Nephrogenic Diabetes Insipidus

Body fluid homeostasis is essential for normal life. In the maintenance of water balance, the most important factor and regulated process is the excretory function of the kidneys. The kidneys are capable to compensate not only the daily fluctuations of water intake but also the consequences of fluid loss (respiration, perspiration, sweating, hemorrhage). The final volume and osmolality of the excreted urine is set in the collecting duct via hormonal regulation. The hormone of water conservation is the vasopressin (AVP), and a large volume of urine is produced and excreted in the absence of AVP secretion or if AVP is ineffective in the kidneys. The aquaporin-2 water channel (AQP2) is expressed in the principal cells, and it plays an essential role in the reabsorption of water in the collecting ducts via type 2 vasopressin receptor (V2R)-mediated mechanism. If neural or hormonal regulation fails to operate the normal function of AVP-V2R-AQP2 system, it can result in various diseases such as diabetes insipidus (DI) or nephrogenic syndrome of inappropriate diuresis (NSIAD). The DI is characterized by excessive production of hyposmotic urine ("insipidus" means tasteless) due to the inability of the kidneys to concentrate urine. In this chapter, we focus and discuss the pathophysiology of nephrogenic DI (NDI) and the potential therapeutic interventions in the light of the current experimental data

Independent beta-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2.

Stimulation of a mutant angiotensin type 1A receptor (DRY/AAY) with angiotensin II (Ang II) or of a wild-type receptor with an Ang II analog ([sarcosine1,Ile4,Ile8]Ang II) fails to activate classical heterotrimeric G protein signaling but does lead to recruitment of beta-arrestin 2-GFP and activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) (maximum stimulation approximately 50% of wild type). This G protein-independent activation of mitogen-activated protein kinase is abolished by depletion of cellular beta-arrestin 2 but is unaffected by the PKC inhibitor Ro-31-8425. In parallel, stimulation of the wild-type angiotensin type 1A receptor with Ang II robustly stimulates ERK1/2 activation with approximately 60% of the response blocked by the PKC inhibitor (G protein dependent) and the rest of the response blocked by depletion of cellular beta-arrestin 2 by small interfering RNA (beta-arrestin dependent). These findings imply the existence of independent G protein- and beta-arrestin 2-mediated pathways leading to ERK1/2 activation and the existence of distinct "active" conformations of a seven-membrane-spanning receptor coupled to each

␤-Arrestin-and Dynamin-Dependent Endocytosis of the AT 1 Angiotensin Receptor

ABSTRACT The major mechanism of agonist-induced internalization of G protein-coupled receptors (GPCRs) is ␤-arrestin-and dynamindependent endocytosis via clathrin-coated vesicles. However, recent reports have suggested that some GPCRs, exemplified by the AT 1 angiotensin receptor expressed in human embryonic kidney (HEK) 293 cells, are internalized by a ␤-arrestinand dynamin-independent mechanism, and possibly via a clathrin-independent pathway. In this study, agonist-induced endocytosis of the rat AT 1A receptor expressed in Chinese hamster ovary (CHO) cells was abolished by clathrin depletion during treatment with hyperosmotic sucrose and was unaffected by inhibition of endocytosis via caveolae with filipin. In addition, internalized fluorescein-conjugated angiotensin II appeared in endosomes, as demonstrated by colocalization with transferrin. Overexpression of ␤-arrestin1(V53D) and ␤-arrestin1(1-349) exerted dominant negative inhibitory effects on the endocytosis of radioiodinated angiotensin II in CHO cells. GTPase-deficient (K44A) mutant forms of dynamin-1 and dynamin-2, and a pleckstrin homology domain-mutant (K535A) dynamin-2 with impaired phosphoinositide binding, also inhibited the endocytosis of AT 1 receptors in CHO cells. Similar results were obtained in COS-7 and HEK 293 cells. Confocal microscopy using fluorescein-conjugated angiotensin II showed that overexpression of dynamin-1(K44A) and dynamin-2(K44A) isoforms likewise inhibited agonist-induced AT 1 receptor endocytosis in CHO cells. Studies on the angiotensin II concentration-dependence of AT 1 receptor endocytosis showed that at higher agonist concentrations its rate constant was reduced and the inhibitory effects of dominant negative dynamin constructs were abolished. These data demonstrate the importance of ␤-arrestin-and dynamin-dependent endocytosis of the AT 1 receptor via clathrin-coated vesicles at physiological angiotensin II concentrations

