Beta-arrestin- and dynamin-dependent endocytosis of the AT1 angiotensin receptor

The major mechanism of agonist-induced internalization of G protein-coupled receptors (GPCRs) is beta-arrestin- and dynamin-dependent endocytosis via clathrin-coated vesicles. However, recent reports have suggested that some GPCRs, exemplified by the AT1 angiotensin receptor expressed in human embryonic kidney (HEK) 293 cells, are internalized by a beta-arrestin- and dynamin-independent mechanism, and possibly via a clathrin-independent pathway. In this study, agonist-induced endocytosis of the rat AT1A 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 beta-arrestin1(V53D) and beta-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 AT1 receptor endocytosis in CHO cells. Studies on the angiotensin II concentration-dependence of AT1 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 beta-arrestin- and dynamin-dependent endocytosis of the AT1 receptor via clathrin-coated vesicles at physiological angiotensin II concentrations.     

Characterization of angiotensin II antagonism displayed by Ib, a novel nonpeptide angiotensin AT(1) receptor antagonist

The pharmacologic profile of Ib, 5-n-butyl-4-{4-[2-(1H-tetrazole-5-yl)-1H-pyrrol-1-yl]phenylmethyl}-2,4-dihydro-2-(2,6-dichloridephenyl)-3H-1,2,4-triazol-3-one, a novel nonpeptide angiotensin AT(1) receptor antagonist, was investigated by receptor-binding studies, functional in vitro assays with rabbit and rat aorta, and in vivo experiments in rats. Ib inhibited [(125)I] angiotensin II binding to AT(1) receptors in rat liver membranes (K(i)=2.5+/-0.5 nM) and did not interact with AT(2) receptors in bovine cerebellar membranes. In functional studies with rat and rabbit aorta, Ib inhibited the contractile response to angiotensin II (pD(2)' value: 7.43 and 7.29, respectively) with a significant reduction in the maximum. In pithed rats, Ib inhibited the angiotensin II induced pressor response in a dose-related manner. After intravenous administration, Ib produced a dose-dependent antihypertensive effects in spontaneously hypertensive rats and renal hypertensive rats. These results suggest that Ib is a potent angiotensin AT(1) selective receptor antagonist with a mode of insurmountable antagonism.     

Molecular mechanisms of constitutive activity: mutations at position 111 of the angiotensin AT1 receptor

Abstract: A possible molecular mechanism for the constitutive activity of mutants of the angiotensin type 1 receptor (AT₁) at position 111 was suggested by molecular modeling. This involves a cascade of conformational changes in spatial positions of side chains along transmembrane helix (TM3) from L112 to Y113 to F117, which in turn, results in conformational changes in TM4 (residues I152 and M155) leading to the movement of TM4 as a whole. The mechanism is consistent with the available data of site‐directed mutagenesis, as well as with correct predictions of constitutive activity of mutants L112F and L112C. It was also predicted that the double mutant N111G/L112A might possess basal constitutive activity comparable with that of the N111G mutant, whereas the double mutants N111G/Y113A, N111G/F117A, and N111G/I152A would have lower levels of basal activity. Experimental studies of the above double mutants showed significant constitutive activity of N111G/L112A and N111G/F117A. The basal activity of N111G/I152A was higher than expected, and that of N111G/Y113A was not determined due to poor expression of the mutant. The proposed mechanism of constitutive activity of the AT₁ receptor reveals a novel nonsimplistic view on the general problem of constitutive activity, and clearly demonstrates the inherent complexity of the process of G protein‐coupled receptor (GPCR) activation.     

Tight binding of the angiotensin AT(1) receptor antagonist

Angiotensin II induces angiotensin AT(1) receptor internalization via Clathrin coated pits formation. We investigated whether insurmountable inhibition by the non-peptide antagonist 2-ethoxy-1-[(2'-(1H-tetrazol-5-yl) biphenyl-4-yl) methyl]-1H-benzimidazoline-7-carboxylic acid (candesartan) was related to receptor internalization. Mild acid treatment can discriminate between internalized and cell surface bound [(3)H]angiotensin II. In contrast, it provides no information about the subcellular localization of bound [(3)H]candesartan since this binding is acid resistant. The internalization of [(3)H]angiotensin II is rapidly inhibited in the presence of 0.4 M sucrose. Yet, no such rapid effect was noticed for [(3)H]candesartan. [(3)H]candesartan displays insurmountable/long lasting binding to the vast majority of both wild type and L(314) truncated rat angiotensin AT(1A) receptors with impaired receptor internalization. In agreement with previously published AT(1) angiotensin receptor visualization experiments, the present data suggest that non-peptide antagonist-angiotensin AT(1) receptor complexes remain at the cell surface. Insurmountable antagonism of candesartan is therefore independent from receptor internalization via clathrin-coated pits.     

