Cytotoxic effects of sigma ligands: Sigma receptor-mediated alterations in cellular morphology and viability

Abstract

The morphological effects of several neuroleptics as well as other novel and prototypic sigma ligands were examined by addition to cultures of C6 glioma cells. Sigma ligands caused loss of processes, assumption of spherical shape, and cessation of cell division. The time course and magnitude of this effect were dependent on the concentration of sigma ligand. Continued exposure to sigma compounds ultimately resulted in cell death. However, the morphological effect was reversible when sigma ligand was removed shortly after rounding. The potency of compounds to produce these effects generally correlated with binding affinity at sigma receptors of C6 glioma cell membranes labeled with [3H](+)-pentazocine. At a concentration of 100 μM, haloperidol, reduced haloperidol, fluphenazine, perphenazine, trifluoperazine, BD737, LR172, BD1008, and SH344 produced significant effects in 3-6 hr of exposure. Other compounds, such as trifluperidol, thioridazine, and (-)-butaclamol, produced significant effects by 24 hr of exposure. Despite the requirement of micromolar concentrations of ligand (some compounds were effective at 30 μM), the effect showed a remarkable specificity for compounds exhibiting sigma receptor binding affinity. Neuroleptics lacking potent sigma affinity [e.g., (-)-sulpiride, (+)- butaclamol, and clozapine] and other compounds that lack significant sigma affinity but that are agonists or antagonists at dopamine, serotonin, adrenergic, glutamate, phencyclidine, GABA, opiate, or muscarinic cholinergic receptors were without effect on cellular morphology at concentrations up to 300 μM over a period of 72 hr. Likewise, blockers and activators of Na+, K+, and Ca2+ channels and a monoamine oxidase inhibitor devoid of sigma affinity were without effect. Interestingly, 1,3-di-o-tolyl-guanidine (DTG), (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [(+)-3-PPP], (+)-pentazocine, (+)-cyclazocine, and other sigma-active benzomorphans and morphinans appeared inactive in up to 72 hr of culture. However, these compounds interacted synergistically with a subeffective dose of BD737 (30 μM) to produce effects usually in 6 hr or less. Also, the pH of the culture medium had a profound effect on the activity of sigma compounds. Increasing the pH from the normal range of 7.2-7.4 to pH 8.3-8.5 shifted the dose curves (30, 100, 300 μM) for all sigma compounds to the left. Under these conditions, DTG, (+)-3-PPP, and benzomorphans produced effects in 24 hr or less. Decreasing the medium pH to 6.5-6.7 markedly reduced the activity of all sigma ligands, producing significant protection from cytotoxic effects. Importantly, compounds that lacked sigma binding affinity showed neither synergism with 30 μM BD737 nor an increase in activity at higher pH. These results confirm the sigma receptor specificity of this effect. Sigma ligands had similar effects on other cells of neuronal and non-neuronal origin, including SK-N-SH and SH- SY5Y neuroblastomas, NCB-20 hybridoma, NG108-15 neuroblastoma-glioma hybrid, COS-7 (kidney), MRS-5 (lung), and PC12 pheochromocytoma. These results suggest that sigma receptors play some vital role in cell function and may have important implications for neurodegenerative disorders and neuroleptic treatment.