Exploring the role of the membrane bilayer in the recognition of candesartan by its GPCR AT1 receptor.

Cardiovascular diseases and hypertension in particular are major health risks worldwide and the improvement on their treatment will be beneficial for the human health. AT1R antagonists belong to the sartans family that targets the renin-angiotensin aldosterone system (RAAS) through blocking the hormone angiotensin II to exert its detrimental effects in pathological states. As a consequence, they are beneficial to treat hypertension, diabetes related kidney failure and hyperaemic episodes. Long unbiased Molecular Dynamics (MD) simulations are performed in order to explore candesartan's possible 2D and 3D diffusion mechanisms towards AT1R receptor. 3D diffusion mechanism is referred to the direct binding of the AT1 antagonist candesartan to the AT1R 3D structure (PDB ID: 4YAY). 2D diffusion mechanism involves first, the incorporation of candesartan in the bilayer core and then its localization on the AT1R binding cavity, through a diffusion mechanism. The obtained results indicate that membranes interact significantly with the neutral form of candesartan, which is indeed approaching the receptors' active site through diffusion via the lipids. On the other hand, the deprotonated form of the drug is interacting with AT1R's extracellular loop and fails to enter the membrane, pointing out the importance of the pH microenvironment around the receptor. To validate the calculated diffusion coefficients of the drug in the lipid bilayers 2D DOSY NMR experiments were recorded and they were in good agreement. Information on the impact that has the interaction of candesartan with the membrane is very important for the rationally design and development of potent ARBs. Thus, its conformational features as well as its localization in the membrane core have to be thoroughly explored.

Characterization of GPCRs in extracellular vesicle (EV).

Extracellular vesicle (EV) are tiny membranous vesicles usually <500nm in size that recently emerged as a new paradigm in human intercellular signaling. EVs have shown a promising role in development of diagnostic markers in many pathophysiological disorders. The presence of chemosensory and therapeutically relevant G protein-coupled receptors (GPCRs) on EV membranes is poorly characterized. Here, we compare different methods including ultracentrifugation and polymer-charge-based separation to isolate EVs from cell culture media and human saliva. The presence of bitter taste GPCRs (T2R4 and T2R38) and a class A GPCR angiotensin II type 1 receptor on these EVs was characterized by qPCR, ELISA, and immunotransmission electron microscopy.

Estimation of the number of angiotensin II AT1 receptors in rat kidney afferent and efferent arterioles.

OBJECTIVE: To examine the effects of the renin-angiotensin system (RAS) on renal arterioles to determine the association between the distribution of angiotensin II AT1 receptors and the morphologic and physiologic heterogeneity of renal arterioles. STUDY DESIGN: To estimate the number of angiotensin II AT1 receptors along the length of the arterioles and per arteriole, we combined immunoelectron microscopy with stereology. RESULTS: The number of AT1 receptor molecules was significantly lower in the renin-positive smooth muscle cells (SMCs) than in the renin-negative SMCs of the afferent and efferent arterioles. There were no significant differences along and between the afferent and efferent arterioles in relative number of AT1 receptors of endothelial cells or SMCs. CONCLUSION: Our results suggest that the heterogeneous activity of angiotensin II in SMCs and the different permeabilities of the endothelium along the afferent arterioles are probably not controlled directly by angiotensin II AT1 receptors. However, the activity of the RAS is possibly involved in the significantly reduced number of receptors in renin-granulated cells. An understanding of how the number of AT1 receptors on the SMC surface is controlled may furnish a new path for pharmacologically changing RAS activity on SMCs.

Detection of angiotensin II mediated nitric oxide release within the nucleus of the solitary tract using electron-paramagnetic resonance (EPR) spectroscopy.

We previously identified an action of nitric oxide (NO) within the nucleus tractus solitarii (NTS) that attenuates the cardiac component of the baroreceptor reflex. In the present study we have tested the hypothesis that angiotensin II (AngII), acting on angiotensin type 1 receptors (AT1R), can release NO within the NTS and that its actions are mediated by soluble guanylate cyclase (sGC). Utilising cryogenic electron paramagnetic resonance (EPR), we have detected NO release in brainstem samples following AngII, but not saline, microinjections into the NTS. In these experiments, we confirmed that both AngII and a NO donor (diethylamine NONOate) in the NTS both depressed the baroreflex bradycardia. In additional studies, we showed that the latter effects were both sensitive to blockade of sGC using 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ). To initiate studies to resolve the cellular source of NO released by angiotensin II in the NTS, we performed immunohistochemical/electron microscopy studies on the distribution of AT1R. We found AT1R located on NTS neurones and blood vessels. Since a rise in intracellular calcium [Ca]i levels is prerequisite for nNOS activation, we imaged responses in [Ca]i in NTS neurones during exposure to AngII in vitro using confocal microscopy. Our data indicate a paucity of neurones showing changes in [Ca]i when exposed to AngII (200 nM). We suggest that AngII-induced release of NO is from non-neuronal sites. With the presence of AT1R on blood vessel endothelial cells we propose that AngII released NO in the NTS is due to activation of endothelial nitric oxide synthase located within the endothelium. The present study supports the novel concept that AngII can trigger NO release in the NTS by a mechanism of vascular-neuronal signalling that affects central neuronal networks regulating cardiovascular function.

Investigation of angiotensin II type 1 receptor by atomic force microscopy with functionalized tip.

Although membrane proteins consist of a substantial amount of the human genome and are the main drug targets, the study of cell membrane proteins in situ is complicated by the technical limitations. The recent development of atomic force microscopy (AFM) opens a new way to study the functions of cell membrane proteins in situ at the single-molecule level. A detailed procedure for investigation of angiotensin II type 1 receptor by AFM with functionalized tip is introduced in this article. Some prospective methods to improve the imaging resolution are also discussed.