Binding of [(3)H]A-778317 to native transient receptor potential vanilloid-1 (TRPV1) channels in rat dorsal root ganglia and spinal cord.
A-778317 (1-((R)-5-tert-butyl-indan-1-yl)-3-isoquinolin-5-yl-urea) is a potent antagonist of human and rat transient receptor potential vanilloid-1 (TRPV1) receptors. We have previously reported that [(3)H]A-778317 is an excellent radioligand to study the recombinant human TRPV1 receptor in a heterologous expression system. These studies were extended to determine the feasibility of using [(3)H]A-778317 to label native TRPV1 channels in rat tissues. Saturable high-affinity binding of [(3)H]A-778317 was detected in membrane preparations of rat dorsal root ganglia (DRG) and spinal cord that was inhibited by TRPV1 receptor agonists and antagonists. [(3)H]A-778317 labeled a single class of high-affinity binding sites in both rat DRG and spinal cord membranes (K(D)=10 and 8.4nM, respectively). The number of binding sites was 10-fold higher in rat DRG membranes than spinal cord membranes (B(max)=3.3 and 0.35pmol/mg protein, respectively). The pharmacology of the high-affinity binding sites was similar in rat DRG and spinal cord, but differed from the recombinant rat TRPV1 (rTRPV1) receptor expressed in transiently transfected HEK293-F cells. In particular, a large disparity in potency (>300-fold) was observed for the TRPV1 receptor agonist resiniferatoxin between native and recombinant rTRPV1 receptors. Our data indicate that the binding of [(3)H]A-778317 to native rTRPV1 channels is pharmacologically distinct, and perhaps more complex, than its binding to the recombinant rTRPV1 receptor.
Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064, USA. bruce.bianchi@abbott.com
Bruce R
BR
Bianchi
Rachid
R
El Kouhen
Jun
J
Chen
Pamela S
PS
Puttfarcken
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002120:Calcium Channel Agonists / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D002461:Cell Line, Transformed; D004305:Dose-Response Relationship, Drug; D005727:Ganglia, Spinal / Q000187:drug effects / Q000378:metabolism*; D007546:Isoquinolines / Q000494:pharmacology*; D011869:Radioligand Assay / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D013116:Spinal Cord / Q000187:drug effects / Q000378:metabolism*; D050916:TRPV Cation Channels / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014508:Urea / Q000031:analogs & derivatives* / Q000494:pharmacology
C523980:1-(5-tertbutylindan-1-yl)-3-isoquinolin-5-ylurea; D002120:Calcium Channel Agonists; D002121:Calcium Channel Blockers; D007546:Isoquinolines; D011994:Recombinant Proteins; D050916:TRPV Cation Channels; C493922:Trpv1 protein, rat; D014508:Urea; D002118:Calcium