Losartan: a selective angiotensin II type 1 (AT1) receptor antagonist for the treatment of heart failure

Losartan (COZAAR) is the prototype of a new class of potent and selective angiotensin II (AII) type 1 (AT(1)) receptor antagonists with the largest published preclinical and clinical data base. Since all of the AII antagonists are selective for the AT(1) receptor, these drugs should exhibit similar cardiovascular effects. However, since the pharmacokinetic/pharmacodynamic profiles of these agents and their degree of affinity for the AT(1) receptor differ, it is likely that differences in clinical profiles between these drugs exist and will require investigation. Losartan (parent compound), has moderate affinity for the AT(1) receptor (competitive inhibition). Losartan is well-absorbed orally as an active drug and is rapidly converted via oxidation in the human liver to a more potent metabolite (designated E3174) with an affinity 20- to 30-times greater for the AT(1) receptor (non-competitive inhibition). E3174 has a half-life of 6 - 9 h; elimination is via renal and hepatic routes. Antihypertensive and, in heart failure patients, haemodynamic activity is observed over a 24 h period with once daily dosing. Over 6 million patients have been treated for hypertension with continued excellent tolerability. Clinical experience in heart failure is growing, and recent data suggest an improved survival with losartan versus captopril, a drug from the angiotensin-converting-enzyme inhibitor class with proven benefit in this population. The current comprehensive losartan clinical end-point programme (4 large scale morbidity/mortality trials) should provide evidence regarding the efficacy of direct blockade of the AT(1) receptor with losartan compared to standard therapy: 1) The Losartan Heart Failure Survival Study - ELITE II, 2) The Losartan Post-Myocardial Infarction Survival Study - OPTIMAAL, 3) The Losartan Hypertension Survival Study - LIFE and 4) The Losartan Renal Protection Study - RENAAL.     

Role of angiotensin II type I receptor agonistic autoantibodies (AT1-AA) in preeclampsia

Despite being one of the leading causes of maternal death and a major contributor of maternal and perinatal morbidity, the mechanisms responsible for the pathogenesis of preeclampsia are unclear. One important initiating event in preeclampsia is thought to be reduced placental perfusion leading to the production of a variety of factors that cause widespread dysfunction of the maternal vasculature. The major objective of this review is to discuss the potential role of a novel agonistic autoantibody to the angiotensin II type I receptor (AT1-AA) in mediating hypertension during pregnancy. Although animal studies suggest that increasing plasma AT1-AA concentration in pregnant rats to levels observed in preeclamptic women or placental ischemic rats result in significant increases in arterial pressure, the quantitative importance of AT1-AA in the pathophysiology of preeclampsia in humans has yet to be fully elucidated.     

Losartan: a selective angiotensin II type 1 (AT1) receptor antagonist for the treatment of heart failure

Losartan (COZAARΟχιρχ^?) is the prototype of a new class of potent and selective angiotensin II (AII) type 1 (AT₁) receptor antagonists with the largest published preclinical and clinical data base. Since all of the AII antagonists are selective for the AT₁ receptor, these drugs should exhibit similar cardiovascular effects. However, since the pharmacokinetic/pharmacodynamic profiles of these agents and their degree of affinity for the AT₁ receptor differ, it is likely that differences in clinical profiles between these drugs exist and will require investigation. Losartan (parent compound), has moderate affinity for the AT₁ receptor (competitive inhibition). Losartan is well-absorbed orally as an active drug and is rapidly converted via oxidation in the human liver to a more potent metabolite (designated E3174) with an affinity 20- to 30-times greater for the AT₁ receptor (non-competitive inhibition). E3174 has a half-life of 6 - 9 h; elimination is via renal and hepatic routes. Antihypertensive and, in heart failure patients, haemodynamic activity is observed over a 24 h period with once daily dosing. Over 6 million patients have been treated for hypertension with continued excellent tolerability. Clinical experience in heart failure is growing, and recent data suggest an improved survival with losartan versus captopril, a drug from the angiotensin-converting-enzyme inhibitor class with proven benefit in this population. The current comprehensive losartan clinical end-point programme (4 large scale morbidity/mortality trials) should provide evidence regarding the efficacy of direct blockade of the AT₁ receptor with losartan compared to standard therapy: 1) The Losartan Heart Failure Survival Study - ELITE II, 2) The Losartan Post-Myocardial Infarction Survival Study - OPTIMAAL, 3) The Losartan Hypertension Survival Study - LIFE and 4) The Losartan Renal Protection Study - RENAAL.     