Compton Scattering Cross Section on the Proton at High Momentum Transfer

Cross-section values for Compton scattering on the proton were measured at 25 kinematic settings over the range s = 5-11 and -t = 2-7 GeV2 with statistical accuracy of a few percent. The scaling power for the s-dependence of the cross section at fixed center of mass angle was found to be 8.0 +/ 0.2, strongly inconsistent with the prediction of perturbative QCD. The observed cross-section values are in fair agreement with the calculations using the handbag mechanism, in which the external photons couple to a single quark.Comment: 5 pages, 5 figure

Measurement of inositol 1,4,5-trisphosphate in living cells using an improved set of resonance energy transfer-based biosensors

Improved versions of inositol-1,4,5-trisphosphate (InsP3) sensors were created to follow intracellular InsP3 changes in single living cells and in cell populations. Similar to previous InsP3 sensors the new sensors are based on the ligand binding domain of the human type-I InsP3 receptor (InsP3R-LBD), but contain a mutation of either R265K or R269K to lower their InsP3 binding affinity. Tagging the InsP3R-LBD with N-terminal Cerulean and C-terminal Venus allowed measurement of Ins P3 in single-cell FRET experiments. Replacing Cerulean with a Luciferase enzyme allowed experiments in multi-cell format by measuring the change in the BRET signal upon stimulation. These sensors faithfully followed the agonist-induced increase in InsP3 concentration in HEK 293T cells expressing the Gq-coupled AT1 angiotensin receptor detecting a response to agonist concentration as low as 10 pmol/L. Compared to the wild type InsP3 sensor, the mutant sensors showed an improved off-rate, enabling a more rapid and complete return of the signal to the resting value of InsP3 after termination of M3 muscarinic receptor stimulation by atropine. For parallel measurements of intracellular InsP3 and Ca2+ levels in BRET experiments, the Cameleon D3 Ca2+ sensor was modified by replacing its CFP with luciferase. In these experiments depletion of plasma membrane PtdIns(4,5)P2 resulted in the fall of InsP3 level, followed by the decrease of the Ca2+-signal evoked by the stimulation of the AT1 receptor. In contrast, when type-III PI 4-kinases were inhibited with a high concentration of wortmannin or a more specific inhibitor, A1, the decrease of the Ca2+-signal preceded the fall of InsP3 level indicating an InsP3-, independent, direct regulation of capacitative Ca2+ influx by plasma membrane inositol lipids. Taken together, our results indicate that the improved InsP3 sensor can be used to monitor both the increase and decrease of InsP3 levels in live cells suitable for high-throughput BRET applications. © 2015, Public Library of Science. All rights reserved

Angiotensin receptors in GtoPdb v.2023.1

The actions of angiotensin II (Ang II) are mediated by AT1 and AT2 receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Angiotensin receptors [63, 155]), which have around 30% sequence similarity. The decapeptide angiotensin I, the octapeptide angiotensin II and the heptapeptide angiotensin III are endogenous ligands. losartan, candesartan, olmesartan, telmisartan, etc. are clinically used AT1 receptor blockers

Angiotensin receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

The actions of angiotensin II (Ang II) are mediated by AT1 and AT2 receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Angiotensin receptors [61, 152]), which have around 30% sequence similarity. The decapeptide angiotensin I, the octapeptide angiotensin II and the heptapeptide angiotensin III are endogenous ligands. losartan, candesartan, telmisartan, etc. are clinically used AT1 receptor blockers

Improved methodical approach for quantitative BRET analysis of G protein coupled receptor dimerization

G Protein Coupled Receptors (GPCR) can form dimers or higher ordered oligomers, the process of which can remarkably influence the physiological and pharmacological function of these receptors. Quantitative Bioluminescence Resonance Energy Transfer (qBRET) measurements are the gold standards to prove the direct physical interaction between the protomers of presumed GPCR dimers. For the correct interpretation of these experiments, the expression of the energy donor Renilla luciferase labeled receptor has to be maintained constant, which is hard to achieve in expression systems. To analyze the effects of non-constant donor expression on qBRET curves, we performed Monte Carlo simulations. Our results show that the decrease of donor expression can lead to saturation qBRET curves even if the interaction between donor and acceptor labeled receptors is non-specific leading to false interpretation of the dimerization state. We suggest here a new approach to the analysis of qBRET data, when the BRET ratio is plotted as a function of the acceptor labeled receptor expression at various donor receptor expression levels. With this method, we were able to distinguish between dimerization and non-specific interaction when the results of classical qBRET experiments were ambiguous. The simulation results were confirmed experimentally using rapamycin inducible heterodimerization system. We used this new method to investigate the dimerization of various GPCRs, and our data have confirmed the homodimerization of V2 vasopressin and CaSR calcium sensing receptors, whereas our data argue against the heterodimerization of these receptors with other studied GPCRs, including type I and II angiotensin, β2 adrenergic and CB1 cannabinoid receptors