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.
Bianchi
Bruce R
BR
Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064, USA. bruce.bianchi@abbott.com
El Kouhen
Rachid
R
Chen
Jun
J
Puttfarcken
Pamela S
PS
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