Characterization of a fluorescent conjugate of the rabbit angiotensin AT(1) receptor

1. The rabbit AT(1) receptor (AT(1)R) for angiotensin II (A(II)) has been conjugated to the yellow fluorescent protein (YFP) in order to establish the pharmacological profile of such a fusion protein and to facilitate the study of ligand-induced regulation. 2. A(II) bound AT(1)R-YFP (K(D) 8.1 nM in transiently transfected cells) and stimulated HEK 293 cells expressing the fusion protein at concentration ranges similar to the ones that stimulate the contraction of the isolated rabbit aorta. Antagonists found to be insurmountable in the latter assay (candesartan and EXP-3174 being the most extreme cases) were also insurmountable in the phospholipase A(2) assay applied to cells expressing AT(1)R-YFP, whereas losartan appeared to be surmountable in both assays. 3. Cells expressing AT(1)R-YFP exhibited a membrane-associated fluorescence that was partly and reversibly translocated into intracellular structures upon A(II) stimulation (confocal microscopy); the nonpeptide antagonists were not active in this respect, but prevented the effect of the agonist. 4. A(II) treatment increased the quantity of the fusion protein in cells, and phorbol 12-myristate 13-acetate (PMA) treatment even more so (immunoblot, confocal microscopy) but, unlike the agonist, the latter drug did not induce receptor endocytosis. A protein kinase C (PKC) inhibitor prevented the effect of either A(II) or PMA on AT(1)R-YFP abundance. 5. The conjugate AT(1)R-YFP retains the pharmacological properties of the parent rabbit AT(1)R. Agonist-induced downregulation was not documented using this system; to the contrary, we have observed a PKC-mediated increased expression AT(1)R-YFP likely to be the result of a decreased breakdown rate of the fusion protein.     

Molecular characterisation of the interactions between olmesartan and telmisartan and the human angiotensin II AT1 receptor

BACKGROUND AND PURPOSE: Whereas some angiotensin II (Ang II) type 1 receptor blockers (ARBs) produce surmountable antagonism of AT(1) receptors, others such as olmesartan and telmisartan display varying degrees of insurmountability. This study compared the molecular interactions of olmesartan and telmisartan with the human AT(1) receptor, using well characterised in vitro methods and model systems. EXPERIMENTAL APPROACH: CHO-K1 cells that stably express human AT(1) receptors (CHO-hAT(1) cells) were used in several pharmacological studies of olmesartan and telmisartan, including direct radioligand binding and inhibition of Ang II-induced inositol phosphate (IP) accumulation. KEY RESULTS: Both ARBs were found to be competitive antagonists that displayed high affinity, slow dissociation, and a high degree of insurmountability for the AT(1) receptor (the latter greater with olmesartan). Their receptor interactions could be described by a two-step process with the initial formation of a loose complex (IR) and subsequent transformation into a tight binding complex (IR*). In washout experiments, [(3)H] telmisartan dissociated from the receptor with a half-life of 29 min and the Ang II-mediated IP accumulation response was 50% maximally restored within 24 min, whereas values for [(3)H] olmesartan were 72 min and 76 min, respectively. CONCLUSIONS AND IMPLICATIONS: The high degree of insurmountability, slow dissociation, and high affinity of olmesartan for its receptor may relate to its ability to stabilise IR* via the carboxyl group of its imidazole core. In comparison, telmisartan displays a less potent interaction with the receptor.     

