In order to verity if the

In order to verity if the N&B method is able to detect any variation in AT1EYFP and ETAEGFP receptors aggregation propensity due to exogenous treatments in living cells, they were treated by the endogenous peptides Ang II and ET-1, as well as the selective receptors antagonist losartan, BQ123 and sitaxentan. It is known for AT1 receptors through Bioluminescence Resonance Energy Transfer (BRET) that homo or oligomercs complexes in living cells are unaffected by the action of both agonist and antagonist [27]. Furthermore, for the ETA receptors using Fluorescence Resonance Eneregy Transfer (FRET), the binding of the agonist ET-1 reduce the FRET efficiency of the dimeres.
Materials and methods
Results
Discussion In this study, the N&B analysis showed new evidence regarding the proportion and the oligomerization state of the AT1 and the ETA receptors near or on the plasma membrane. Althought, preliminary studies suggest that the AT1 and the ETA receptors occurred only as monomers and dimers [39,40], this study showed that for both receptors the majority of the particles appeared as monomers, a small amount as dimers and a much smaller proportion as tetramers. Preliminary studies suggest that dimerization of the AT1 receptor occurred during the biosynthesis in the endoplasmic reticulum before surface expression [27,40], which may explain the presenece of these all the way before the addition of any ligand. Our results showed that the presence of Ang II did not change the proportion of AT1 receptor monomers, dimers, and tetramers, which is in agreement with other studies using FRET, BRET and co-immunoprecipitation techniques [27,41]. Regarding ETA receptors, preliminary studies using FRET showed that both ET-1 and BQ123 reduced ETA receptor dimers [40,42], while the present study showed that BQ123 reduced receptor-dimers and ET-1 has no influence over receptor populations. Little is still known about the effect of antagonists on the population of monomeric, dimeric and higher order aggregates of these two receptors. For instance, the AT1 receptor antagonist telmisartan did not modify the population of AT1 dimers [27], while the ETA receptor antagonist BQ123 reduced the proportion of ETA dimers [42]. Our results showed that the effect of BQ123 is similar to that found in literature, however the effect shown by the AT1 receptor antagonist losartan is different to that of telmisartan. Additionally, the ETA receptor antagonist sitaxentan had no effect on any of the ETA receptor populations. The question arises whether losartan and BQ123 have a different pharmacological effect which is responsible for the differences seen at the level of receptor organization. In this line, there is evidence that suggests that both, losartan and BQ123 act as inverse agonists [43]. Regarding to the variations in the monomer/dimer/tetramer rate among cells of CHO-ETAEGFP receptors before adding the ligands, we hypothesize the used of transient transfection increase the variability further experiments need to be done to clarify this observations.