title: Investigation of the inhibitory effects of the benzodiazepine derivative, 5-BDBD on P2X4 purinergic receptors by two complementary methods.
abstract: OBJECTIVE
  ATP-gated P2X4 purinergic receptors (P2X4Rs) are cation channels with important roles in diverse cell types. To date, lack of specific inhibitors has hampered investigations on P2X4Rs. Recently, the benzodiazepine derivative, 5-BDBD has been proposed to selectively inhibit P2X4Rs. However, limited evidences are currently available on its inhibitory properties. Thus, we aimed to characterize the inhibitory effects of 5-BDBD on recombinant human P2X4Rs.
  
  
  METHODS
  We investigated ATP-induced intracellular Ca(2+) signals and whole cell ion currents in HEK 293 cells that were either transiently or stably transfected with hP2X4Rs.
  
  
  RESULTS
  Our data show that ATP (< 1 μM) stimulates P2X4R-mediated Ca(2+) influx while endogenously expressed P2Y receptors are not activated to any significant extent. Both 5-BDBD and TNP-ATP inhibit ATP-induced Ca(2+) signals and inward ion currents in a concentration-dependent manner. Application of two different concentrations of 5-BDBD causes a rightward shift in ATP dose-response curve. Since the magnitude of maximal stimulation does not change, these data suggest that 5-BDBD may competitively inhibit the P2X4Rs.
  
  
  CONCLUSIONS
  Our results demonstrate that application of submicromolar ATP concentrations allows reliable assessment of recombinant P2XR functions in HEK 293 cells. Furthermore, 5-BDBD and TNP-ATP have similar inhibitory potencies on the P2X4Rs although their mechanisms of actions are different.
authors.affiliation: Institute of Human Physiology and Clinical Experimental Research, Semmelweis University, Budapest, Hungary.
authors.forename: Bernadett
authors.initials: B
authors.lastname: Balázs
authors.forename: Tamás
authors.initials: T
authors.lastname: Dankó
authors.forename: Gergely
authors.initials: G
authors.lastname: Kovács
authors.forename: László
authors.initials: L
authors.lastname: Köles
authors.forename: Matthias A
authors.initials: MA
authors.lastname: Hediger
authors.forename: Akos
authors.initials: A
authors.lastname: Zsembery
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000494:Allosteric Regulation; D001569:Benzodiazepines / Q000737:chemistry / Q000494:pharmacology*; D001570:Benzodiazepinones / Q000737:chemistry / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D057809:HEK293 Cells; D006801:Humans; D018408:Patch-Clamp Techniques; D058920:Purinergic P2X Receptor Antagonists / Q000737:chemistry / Q000494:pharmacology*; D058848:Receptors, Purinergic P2X4 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D014162:Transfection
chemical_list: C587048:5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro(3,2-e)-1,4-diazepin-2-one; D001570:Benzodiazepinones; D058920:Purinergic P2X Receptor Antagonists; D058848:Receptors, Purinergic P2X4; D011994:Recombinant Proteins; D001569:Benzodiazepines; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D002118:Calcium