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Since impaired mitochondrial ATP production in cardiomyocytes is thought to lead to heart failure, a drug that protects mitochondria and improves ATP production under disease conditions would be an attractive treatment option. In this study, we identified small-molecule drugs, including the anti-parasitic agent, ivermectin, that maintain mitochondrial ATP levels under hypoxia in cardiomyocytes. Mechanistically, transcriptomic analysis and gene silencing experiments revealed that ivermectin increased mitochondrial ATP production by inducing Cox6a2, a subunit of the mitochondrial respiratory chain. Furthermore, ivermectin inhibited the hypertrophic response of human induced pluripotent stem cell-derived cardiomyocytes. Pharmacological inhibition of importin β, one of the targets of ivermectin, exhibited protection against mitochondrial ATP decline and cardiomyocyte hypertrophy. These findings indicate that maintaining mitochondrial ATP under hypoxia may prevent hypertrophy and improve cardiac function, providing therapeutic options for mitochondrial dysfunction., Highlights • High-throughput image-based phenotypic screens based on mitochondrial ATP under hypoxia were developed. • Mitochondrial ATP protectors, including ivermectin, were identified in cardiomyocytes under hypoxia. • Ivermectin increased mitochondrial ATP production by inducing Cox6a2. • Mitochondrial ATP protectors inhibited hypertrophic responses in human iPSC-derived cardiomyocytes. Impaired mitochondrial ATP production in cardiomyocytes is one of the major causes of heart failure. A drug that maintains ATP production would, therefore, be an attractive heart failure treatment option. We performed high-throughput image-based screens under hypoxia, and identified mitochondrial ATP protectors, including the anti-parasite drug, ivermectin. We show that ivermectin increased mitochondrial ATP production by inducing Cox6a2 expression and inhibited the hypertrophic response of human iPSC-derived cardiomyocytes. Pharmacological inhibition of importin β, a target of ivermectin, exhibited similar protective effects. Our studies indicate that compounds maintaining mitochondrial ATP under hypoxia may prevent pathological hypertrophy and improve cardiac function.

The cardiac sarco/endoplasmic reticulum Ca2+-dependent ATPase 2a (SERCA2a) plays a central role in Ca2+ handling within cardiomyocytes and is negatively regulated by phospholamban (PLN), a sarcoplasmic reticulum (SR) membrane protein. The activation of SERCA2a, which has been reported to improve cardiac dysfunction in heart failure, is a potential therapeutic approach for heart failure. Therefore, we developed a novel small molecule, compound A and characterized it both in vitro and in vivo. Compound A activated the Ca2+-dependent ATPase activity of cardiac SR vesicles but not that of skeletal muscle SR vesicles that lack PLN. The surface plasmon resonance assay revealed a direct interaction between compound A and PLN, suggesting that the binding of compound A to PLN attenuates its inhibition of SERCA2a, resulting in SERCA2a activation. This was substantiated by inhibition of the compound A-mediated increase in Ca2+ levels within the SR of HL-1 cells by thapsigargin, a SERCA inhibitor. Compound A also increased the Ca2+ transients and contraction and relaxation of isolated adult rat cardiomyocytes. In isolated perfused rat hearts, the compound A enhanced systolic and diastolic functions. Further, an infusion of compound A (30mg/kg, i.v. bolus followed by 2mg/kg/min, i.v. infusion) significantly enhanced the diastolic function in anesthetized normal rats. These results indicate that compound A is a novel SERCA2a activator, which attenuates PLN inhibition and enhances the systolic and diastolic functions of the heart in vitro and in vivo. Therefore, compound A might be a novel therapeutic lead for heart failure.

