compounds, ocytoma. These results suggest that sigma receptors play such as trifluperidol, thioridazine, and (-)-butaclamol, pro- some vital role in cell function and may have important im- duced significant effects by 24 hr of exposure. Despite the plications for neurodegenerative disorders and neuroleptic requirement of micromolar concentrations of ligand (some treatment. compounds were effective at 30 PM), the effect showed a [Key words: sigma receptors, neurolepfics, cytotoxicity, remarkable specificity for compounds exhibiting sigma re- neurodegeneration, C6 glioma, haloperidol] ceptor binding affinity. Neuroleptics lacking potent sigma affinity [e.g., (-)-sulpiride, (+)-butaclamol, and clozapine] Sigma receptorsare saturable, high-affinity binding sitesfor sev- and other compounds that lack significant sigma affinity but eral important classesof psychotropic drugs, and have received that are agonists or antagonists at dopamine, serotonin, ad- intensive study in recent years (Musacchio et al., 1989a;Itzhak renergic, glutamate, phencyclidine, GABA, opiate, or mus- and Stein, 1990; Walker et al., 1990, 1993b; Ferris et al., 1991; carinic cholinergic receptors were without effect on cellular Su, 1991; Bowen, 1993; Debonnel, 1993). These receptors are morphology at concentrations up to 300 PM over a period of distinct in pharmacologicalprofile, tissuedistribution, and sub- 72 hr. Likewise, blockers and activators of Na+, K+, and Ca2+ cellular localization from any known neurotransmitter or hor- channels and a monoamine oxidase inhibitor devoid of mone receptor. Sigma sitesare located not only in the CNS, but sigma affinity were without effect. Interestingly, 1,3-di-etolyl- also occur in high density in severalperipheral tissues,including guanidine (DTG), (+)-3-(3-hydroxyphenyI)-N-( 1 -pro- liver and endocrine tissuesuch as testis and ovary. At least two pyl)piperidine [( +)3-PPP], (+)-pentazocine, (+)-cyclazo- classesof sigma sites, termed sigma-l and sigma-2,have been tine, and other sigma-active benzomorphans and morphinans identified (Walker et al., 1990; Quirion et al., 1992). These sites appeared inactive in up to 72 hr of culture. However, these are distinguishable by their affinity for dextrorotatory benzo- compounds interacted synergistically with a subeffective morphans and by molecular size (Hellewell and Bowen, 1990; dose of BD737 (30 AM) to produce effects usually in 6 hr or Di Paolo et al., 1991; Bowen et al., 1993; Hellewell et al., 1994. less. Also, the pH of the culture medium had a profound Though the exact role of sigma receptors in synaptic transmis- sion and cell function is still largely unknown, sigmasites have Received Dec. 3 I, 1993; revised Apr. 20, 1994; accepted June 9, 1994. been implicated in a number of biochemical, physiological, and We acknowledge Dr. F. 1. Carroll, Research Triangle Institute, and Dr. Kenner behavioral processesincluding motor control, neurotransmitter C. Rice, NIDDK, for providing some of the opiate-related compounds used in synthesisand release,ileal smooth musclecontraction, duodenal this study. We also acknowledge Dr. Xiao-shu He, NIDDK, for the synthesis of SH322, SH344, and SH384. bicarbonate secretion,and modulation of muscarinicand N-me- Correspondence should be addressed to Wayne D. Bowen, Ph.D., Chief, Unit thyl-D-aspartate (NMDA) receptor function (Walker et al., 1990, on Receptor Biochemistry and Pharmacology, Laboratory of Medicinal Chem- 1993b; Bowen, 1993; Debonnel, 1993). istry, NIDDK, Building 8, Room BI-23, 9000 Rockville Pike, Bethesda, MD 20892. Much of the initial interest in sigma siteshas stemmedfrom Copyright 0 1995 Society for Neuroscience 0270-6474/95/150117-18$05.00/O their high affinity for most of the typical antipsychotic drugs. 