Introduction Angiotensin II Ang II and Endothelin ET
Introduction Angiotensin II (Ang II) and Endothelin 1 (ET-1) are potent vasoconstrictive peptides recognized as key players in many cardiovascular diseases . Cardiac hypertrophy, ischemic arrhythmia, and stroke have been associated to an overstimulation of the angiotensin II type 1 (AT1) receptor . Other diseases like atherosclerosis, and pulmonary hypertension have been associated to an increase in ET-1 synthesis and the consequent overactivation of endothelin 1 type A (ETA) receptors . Additionally, activation of both receptors lead to hypertension, chronic heart failure and renal diseases . Both receptors belong to the large family of G protein-coupled receptors (GPCRs) [4,5], which often appeared as monomers, dimers and higher order aggregates [6,7]. There is evidence that these receptor-complexes can modulate the functionality and the pharmacology of the receptors [8,9] by sequestering the receptors, and changing the activation and binding properties of the G-protein [10,11]. Moreover, these processes of oligomerization can influence properties of the receptor such as ligand-binding, receptor activation, receptor desensitization and membrane trafficking . For example, heterodimerization of ϒ-aminobutyric paf receptor B receptor (GABAB) requires the formation of dimers between GABAB1 and GABAB2 to be effectively trafficked to the cell surface , where ligand-binding occurs . Another example, is the heterodimerization of β1/β2-adrenergic receptors which inhibits the activation and internalization of β2-adrenergic receptor after agonist-binding . Available biophysical techniques, currently used to study receptor oligomerization in intact cells, [6,8,, , ] have two major limitations: background fluorescence and photobleaching [19,20]. These two limitations are drastically reduced by Total Internal Reflection Fluorescence Microscopy (TIRF), which exploits the behavior of light at the interface between two media of differing refractive index, a component of the light energy, called the evanescent wave, does penetrate into the second medium at a perpendicular angle and, importantly, decays away exponentially with distance . At a glass/water interface typical of living cells in solution, and using a fluorescent microscope objective with numerical aperture (N.A.) of typically 1.45, the evanescent wave, and thus the excitation of the cell, only penetrates to a depth of ~100 nm. Thus, if one is performing experiments under TIRF illumination, only fluorophores located within or close to the plasma membrane are selectively detected within the evanescent field [20,21]. TIRF microscopy has been successfully used to detect and measure the aggregation state of GPCRs like the GABAB and the muscarinic M1 receptors [22,23]. In this work, TIRF coupled to the number and brightness (N&B) method of analysis were used, this method uses the fast statistical fluctuations of the intensity at each pixel allowing the determination of the number of diffusing particles within the focal point and the intrinsic brightness of each particle without the need of single-molecule imaging [, , ]. The advantage of the N&B method proposed in this study is that a signal of a fluorophore may be detected by its brightness and for a prolonged measuring time due to the absence of photoblechiung in our measurements. 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 . 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.