Yoshihiro Kiya

Conformational switch of angiotensin II type 1 receptor underlying mechanical stress-induced activation

The angiotensin II type 1 (AT1) receptor is a G protein‐coupled receptor that has a crucial role in the development of load‐induced cardiac hypertrophy. Here, we show that cell stretch leads to activation of the AT1 receptor, which undergoes an anticlockwise rotation and a shift of transmembrane (TM) 7 into the ligand‐binding pocket. As an inverse agonist, candesartan suppressed the stretch‐induced helical movement of TM7 through the bindings of the carboxyl group of candesartan to the specific residues of the receptor. A molecular model proposes that the tight binding of candesartan to the AT1 receptor stabilizes the receptor in the inactive conformation, preventing its shift to the active conformation. Our results show that the AT1 receptor undergoes a conformational switch that couples mechanical stress‐induced activation and inverse agonist‐induced inactivation.

Molecular Mechanism Underlying Inverse Agonist of Angiotensin II Type 1 Receptor

To delineate the molecular mechanism underlying the inverse agonist activity of olmesartan, a potent angiotensin II type 1 (AT1) receptor antagonist, we performed binding affinity studies and an inositol phosphate production assay. Binding affinity of olmesartan and its related compounds to wild-type and mutant AT1 receptors demonstrated that interactions between olmesartan and Tyr113, Lys199, His256, and Gln257 in the AT1 receptor were important. The inositol phosphate production assay of olmesartan and related compounds using mutant receptors indicated that the inverse agonist activity required two interactions, that between the hydroxyl group of olmesartan and Tyr113 in the receptor and that between the carboxyl group of olmesartan and Lys199 and His256 in the receptor. Gln257 was found to be important for the interaction with olmesartan but not for the inverse agonist activity. Based on these results, we constructed a model for the interaction between olmesartan and the AT1 receptor. Although the activation of G protein-coupled receptors is initiated by anti-clockwise rotation of transmembrane (TM) III and TM VI followed by changes in the conformation of the receptor, in this model, cooperative interactions between the hydroxyl group and Tyr113 in TM III and between the carboxyl group and His256 in TM VI were essential for the potent inverse agonist activity of olmesartan. We speculate that the specific interaction of olmesartan with these two TMs is essential for stabilizing the AT1 receptor in an inactive conformation. A better understanding of the molecular mechanisms of the inverse agonism could be useful for the development of new G protein-coupled receptor antagonists with inverse agonist activity.

Molecular mechanisms of the antagonistic action between AT1 and AT2 receptors.

Although angiotensin II (Ang II) binds to Ang II type 1 (AT(1)) and type 2 (AT(2)) receptors, AT(1) and AT(2) receptors have antagonistic actions with regard to cell signaling. The molecular mechanisms that underlie this antagonism are not well understood. We examined AT(1) and AT(2) receptor-induced signal cross-talk in the cytoplasm and the importance of the hetero-dimerization of AT(1) receptor with AT(2) receptor on the cell surface. AT(1) and AT(2) receptors showed antagonistic effects toward inositol phosphate production. AT(1) receptors mainly formed homo-dimers, rather than hetero-dimers with AT(2) receptor, on the cell surface as determined by immunoprecipitation, and subsequently induced cell signals. AT(2) receptor mainly formed homo-dimers, rather than hetero-dimers with AT(1) receptor, on the cell surface. The expression levels of homo-dimerized AT(1) receptor or AT(2) receptor on the cell surface did not change after treatment with Ang II, the AT(1) receptor antagonist telmisartan or the AT(2) receptor antagonist PD123319. Finally, AT(1) and AT(2) receptor-induced signals antagonized phospholipase C-beta(3) phosphorylation. In conclusion, Ang II-induced AT(1) receptor signals may be mainly blocked by AT(2) receptor signals through their negative cross-talk in the cytoplasm rather than by the hetero-dimerization of both receptors on the cell surface. The proper balance of the expression levels of AT(1) and AT(2) receptors might be critical for the antagonistic action between these receptors.

Antiarrhythmogenic Effect of Reconstituted High-Density Lipoprotein Against Ischemia/Reperfusion in Rats