Plasminogen activator inhibitor-1 deficiency retards diabetic nephropathy. Background Plasminogen activator inhibitor-1 (PAI-1) is increased in kidneys of humans and animals with diabetic nephropathy and is associated with extracellular matrix (ECM) accumulation. PAI-1 may promote ECM buildup by preventing plasmin and matrix metalloproteinase (MMP) activation. However, the importance and mechanism of PAI-1 action in the pathogenesis of diabetic nephropathy is unknown. Methods We investigated the effect of streptozotocin (STZ)-induced diabetes in wild-type (PAI-1 +/+ ) mice and mice null for PAI-1 (PAI-1 −/− ). After 1 month of diabetes, animals were placed in metabolic cages for 24-hour urine collection. Total RNA was isolated from kidney cortex for reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis, and Western blots were quantitated from cortical protein. Primary mesangial cells were grown from Sprague-Dawley rats and used in signal transduction studies. Results Urinary albumin excretion (UAE) in diabetic PAI-1 +/+ mice increased >threefold, but remained unchanged in PAI-1 −/− mice. Transforming growth factor-β (TGF-β) and fibronectin message and protein levels were lower in diabetic PAI-1 −/− vs. PAI-1 +/+ mice, suggesting that PAI-1 deficiency impaired TGF-β expression despite diabetes. Indeed, recombinant PAI-1 directly stimulated TGF-β message and protein via mitogen-activated protein kinase (MAPK) signal transduction in cultured mesangial cells. Urokinase plasminogen activator (uPA) inhibited this PAI-1 action in a dose-dependent manner. The inhibitory effect of antibody to uPA receptor (uPAR) on PAI-1–induced TGF-β function suggested that uPAR mediated the cellular effect of PAI-1. Conclusion PAI-1 can regulate TGF-β expression by binding to uPAR and activating the extracellular-regulated signal kinase (ERK)/MAPK pathway. Therefore, PAI-1 contributes to diabetic nephropathy by regulating TGF-β and renal ECM production and may be a therapeutic target in diabetic nephropathy.

Background. Recently, inhibition of the renin-angiotensin system has been reported to be effective for patients with diabetic nephropathy. In this study, we examined the renal protective effects of candesartan cilexetil (TCV-116) in Wistar fatty rats, a model of non-insulin-dependent diabetes mellitus (NIDDM) with renal injuries. Methods. Twelve-week-old Wistar fatty rats received candesartan cilexetil (0.3 or 1 mg/kg) or enalapril (10 mg/kg) daily, administered orally, for 16 weeks. Routine laboratory parameters, such as urinary albumin and total protein excretion, were measured every 4 weeks, and renal function tests and histopathological studies were carried out at the end of the experiment. Results. In the 12-week-old Wistar fatty rats, plasma glucose and insulin levels were significantly higher than those in age-matched Wistar lean rats (normal controls). Urinary albumin and total protein excretion was slightly but significantly increased as compared to these parameters in Wistar lean rats, and increased further with time. Daily administration of candesartan cilexetil or enalapril inhibited the increase in urinary albumin and total protein excretion without affecting plasma glucose and insulin levels. In histopathological studies, candesartan cilexetil (0.3 and 1 mg/kg) and enalapril (10 mg/kg) prevented the glomerular injury observed in vehicle-treated rats. Candesartan cilexetil (1 mg/kg) and enalapril also inhibited tubular changes. Systolic blood pressure in the drug-treated groups was significantly decreased compared with that in vehicle-treated rats. Conclusions. Inhibitors of the renin-angiotensin system ameliorated proteinuria and the pathological changes of renal injuries in this NIDDM model. Candesartan cilexetil may be useful for the treatment of NIDDM patients with renal damage.

Effect of candesartan cilexetil (TCV-116) in rats with chronic renal failure. Background Inhibition of the renin-angiotensin system by both angiotensin II type 1 receptor antagonists (AT 1 As) and angiotensin I-converting enzyme inhibitors (ACEIs) shows renoprotective effects in rats with chronic renal failure when treatment is started in the early phase of renal injury. In this study, we examined the renal protective effects of candesartan cilexetil (TCV-116), an AT 1 A, and enalapril, an ACEI, in the progressive phase of renal injury in 5/6 nephrectomized rats. Methods Candesartan cilexetil (1 mg/kg/day) and enalapril (10 mg/kg/day) were orally administered once a day for 4 weeks (the short-term experiment) or 16 weeks (the long-term experiment) to 5/6 nephrectomized rats beginning 15 weeks after the nephrectomy, that is, after they had already showed marked proteinuria. Results In vehicle-treated rats, proteinuria, glomerulosclerosis, and interstitial fibrosis developed. Moreover, enhanced expression of transforming growth factor-β 1 (TGF-β 1 ) in the injured glomeruli was observed. These adverse changes progressed with time, and in the short-term experiment, both drugs inhibited them. In the long-term experiment, the progressive proteinuria and the elevation of blood pressure were similarly attenuated by both drugs. However, candesartan cilexetil significantly inhibited the progression of glomerulosclerosis, the expression of TGF-β 1 , and interstitial fibrosis, whereas enalapril did not. Conclusion These results indicate that candesartan cilexetil shows potent and long-term preventive effects against the progression of previously developed renal injury.

