TY - JOUR
T1 - Methylene blue inhibits function of the 5-HT transporter
AU - Oz, Murat
AU - Isaev, Dmytro
AU - Lorke, Dietrich E.
AU - Hasan, Muhammed
AU - Petroianu, Georg
AU - Shippenberg, Toni S.
PY - 2012/5
Y1 - 2012/5
N2 - 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.
AB - 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.
KW - 5-HT transporter
KW - HEK-293 cells
KW - methylene blue
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U2 - 10.1111/j.1476-5381.2011.01462.x
DO - 10.1111/j.1476-5381.2011.01462.x
M3 - Article
C2 - 21542830
AN - SCOPUS:84859715722
SN - 0007-1188
VL - 166
SP - 168
EP - 176
JO - British Journal of Pharmacology
JF - British Journal of Pharmacology
IS - 1
ER -