The role of sulfhydryl groups in d-aspartate and rubidium release from neonatal rat primary astrocyte cultures

We have recently demonstrated that both methylmercury (MeHg) and mercuric chloride (MC) induce d-aspartate release from neonatal rat primary astrocyte cultures maintained in isotonic conditions [1,3,21,22]. In the present study, we compare several other sulfhydryl-(-SH) selective alkylating reagents [methyl methanethiosulfonate (MMTS), N-ehtylmaleimide (NEM), and iodoacetamide (IA)] in isotonic, as well as hypotonic conditions to discern the functional importance of -SH groups in [³H]d-aspartate and ⁸⁶rubidium (⁸⁶Rb) release from astrocytes. Treatment of astrocytes (5 min) in isotonic buffer with the hydrophobic reagent NEM (10 μM) caused a marked increase in ⁸⁶Rb release but had no effect of [³H]d-aspartate release. Neither IA-, nor MMTS-treatment (both at 10 μM) induced increase in [³H]d-aspartate of ⁸⁶Rb release in isotonic buffer. In hypotonic condition (-50 mM Na⁺), astrocytes were most sensitive to MC exposure (5 μM), exhibiting an increase in both [³H]d-aspartate and ⁸⁶Rb efflux. The hydrophobic compounds MMTS and NEM, and the hydrophilic -SH modifying reagent, IA, attenuated the hypotonic-induced efflux of [³H]d-aspartate, in the absence of an effect of ⁸⁶Rb release. These observations are consistent with a critical role for -SH groups both in basal (i.e. isotonic) and hypotonic-induced release of d-aspartate and Rb from astrocytes. Lack of uniformity of these effects may be attributed to site-specificity, related to the physicochemical properties of these -SH alkylating reagents.