title: Methylene blue inhibits function of the 5-HT transporter.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  Live cell imaging, in conjunction with the fluorescent SERT substrate 4-(4-(dimethylamino)-styryl)-N-methylpyridinium (ASP(+) ), [(3) H]5-HT uptake and whole-cell patch-clamp techniques were employed to examine the effects of MB on SERT function.
  
  
  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 [(3) H]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 [(125) I]RTI-55.
  
  
  CONCLUSIONS
  MB modulated SERT function and suggested that SERT may be an additional target upon which MB acts to produce 5-HT toxicity.
authors.affiliation: Department of Pharmacology, UAE University, Al Ain, UAE. Murat_Oz@uaeu.ac.ae
authors.forename: Murat
authors.initials: M
authors.lastname: Oz
authors.forename: Dmytro
authors.initials: D
authors.lastname: Isaev
authors.forename: Dietrich E
authors.initials: DE
authors.lastname: Lorke
authors.forename: Muhammed
authors.initials: M
authors.lastname: Hasan
authors.forename: Georg
authors.initials: G
authors.lastname: Petroianu
authors.forename: Toni S
authors.initials: TS
authors.lastname: Shippenberg
mesh_terms: D002118:Calcium / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000008:administration & dosage / Q000633:toxicity*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D008751:Methylene Blue / Q000008:administration & dosage / Q000633:toxicity*; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D011726:Pyridinium Compounds / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins / Q000187:drug effects* / Q000378:metabolism
chemical_list: C032315:4-(4-dimethylaminostyryl)-1-methylpyridinium; D004791:Enzyme Inhibitors; D011726:Pyridinium Compounds; D050486:Serotonin Plasma Membrane Transport Proteins; D049452:Green Fluorescent Proteins; D012701:Serotonin; D002118:Calcium; D008751:Methylene Blue