OBJECTIVES This study analyzed the antiarrhythmogenic effect of reconstituted high-density lipoprotein (rHDL) against ischemia/reperfusion in vivo. BACKGROUND Recent studies have suggested that a reduction in the plasma HDL level may contribute to cardiac sudden death. Although there are currently only a few therapeutic strategies for increasing HDL, an exciting new therapeutic option, rHDL, has recently been developed to prevent coronary artery disease. METHODS To analyze the suppression of reperfusion arrhythmia by rHDL (apolipoprotein A-I with 1-palmitoyl-2-oleoyl-phosphatidyl-choline), 92 male Wistar rats were divided into 10 groups: rats that had been pre-treated with or without rHDL, apolipoprotein A-I, or 1-palmitoyl-2-oleoyl-phosphatidyl-choline in the presence or absence of inhibitors of Akt protein kinase, nitric oxide (NO), or extracellular-signal-regulated kinase (ERK) administered intravenously before left coronary artery occlusion. We also used human coronary artery endothelial cells and adenosine triphosphate-binding cassette transporter (ABC) A1-, ABCG1-, or scavenger receptor class B, type I-transfected ldlA7 cells systems. RESULTS The duration of ventricular tachycardia or ventricular fibrillation after reperfusion in rHDL-pre-treated rats was much shorter than that in untreated rats. Apolipoprotein A-I or 1-palmitoyl-2-oleoyl-phosphatidyl-choline alone had no effect. The effect of rHDL was blocked by inhibitors of Akt, NO, and ERK. Plasma NO concentration in the rHDL group was significantly higher. In addition, rHDL activated phospho(p)-Akt, p-ERK, and p-endothelial NO synthesis in endothelial cells. The rHDL activated p-ERK in ABCA1- or ABCG1-transfected but not scavenger receptor class B, type I-transfected ldlA7 cells. CONCLUSIONS The rHDL-induced NO production, probably mediated by ABCA1 or ABCG1 through an Akt/ERK/NO pathway in endothelial cells, may suppress reperfusion-induced arrhythmias. The HDL-based therapy may hold the promise of reducing the incidence of such arrhythmias after ischemia/reperfusion.

A small difference in the molecular structure of angiotensin II receptor blockers induces AT1 receptor-dependent and -independent beneficial effects

Angiotensin II (Ang II) type 1 (AT1) receptor blockers (ARBs) induce multiple pharmacological beneficial effects, but not all ARBs have the same effects and the molecular mechanisms underlying their actions are not certain. In this study, irbesartan and losartan were examined because of their different molecular structures (irbesartan has a cyclopentyl group whereas losartan has a chloride group). We analyzed the binding affinity and production of inositol phosphate (IP), monocyte chemoattractant protein-1 (MCP-1) and adiponectin. Compared with losartan, irbesartan showed a significantly higher binding affinity and slower dissociation rate from the AT1 receptor and a significantly higher degree of inverse agonism and insurmountability toward IP production. These effects of irbesartan were not seen with the AT1-Y113A mutant receptor. On the basis of the molecular modeling of the ARBs–AT1 receptor complex and a mutagenesis study, the phenyl group at Tyr113 in the AT1 receptor and the cyclopentyl group of irbesartan may form a hydrophobic interaction that is stronger than the losartan–AT1 receptor interaction. Interestingly, irbesartan inhibited MCP-1 production more strongly than losartan. This effect was mediated by the inhibition of nuclear factor-kappa B activation that was independent of the AT1 receptor in the human coronary endothelial cells. In addition, irbesartan, but not losartan, induced significant adiponectin production that was mediated by peroxisome proliferator-activated receptor-γ activation in 3T3-L1 adipocytes, and this effect was not mediated by the AT1 receptor. In conclusion, irbesartan induced greater beneficial effects than losartan due to small differences between their molecular structures, and these differential effects were both dependent on and independent of the AT1 receptor.

Safety and efficacy of antihypertensive therapy with add-on angiotensin II type 1 receptor blocker after successful coronary stent implantation

This study was performed to evaluate the safety and efficacy of additional antihypertensive therapy with angiotensin II type 1 receptor blocker (ARB; olmesartan or valsartan) after successful stent implantation in patients with coronary artery disease (CAD). Fifty patients with CAD after successful stent implantation were included in this study. They were divided into an ARB group, which initially received olmesartan (n=20, 14±8 mg day−1) or valsartan (n=20, 60±23 mg day−1) immediately after stent implantation, and a non-ARB group (n=10) according to their blood pressure (BP). Follow-up coronary angiography, measurement of BP and blood sampling were performed before (at baseline) and 6–8 months after stent implantation (at follow-up). There were no significant differences in the baseline characteristics between the groups, except for BP. Although there were no changes in % diameter restenosis between the groups, the BP level in the ARB group at follow-up showed a significant reduction (125±12/69±9 mm Hg) and reached the target BP. There were no critical adverse effects in the ARB group throughout the study period. In addition, serum high-sensitive C-reactive protein (hs-CRP) and pentraxin 3 were significantly decreased in the ARB group but not in the non-ARB group. Although olmesartan and valsartan induced similar BP-lowering effects, olmesartan but not valsartan induced a significant decrease in hs-CRP, but did not increase serum uric acid. In conclusion, antihypertensive therapy with add-on low-dose ARB after stent implantation was safe and achieved the target BP. In particular, olmesartan had an anti-inflammatory effect.

Reconstituted high-density lipoprotein attenuates postinfarction left ventricular remodeling in rats.

