Comparison of effects of anandamide at recombinant and endogenous rat vanilloid receptors.
BACKGROUND
Anandamide, an endogenous lipid, activates both cannabinoid (CB(1)) and vanilloid (VR1) receptors, both of which are co-expressed in rat dorsal root ganglion (DRG) cells. Activation of either receptor results in analgesia but the relative contribution of CB(1) and VR1 in anandamide-induced analgesia remains controversial. Here we compare the in vitro pharmacology of recombinant and endogenous VR1 receptors using calcium imaging, in clonal and DRG cells, respectively. We also consider the contribution of CB(1) and VR1 receptors to anandamide-induced analgesia.


METHODS
Using a Flurometric Imaging Plate Reader (FLIPR), calcium imaging has been used to study the effects of several vanilloid and cannabinoid ligands in rat VR1-transfected HEK293 (rVR1-HEK) cells and in DRG cells. The effect of pre-exposure of several vanilloid and cannabinoids has also been compared in DRG cells.


RESULTS
The VR1 agonists capsaicin, olvanil, (N-(4-hydroxyphenyl-arachinoylamide) (AM404) and anandamide caused a concentration-dependent increase in intracellular calcium concentration ([Ca(2+)](i)), with similar temporal profiles in both rVR1-HEK and DRG cells, and potency (pEC(50)) values of 8.25 (SEM 0.11), 8.37 (0.04), 6.96 (0.06), 5.85 (0.01) and 7.45 (0.10), 7.55 (0.07), 6.10 (0.13), approximately 5.5, respectively. These responses were inhibited by the VR1 antagonist capsazepine (1 micro M). In contrast, application of synthetic cannabinoid antagonists failed to inhibit the anandamide-induced increase in [Ca(2+)](i). Reapplication of VR1 agonists significantly inhibited a subsequent challenge to either capsaicin or anandamide in either cell type, whilst pre-exposure to cannabinoid agonists were without effect.


CONCLUSIONS
Here we provide evidence that the pharmacology of recombinant rVR1 receptors is similar to those endogenously expressed in DRG cells. Moreover, we have shown that VR1, but not CB(1), receptors are involved in anandamide-induced responses in dorsal root primary neurones in vitro. Therefore, the analgesic properties of anandamide are likely to be mediated, at least in part, by VR1 activation in DRG cells in vivo.
Jerman
J C
JC
Department of Discovery Research and Department of Neurology, GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK. jeff_jerman@gsk.com
Gray
J
J
Brough
S J
SJ
Ooi
L
L
Owen
D
D
Davis
J B
JB
Smart
D
D
D000818:Animals; D001095:Arachidonic Acids / Q000494:pharmacology*; D002118:Calcium / Q000032:analysis; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D002211:Capsaicin / Q000031:analogs & derivatives* / Q000494:pharmacology; D002478:Cells, Cultured / Q000187:drug effects; D002999:Clone Cells; D063388:Endocannabinoids; D005727:Ganglia, Spinal / Q000166:cytology / Q000187:drug effects*; D053284:Polyunsaturated Alkamides; D051381:Rats; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug / Q000819:agonists / Q000037:antagonists & inhibitors / Q000187:drug effects*
D001095:Arachidonic Acids; D002121:Calcium Channel Blockers; D063388:Endocannabinoids; D053284:Polyunsaturated Alkamides; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug; C054256:olvanil; D002211:Capsaicin; D002118:Calcium; C078814:anandamide; C107594:N-(4-hydroxyphenyl)arachidonylamide