Abstract We have shown recently that mechanical stretch of cultured rat aortic smooth muscle cells induces a marked increase in gene expression of the vasorelaxant parathyroid hormone–related peptide. In the present study, we investigated whether mechanical force affected the in vivo parathyroid hormone–related peptide gene expression in blood vessels. Northern blot analysis revealed that stretch of isolated rat aortic strips increased the expression level of parathyroid hormone–related peptide mRNA. The parathyroid hormone–related peptide transcript level in aorta and mesenteric vessels from 18-week-old spontaneously hypertensive rats (SHR) was 2.5- and 2.2-fold higher, respectively, compared with age-matched Wistar-Kyoto (WKY) controls, whereas the parathyroid hormone–related peptide mRNA level in aorta from normotensive 4-week-old SHR was similar to that of age-matched WKY controls. The aortic parathyroid hormone–related peptide content was higher in 18-week-old SHR than in age-matched WKY controls. Moreover, treatment of mature SHR with an angiotensin II type 1 receptor antagonist or hydralazine caused a concomitant decrease in the parathyroid hormone–related peptide transcript level in aorta with lowering of blood pressure. These results suggest that the in vivo parathyroid hormone–related peptide gene expression in blood vessels is under the control of mechanical force, pointing to a role of parathyroid hormone–related peptide in the regulation of vascular tone.

Background Growth factors such as platelet-derived growth factor (PDGF) have been postulated to be important mediators of neointimal formation in balloon-injured artery. Binding of growth factors to their receptors activates intrinsic receptor tyrosine kinase, resulting in tyrosine phosphorylation of receptors themselves and cellular substrate proteins. We investigated in vivo activities of growth factors by determining the extent of tyrosine phosphorylation of growth factor receptors and substrate proteins in injured artery. Methods and Results Rat balloon-injured carotid artery was analyzed for phosphotyrosine content of PDGF α- and β-receptors, epidermal growth factor (EGF) receptors, and insulin receptor substrate-1 (IRS-1) by immunoprecipitation and anti-phosphotyrosine Western blot. The development of intimal thickening after deendothelializing balloon catheterization of rat carotid artery was accompanied by transient twofold to threefold increases in the extent of tyrosyl phosphorylation of PDGF α- and β-receptors but not EGF receptor or IRS-1. The AT 1 angiotensin II (Ang II) receptor antagonist TCV-116 markedly inhibited both tyrosyl phosphorylation of PDGF α- and β-receptors and intimal thickening. The AT 1 antagonist reduced mRNA levels of both PDGF-A and -B chains in injured arteries. Conclusions The present study provides direct evidence for increased PDGF activities in injured artery in situ and the involvement of Ang II in stimulated activation of PDGF receptors. These results are consistent with the pathogenetic role for PDGF in intimal thickening.

Tyrosine kinase dependent expression of TGF-β induced by stretch in mesangial cells. Increased glomerular hydraulic pressure has been suggested as a major causative factor in the development of glomerular sclerosis. The elevation of glomerular pressure increases the magnitude of stretch to mesangial cells. The study was, therefore, designed to investigate the effect of mechanical stretch on expression of TGF-β and extracellular matrix components in cultured rat mesangial cells. The results showed that mechanical stretch stimulated mRNA expression for TGF-β1 and TGFβ3 in a time dependent manner, and that mesangial cells secreted substantial amounts of TGF-β proteins in response to stretch. Stretch was also shown to stimulate mRNA expression for collagen types I and IV, and fibronectin, major components of mesangial extracellular matrix. The stretch-induced mRNA expression for extracellular matrix components was inhibited by neutralizing antibody to TGF-β. Moreover, stretch-induced mRNA expression of TGF-β was inhibited by tyrosine kinase inhibitors, genistein or herbimycin A, whereas Ca channel blockers nitrendipine or Gd3+, and inhibitors for protein kinase A or C had no effect. These findings indicate that stretch induced TGF-β mRNA primarily through tyrosine kinase-dependent mechanisms in cultured rat mesangial cells, and the secreted TGF-β may play a significant role for the stretch-induced expression of extracellular matrix proteins. Our results suggest that stretch-induced TGF-β of mesangial cells might be a mediator in the progression of glomerular sclerosis as an autocrine/paracrine factor.