Since little is known about the effects of reconstituted high-density lipoprotein (rHDL) in left ventricular (LV) remodeling, these effects were examined in rats after acute myocardial infraction (MI). Sixteen male Wistar rats were randomly divided into three groups: Sham-operated (n=6), and MI rats that received a permanent ligation around the proximal left coronary artery and infusions of placebo (MI group, n=5) or rHDL (containing as apolipoproteinA-I 6mg/kg) administered intravenously (MI+rHDL group, n=5). rHDL was infused once a week for 4 weeks. In addition, in vitro assays were performed to examine the effect of rHDL. The MI+rHDL group showed a significant increase in LV ejection fraction (EF) between weeks 1 and 4, a decrease in LV end-systolic diameter, compared with the progressive deterioration of LV size and function in the MI group. In addition, the MI+rHDL group showed a significant decrease in fibrotic area of MI in LV compared to that in the MI group, while there were no significant increases in capillary density or cell size in LV in the MI+rHDL group. Interestingly, the MI+rHDL group showed a significant activation of retinoblastoma and ERK (extracellular-signal-regulated kinase) but not cleaved caspase-3, p38 MAPK or Jun N-terminal kinase. rHDL suppressed H(2)O(2)-induced arrest of cell growth in myocytes. This effect was blocked by PD98059, an ERK inhibitor. In conclusions, rHDL-promoted cell survival has beneficial morphological effects that help to prevent LV remodeling and improve function after MI, and may prevent arrest of cell growth through ERK pathway in myocytes.

Small Molecules with Similar Structures Exhibit Agonist, Neutral Antagonist or Inverse Agonist Activity toward Angiotensin II Type 1 Receptor

Small differences in the chemical structures of ligands can be responsible for agonism, neutral antagonism or inverse agonism toward a G-protein-coupled receptor (GPCR). Although each ligand may stabilize the receptor conformation in a different way, little is known about the precise conformational differences. We synthesized the angiotensin II type 1 receptor blocker (ARB) olmesartan, R239470 and R794847, which induced inverse agonism, antagonism and agonism, respectively, and then investigated the ligand-specific changes in the receptor conformation with respect to stabilization around transmembrane (TM)3. The results of substituted cysteine accessibility mapping studies support the novel concept that ligand-induced changes in the conformation of TM3 play a role in stabilizing GPCR. Although the agonist-, neutral antagonist and inverse agonist-binding sites in the AT1 receptor are similar, each ligand induced specific conformational changes in TM3. In addition, all of the experimental data were obtained with functional receptors in a native membrane environment (in situ).

Clinical and pharmacotherapeutic relevance of the double-chain domain of the angiotensin II type 1 receptor blocker olmesartan.

We previously reported that the angiotensin II type 1 (AT1) receptor blocker (ARB) olmesartan has two important interactions to evoke inverse agonism (IA). We refer to these interactions as the “double-chain domain (DCD).” Since the clinical pharmacotherapeutic relevance of olmesartan is still unclear, we examined these effects in rats and humans. We analyzed the effects at an advanced stage of renal insufficiency in Dahl salt-sensitive hypertensive rats (Study 1). Rats were fed a high-salt diet from age 9 weeks and arbitrarily assigned to three treatment regimens at age 16 to 21 weeks: olmesartan (2 mg/kg/day) with DCD, a compound related to olmesartan without DCD (6 mg/kg/day, R-239470) or placebo. We also compared the depressor effects of olmesartan to those of other ARBs in patients with essential hypertension (Study 2). Thirty essential hypertensive outpatients who had been receiving ARBs other than olmesartan were recruited for this study. Our protocol was approved by the hospital ethics committee and informed consent was obtained from all patients 12 weeks prior to switching from ARBs other than olmesartan to olmesartan. In Study 1, olmesartan induced a more prominent suppression of the ratio of urinary protein excretion to creatinine at age 21 weeks without lowering blood pressure among the three groups. In Study 2, the depressor effect of olmesartan was significantly stronger than those of other ARBs, which do not contain the DCD. These additive effects by olmesartan may be due to DCD.

Possibility of increasing cholesterol efflux by adiponectin and its receptors through the ATP binding cassette transporter A1 in HEK293T cells

A decrease in adiponectin secretion leads to the early stage of atherosclerosis. Discoidal high-density lipoproteins (HDL) accept the cholesterol that effluxes from cells expressing the ATP binding cassette transporter A1 (ABCA1) in the first step of reverse cholesterol transport (RCT). Recently, a new therapeutic strategy involving reconstituted (r)HDL has been shown to enhance RCT. Therefore, we hypothesized that adiponectin may increase the efflux associated with ABCA1 and also enhance rHDL-induced efflux in human embryonic kidney 293 (HEK293T) cells. We transfected adiponectin receptor 1 and 2 (AdipoR1 and AdipoR2) cDNA into cells. The transfected cells were labeled with [(3)H]cholesterol following cholesterol loading with or without adiponectin for 24h. The levels of cholesterol efflux were analyzed using a liquid scintillation counter. Treatment with adiponectin was associated with significantly higher levels of efflux in AdipoR1- and AdipoR2-transfected cells. Interestingly, rHDL-induced cholesterol efflux was enhanced in the presence of adiponectin. The down-regulation of adiponectin receptors using short-hairpin RNA decreased rHDL-induced cholesterol efflux with the down-regulation of ABCA1. In summary, adiponectin and its receptors increased cholesterol efflux and also enhanced rHDL-induced efflux at least partially through an ABCA1 pathway. These results suggest that adiponectin may enhance the RCT system and induce an anti-atherogenic effect.