Arachidonyl dopamine as a ligand for the vanilloid receptor VR1 of the rat

The vanilloid receptor VR1 is a nonspecific Ca2+ channel, expressed in sensory neurons in the peripheral nervous system and in various brain regions, which is believed to be an important molecular integrator of several chemical (acid, vanilloids) and physical stimuli (heat) that cause pain. Recently, several endogenous ligands for VR1 have been identified such as arachidonyl ethanolamide (anandamide) and the more potent arachidonyl dopamine (AA-DO). Here, we further characterize AA-DO As a ligand for rat VR1, heterologously expressed in CHO and HEK293 cells. AA-DO inhibited the binding of [H-3]RTX to VR1 with a K-d value of 5.49 +/- 0.68 muM and with positive cooperativity (p = 1.89 +/- 0.27), indicating that AA-DO was about 5-fold more potent than anandamide in this system. The K-d (9.7 +/- 3.3 muM), and p values (1.54 +/- 0.04) for the binding of AA-DO to spinal cord membranes are in good correlation with the CHO-VR1 data. AA-DO stimulated Ca-45(2+) uptake on CHO-VR1 and HEK-VR1 cells with EC50 values of 4.76 +/- 1.43 and 7.17 +/- 1.64 muM and Hill coefficients of 1.28 +/- 0.11 and 1.13 +/- 0.13, respectively, consistent with the binding measurements. In contrast to anandamide, AA-DO induced virtually the same level of Ca-45(2+) uptake as did capsaicin (90 +/- 6.6% in the CHO cells expressing VR1 and 89.3 +/- 9.4% in HEK293 cells expressing VR1). In a time dependent fashion following activation, AA-DO partially desensitized VR1 both in 45Ca2+ uptake assays (IC50 = 3.24 +/- 0.84 muM, inhibition is 68.5 +/- 6.85%) as well as in Ca2+ imaging experiments (35.8 +/- 5.1% inhibition) using the CHO-VR1 system. The extent of desensitization was similar to that caused by capsaicin itself We conclude that AA-DO is a full agonist for VR1 with a potency in the low micromolar range and is able to significantly desensitize the cells in a time and dose dependent manner. Published by Elsevier Science Inc.