In vitro phamacology of MK-996, a new potent and selective angiotensin II (AT1) receptor antagonist

MK-996 (N-((4′-((5,7-Dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridin-3-yl)methyl) (1,1′-biphenyl)-2-yl) sulfonylbenzamide) interacted in a competitive manner with rabbit aortic angiotensin II (All) receptors as determined by Scatchard analysis of specific binding of [¹²⁵l]-Sar¹lle⁸-All. MK-996 also exhibited high affinity at All receptors in several tissues from different animal species (K_i = 0.1–0.4 nM). In vitro functional assays utilizing All-induced aldosterone release in rat adrenal cortical cells demonstrated further that MK-996 acts as a competitive, high affinity antagonist of All (pA₂ = 10.3) and lacks agonist activity. MK-996 also potently inhibited All-induced contractile response in isolated rabbit aorta and pulmonary artery with a reduction in maximal response. The specificity of MK-996 for All receptors was demonstrated by its lack of activity (IC₅₀> 1 μM) in several other receptor binding assays and its inability to affect in vitro functional responses produced by other agonists. MK-996 demonstrated a very high selectivity for the AT₁ compared to AT₂ receptor subtype (AT₂ IC₅₀ ≥ 2 μM). Direct binding studies using [³H]-MK-996 in rat adrenal indicated specific binding of [³H]-MK-996 is saturable and of high affinity (K_d = 0.47 nM). The specific [³H]-MK-996 binding in rat adrenal represents binding to pharmacologically relevant AT₁ receptors as demonstrated by the similar K_i values for various All agonists and antagonists in inhibiting specific ³H-MK-996 and [¹²⁵l]-All binding to AT₁ receptors. Dissociation rate studies of specific [³H]-MK-996 binding indicated a t1/2 of 103 min. This slow dissociation may account for the reduction in maximal responses to All in MK-996 treated isolated blood vessels.     

Quantitative structure activity relationship analysis of angiotensin II AT1 receptor antagonists

The quantitative structure activity relationship (QSAR) models were developed using multiple linear regression (MLR) and partial least square (PLS) for a set of 85 AT₁ receptor antagonists of hydantoin series. The MLR and PLS generated comparable models with good predictive ability and all the other statistical values, such as r, r², \textr_(\textcv)² , {\text{r}}_{{({\text{cv}})}}^{2} , and F and s values, were satisfactory. The results obtained from this study indicate the importance of steric (K-alpha3), hydrophobic (log P, and total lipole), and total energy (Cosmic total energy) in determining the activity of AT₁ receptor antagonists. The results clearly explained that optimum hydrophobicity of substituent at R₂ position is favorable for the activity and presence of a substituent of particular size and shape on phenyl ring at R₃ position is essential for the activity. This information is pertinent to the further design of new AT₁ receptor antagonist containing the hydantoin nucleus.     

Specific nonpeptide photoprobes as tools for the structural study of the angiotensin II AT(1) receptor

The aim of this work was to obtain photoactivatable nonpeptide antagonists of the angiotensin II AT(1) receptor. Based on structure-function relationships, two chemical structures as well as appropriate synthetic schemes were chosen as a frame for the design of radiolabeled azido probes. The feasibility of the strategy was first assessed by the synthesis of two tritiated ligands 21 and 22 possessing a high affinity for the AT(1) receptor and a low nonspecific binding to membrane or cell preparations. We then prepared two unlabeled azido derivatives 7 and 14 which retained a fairly high affinity for the AT(1) receptor. The latter compound proved to be suitable for receptor irreversible labeling and was prepared in its tritiated form 28. This tritiated azido nonpeptide probe displayed a K(d) value of 11.8 nM and a low nonspecific binding. It was suitable for specific and efficient covalent labeling of the recombinant AT(1A) receptor stably expressed in CHO cells. The electrophoretic pattern of the specifically labeled entity was strictly identical to that of purified receptor photolabeled with a biotinylated peptidic photoactivatable probe. This new tool should be useful for the mapping of the nonpeptide receptor binding site. These potential applications are discussed in light of the current knowledge of molecular mechanisms of G-protein coupled receptor activation and inactivation.     

pK(a) determination of angiotensin II receptor antagonists (ARA II) by spectrofluorimetry

