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UNIVERSITY OF BUCHAREST
FACULTY OF PHYSICS Guest
2024-11-22 1:42 |
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Conference: Bucharest University Faculty of Physics 2010 Meeting
Section: Biophysics and Medical Physics;Electricity and Magnetism
Title:
Multiple effects of omega-3 polyunsaturated fatty acids on vanilloid receptors (rTRPV1wt) expressed in HEK293 cells
Authors:
T. Selescu, B. Amuzescu, S. Dumitru, G. Cocina, V. Ristoiu, M.L. Flonta
Affiliation:
Dept. Biophysics & Physiology, Faculty of Biology, University of Bucharest
E-mail
bogdan@biologie.kappa.ro
Keywords:
omega-3 PUFA, TRPV1, HEK293, patch-clamp, Ca imaging
Abstract:
Omega-3 polyunsaturated fatty acids (PUFA) have been known for long as essential nutrients, which cannot be synthesized or converted one to the other by human metabolic pathways. Their concentration is extremely high in the brain, and their presence in maternal milk during infancy is vital for the normal development of the central nervous system and cognitive functions. Recently, they have been also attributed protective cardiovascular roles. Foods rich in omega-3 PUFA are rapeseed (canola) and walnut oils, oily fish, and certain eggs. Their structural similarity to anandamide (AEA), an arachidonic acid precursor and main transmitter of the endocannabinoid and endovanilloid signaling systems, the receptors of which are cannabinoid receptors CB1 and CB2, and the vanilloid receptor TRPV1 (transient receptor potential-vanilloid 1), respectively, emerged in the hypotehsis of the present study, namely to assess whether structural resemblance is translated into similar activatory effects. The interaction of AEA with TRPV1 occurs at a cytosolic binding site (De Petrocellis et al. 2001, Jordt & Julius, 2002), triggering a variety of intracellular responses leading ultimately to apoptosis.
In a series of preliminary experiments of whole-cell patch clamp and simultaneous intracellular Ca2+ imaging with deesterified Ca Green-1 applied via the pipette on HEK293 cells transfected with rTRPV1wt and GFP inserted in pcDNA3.1 we titrated the effect of capsaicin, obtaining a half-activatory concentration of 0.39 {micro}M and a Hill coefficient of 1.69. We proved that capsaicin-activated current inhibition at high concentrations (10 {micro}M) is mainly due to activation of phosphatase 2B, which can be inhibited by sodium orthovanadate 1 {micro}M in the pipette solution. We also computed a conversion function between relative peak capsaicin currents and intracellular Ca2+ transients triggered by them. Further, we applied eicosapentaenoic acid (EPA) 20 {micro}M, either alone or in the presence of capsaicin 0.03 {micro}M, on cells with high levels of transfection, approached in whole-cell voltage clamp using a double ramp protocol. EPA alone exerted strong TRPV1 activation (up to 211.12% current increase at +80 mV, 73,3+/-104.9 %, n = 6 cells), while in the presence of capsaicin there was either a mild activation (up to 8.4 % at +80 mV) or mild inhibition (up to –10.2 % at +80 mV), presumably due to EPA oxidation metabolites. Both capsaicin and EPA induced a region of negative slope conductance towards the end of the descending ramp.
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