118 Vilner et al. Sigma Receptor-mediated Morphologic and Cytotoxic Effects Among these are haloperidol, fluphenazine, and perphenazine. 44 mM sodium bicarbonate and 10% fetal bovine serum (FBS) (Ad- In view of this, sigma sites have been implicated in the etiology vanced Biotechnologies, Inc., Columbia, MD) in 24-well plastic plates (Costar, Cambridge, MA) in a humidified atmosphere of 5% CO,/95% of psychosis and have been viewed as an alternative target to air (normal conditions, pH 7.2-7.4). For all drug additions, FBS was dopamine D, receptors for antipsychotic drug design. Because reduced from 10% to 1%. Where specified, the pH of culture medium sigma receptors occur in very high density in motor regions of was altered from normal during treatment of cells with drugs. For al- the brain, such as brainstem motor nuclei, substantia nigra, and kaline conditions (pH 8.3-8.5), cells were cultured in the above medium cerebellum, sigma sites have also been proposed to contribute in humidified air only. This was accomplished by placing plates inside of a sealed desiccator that contained normal room air and water (for to the untoward motor side effects resulting from acute and humidity), and maintaining the desiccator at 37°C inside ofan incubator. chronic neuroleptic treatment (Walker et al., 1988). Consistent For acidic conditions (pH 6.5-6.7) cells were cultured in humidified 5% with this, microinjection of haloperidol, 1,3-di-o-tolylguanidine CO,/95%air in eitherDulbecco’s phosphate-buffered saline (Life Tech- (DTG), (+)-pentazocine, and other sigma ligands into the rat nologies, Inc.-GIBCO BRL, Grand Island, NY) supplemented with l- 2% FBS or L-l 5 medium (GIBCO-Bethesda Research Labs, Grand red nucleus causes acute dystonic reactions, reminiscent of the Island, NY) supplemented with 1% FBS, both without sodium bicar- acute reactions observed in patients receiving neuroleptic treat- bonate. Cells were preplated 1-2 d before the start of the experiment, ment (Walker et al., 1988; Matsumoto et al., 1990). In the course at a density of 5-6 x lo3 or 1 x IO4 cells/cm’ to avoid contact inhibition of investigating the behavioral effects of haloperidol and its of dividing cells during the 3 d observation period. metabolites by microinjection into the red nucleus of the rat, SK-N-SH human neuroblastoma (ATCC, Rockville, MD), SH-SYSY subclone of SK-N-SH (from Dr. Tommaso Costa, NICHD/NIH, Be- we discovered that reduced haloperidol, a major metabolite and thesda, MD), NCB-20 mouse neuroblastoma x Chinese hamster brain potent sigma ligand, was neurotoxic (Bowen et al., 1990). The hybrid (from Dr. Richard Miller, University of Chicago, Chicago, IL), compound caused extensive gliosis and loss of magnocellular NG 1 OS- I5 mouse neuroblastoma x rat glioma hybrid (from Dr. Rich- neurons in and around the area of microinjection. These patho- ard Weber, NIDDK, Bethesda, MD), COS-7 African green monkey SV- transformed kidney (ATCC, Rockville, MD), and MRS-5 human lung, logic changes were accompanied by long-lasting (> 3 d) postural diploid, x-irradiated (ATCC, Rockville, MD), were cultured as de- abnormalities. Similar results were observed with BD614, a scribed above for C6 glioma cells. PC1 2 pheochromocytoma were cul- novel and highly selective sigma ligand (Bowen et al., 1992). It tured in RPM1 I640 (Siama- Chemical Co.. St. Louis. MO1, SuDDlemented. was difficult, initially, to link these effects with action at sigma with 23 mM sodium bicarbonate, 10% horse serum (Advanced Bio- receptors since other sigma ligands tested had produced only technologies, Inc.), and 5% fetal