The acid-base equilibrium constants of a new family of antihypertensive drugs, the angiotensin II receptor antagonists (ARA II), Losartan, Irbesartan, Valsartan, Candesartan cilexetil, its metabolite Candesartan M1 and Telmisartan were determined by spectrofluorimetry. Relative fluorescent intensity (I(F,rel))-pH data were treated by graphical (derivatives and curve-fitting) and numerical methods (LETAGROP SPEFO). The resultant pK(a) values at an ionic strength of 0.5 M were (3.15+/-0.07) for Losartan, (4.70+/-0.06) for Irbesartan, (4.90+/-0.09) for Valsartan, (6.0+/-0.1) for Candesartan cilexetil, (3.9+/-0.1) for Candesartan M1, and (4.45+/-0.09) for Telmisartan.     

Synthesis and pharmacological evaluation of new pyrazolidine-3, 5-diones as AT(1) angiotensin II receptor antagonists

On the basis of the structure of the non-peptide receptor antagonist irbesartan, a new series of AT(1) ligands was designed. In these compounds the central imidazolone nucleus of irbesartan was replaced by a pyrazolidine-3,5-dione structure. The key intermediate N-alkylpyrazolidine-3,5-diones were synthesized according to a new and general method. The most active compounds possess a spirocyclopentane ring at position 4, a linear butyl chain at position 1, and the [2'-(5-tetrazolyl)biphenyl-4-yl]methyl or [2'-(benzoylaminosulfonyl)biphenyl-4-yl]methyl group at position 2. Affinity toward the AT(1) and AT(2) receptors was assessed by the ability of the compounds to competitively displace [(3)H]AII from its specific binding sites. The most active compounds, 28 and 48, displayed high affinity for the AT(1) receptor, good selectivity AT(1) versus AT(2), and potent in vitro antagonist activity.     

Dynamic mechanisms of non-classical antagonism by competitive AT(1) receptor antagonists

Selective competitive angiotensin AT(1) receptor antagonists exhibit diverse patterns of antagonism of angiotensin-II-mediated responses in functional assays. These range from the classical parallel rightward shift of agonist concentration-response curves with no depression of the maximum response to an apparently straightforward insurmountable antagonism with complete depression of the maximum response and no rightward shift. This article reviews some earlier equilibrium-based models that have been used to explain the insurmountable antagonism, and suggests that a kinetic model might provide a more satisfactory account of the observations. Such a model might provide deeper insights into the pharmacology of G-protein-coupled receptors than the more popular equilibrium models.     

The effects of KT3-671, a new angiotensin II (AT 1) receptor blocker in mild to moderate hypertension

AIMS: To compare the antihypertensive effect, and tolerability and safety of once daily doses of KT3-671 with that of placebo in patients with mild to moderate uncomplicated essential hypertension. METHODS: A randomised, multicentre, double blind, parallel-group comparison of KT3-671 with placebo. Hypertensive patients [Ambulatory Blood Pressure Monitoring (ABPM), mean daytime DBP > 90 mmHg, Office sitting mean DBP 95-114 after a 7-28 day washout period] entered a 2-week, single blind, run-in phase. Patients eligible for the double-blind phase were randomised to receive KT3-671 40 mg, 80 mg, 160 mg or placebo once daily over 4 weeks. The primary end-point was trough mean sitting office DBP. The study had 90% power to detect a 5 mmHg change between treatments and placebo at the 5% level of significance. The secondary end-points were 24 hour, daytime and night time mean ABPM. RESULTS: Office DBP was significantly lower with KT3-671 40 mg but not the other 2 dosage groups (-3.2; 95% CL -6.1 : -0.3 P < 0.03). Office SBP was significantly reduced with all dosage groups (40 mg -5.9, 95% CL -11 : -0.9; 80 mg -4.9, 95% CL -9.9 : 0.1 and 160 mg -5.7, 95% CL -10.8 : -0.7 P < 0.05). All doses of KT3-671 reduced systolic and diastolic ABPM. The number of patients with treatment related adverse events were comparable to placebo (38.8% KT3-671 vs 32.8% placebo). There was some evidence of a dose-response relationship with fall in nocturnal ABPM. CONCLUSIONS: Oral KT3-671 was well tolerated. KT3-671 reduced office systolic BP at all doses and diastolic BP at some of the doses. Due to greater precision and power, the falls in mean ambulatory systolic and diastolic pressure were all significantly lower than placebo.