Altered pharmacology of native rodent spinal cord TRPV1 after phosphorylation.
OBJECTIVE
Evidence suggests that phosphorylation of TRPV1 is an important component underlying its aberrant activation in pathological pain states. To date, the detailed pharmacology of diverse TRPV1 receptor agonists and antagonists has yet to be reported for native TRPV1 under phosphorylating conditions. Our goal was to optimize a relatively high-throughput methodology to allow pharmacological characterization of the native TRPV1 receptor using a spinal cord neuropeptide release assay under naive and phosphorylating states.


METHODS
Herein, we describe characterization of rodent TRPV1 by measurement of CGRP release from acutely isolated lumbar (L1-L6) spinal cord using a 96-well technique that combines use of native, adult tissue with quantitation of CGRP release by ELISA.


RESULTS
We have studied a diverse panel of TRPV1 agonists and antagonists under basal and phosphorylating conditions. We show that TRPV1-mediated CGRP release is evoked, in a temperature-dependent manner, by a PKC activator, phorbol 12,13-dibutyrate (PDBu); and that treatment with PDBu increases the potency and efficacy of known TRPV1 chemical agonists, in an agonist-specific manner. We also show that the pharmacological profile of diverse TRPV1 antagonists is dependent on whether the stimulus is PDBu or capsaicin. Of note, HPPB was identified as an antagonist of capsaicin-evoked, but a potentiator of PDBu-evoked, CGRP release.


CONCLUSIONS
Our findings indicate that both TRPV1 agonist and antagonist profiles can be differentially altered by PKC activation. These findings may offer new insights for targeting TRPV1 in pain states.
Neuroscience Research Division, Lilly Research Centre, Eli Lilly & Co. Ltd, Windlesham, Surrey, UK. mogg_adrian@lilly.com
A J
AJ
Mogg
C E J
CE
Mill
E A
EA
Folly
R E
RE
Beattie
M J
MJ
Blanco
J P
JP
Beck
L M
LM
Broad
D000818:Animals; D015740:Calcitonin Gene-Related Peptide / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002211:Capsaicin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011719:Pyrazines / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013116:Spinal Cord / Q000187:drug effects* / Q000378:metabolism; D050916:TRPV Cation Channels / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics
C475772:N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide; D011719:Pyrazines; D011725:Pyridines; D050916:TRPV Cation Channels; C493921:TRPV1 protein, mouse; C493922:Trpv1 protein, rat; D011493:Protein Kinase C; D015740:Calcitonin Gene-Related Peptide; D002211:Capsaicin; D002118:Calcium