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T Bisogno, L De Petrocellis, V Di Marzo; Studies on the Interactions of Prostamides With Fatty Acid Amide Hydrolase, the Anandamide Transporter, and VR1 Receptors . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1976.
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Purpose: The prostamides are COX-2 products of anandamide. These studies were performed to determine if prostamides exhibited similar properties to their precursor anandamide with respect to inactivation mechanisms. Anandamide is transported into cells by facilitated diffusion. This is important since the anandamide recognition site on the VR1 receptor has been reported as intracellular (De Petrocellis, L. et al., J. Biol. Chem. 276, 12856, 2001). Following transport into cells, anandamide may also be rapidly hydrolyzed to arachidonic acid and ethanolamine. Thus, the affinity of prostamides for the putative anandamide transporter and Fatty Acid Amide Hydrolase was determined. Finally, prostamide activity at the VR1 receptor was evaluated since anandamide is a VR1 receptor stimulant. Methods: Affinity of prostamides D2, E2 and F2α for the anandamide transporter and Fatty Acid Amide Hydrolase was determined by competition studies vs. [14-C] anandamide. VR1 receptor stimulation was determined by measuring [Ca2+]i in cells over-expressing the recombinant VR1 receptor. Results: The prostamides did not inhibit uptake of anandamide by RBL-2H3 cells. Prostamides D2, E2 and F2α did not attenuate anandamide hydrolysis by rat brain homogenates, at doses up to and including 10-4M. At high concentrations, prostamides stimulated the VR1 receptor according to the following potency rank order prostamide F2α ≷ prostamide D2 ≷ prostamide E2. The effect of prostamide F2α was blocked by 10-5M capsacian treatment. Prostamide potency was not increased by using pluronic to increase cell permeability. Conclusion: The prostamides are not substrates for the anandamide transporter or Fatty Acid Amide Hydrolase. They exhibit no meaningful effects on the VR1 receptor and an EC50 of 10-5M for prostamide F2α indicates that this receptor is not its primary receptor target.
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