Methylene blue inhibits function of the 5-HT transporter

Background and Purpose Methylene blue (MB) is commonly employed as a treatment for methaemoglobinaemia, malaria and vasoplegic shock. An increasing number of studies indicate that MB can cause 5-HT toxicity when administered with a 5-HT reuptake inhibitor. MB is a potent inhibitor of monoamine oxidases, but other targets that may contribute to MB toxicity have not been identified. Given the role of the 5-HT transporter (SERT) in the regulation of extracellular 5-HT concentrations, the present study aimed to characterize the effect of MB on SERT. Experimental Approach Live cell imaging, in conjunction with the fluorescent SERT substrate 4-(4-(dimethylamino)-styryl)-N-methylpyridinium (ASP ⁺), [ ³H]5-HT uptake and whole-cell patch-clamp techniques were employed to examine the effects of MB on SERT function. Key Results In EM4 cells expressing GFP-tagged human SERT (hSERT), MB concentration-dependently inhibited ASP ⁺ accumulation (IC ₅₀: 1.4 ± 0.3 μM). A similar effect was observed in N2A cells. Uptake of [ ³H]5-HT was decreased by MB pretreatment. Furthermore, patch-clamp studies in hSERT expressing cells indicated that MB significantly inhibited 5-HT-evoked ion currents. Pretreatment with 8-Br-cGMP did not alter the inhibitory effect of MB on hSERT activity, and intracellular Ca ²⁺ levels remained unchanged during MB application. Further experiments revealed that ASP ⁺ binding to cell surface hSERT was reduced after MB treatment. In whole-cell radioligand experiments, exposure to MB (10 μM; 10 min) did not alter surface binding of the SERT ligand [ ¹²⁵I]RTI-55. Conclusions and Implications MB modulated SERT function and suggested that SERT may be an additional target upon which MB acts to produce 5-HT toxicity.