In summary, we have shown that adenovirus vector efficiently introduced foreign gene into cultured renal cells both of mesangial and tubular origin. Genes transferred were properly expressed to produce the molecules of expected function. It was possible to introduce the gene into nearly 100% of the cells treated. Expression of the gene began as early as 12 hours after the infection, increased until 48 hours and persisted at least up to eight days. Finally, the vector was non-toxic to the cells, as judged from simple toxicity tests. Successful application of adenovirus vector enables for us to study function of pertinent molecules in suitable host cells and opens a new way for examining renal cellular physiology and pathophysiology.

Wistar fatty (WF) rats have a genetic predisposition to hyperglycemia, polyuria, hyperinsulinemia, hyperlipidemia, obesity and nephropathy. These phenotypic characteristics are similar to those observed in obese patients with non-insulin-dependent diabetes mellitus (NIDDM) nephropathy. In this study, the effects of two types of renin-angiotensin system inhibitors, an angiotensin II type 1-receptor antagonist (AT1A) and an angiotensin I-converting enzyme inhibitor (ACEI), on renal injury in WF rats were studied during the progressive phase of diabetic nephropathy. An AT1A, candesartan cilexetil (1 mg/kg), and an ACEI, enalapril (10 mg/kg), were administered orally once a day for 12 weeks, beginning when the rats were 27-week-old and already showed diabetic nephropathy and obesity. Both drugs prevented an increase in proteinuria during the experimental period. Furthermore, after 4-week intervention, the levels of proteinuria were markedly lower in drug-treated rats. At the end of the experiment, both drugs prevented the development of glomerular lesions without affecting glucose metabolism and obesity. In conclusion, the inhibition of angiotensin II activity ameliorated both existing proteinuria and the progression of proteinuria, resulting in preservation of glomerular structure. Thus angiotensin II plays important roles in the development and the progression of nephropathy in genetically obese diabetic WF rats.

Quantitative sandwich enzyme immunoassay (EIA) systems, that can distinguish between active-form subtypes of mitogen-activated protein kinases (p44 and p42 MAP kinase, also called ERK1 and ERK2), were developed employing subtype-specific antibodies as a solid phase and an antibody specific for the phosphorylated region of MAP kinases as the detector. Using these systems, we investigated the dynamic changes in the activity of ERK1 and ERK2 in platelet-derived growth factor (PDGF)-treated rat mesangial cells and nerve growth factor (NGF)-treated PC12. Both ERK1 and ERK2 were activated immediately after stimulation, and the activity reached a maximum at 5–10 min. The total activity of both subtypes correlated well with that obtained using the conventional method. Compared with the usual methods, these systems should have a higher specificity and be more convenient and suitable for experiments with multiple samples. Moreover, as these EIA systems can be applied not only to rat MAP kinases but also to human, mouse and rabbit MAP kinases, they are potentially very useful for a range of investigations.

AT1 receptor antagonists improve capillary hyperIntroduction tension of the glomerulus via reduction of glomerular efferent arteriolar tone. Thus they interfere with the Progressive loss of renal function is associated not only development of glomerular injury and proteinuria [3]. with development of glomerulosclerosis, but also with In addition, disturbed glomerular barrier function secthat of interstitial fibrosis. Interstitial fibrosis is characondary to activation of AT1 receptor by AII permits terized by the destruction of renal tubules and interproteins to escape into the urinary space and to exert stitial capillaries as well as by the accumulation of toxic effects upon tubular epithelial cells [1,7]. Immune extracellular matrix proteins. The severity of tubuloinresponses of tubular epithelial cells are induced by terstitial fibrosis has long been considered as a crucial exposure to proteins, e.g. stimulation of the release of determinant of progressive renal injury in both human various cytokines, including transforming growth and experimental glomerulonephritis. Moreover, previ1 1 [1]. The release of such cytokines ous studies have shown that decrease in glomerular causes differentiation of interstitial fibroblasts (and filtration rate is better correlated with tubulointerstitial possibly tubular epithelial cells) into myofibroblasts. injury than with glomerulosclerosis [1,2]. This change of phenotype is characterized by de novo Many studies have been published on the mechanisms and treatment of glomerulosclerosis. In contrast, expression of alpha-smooth-muscle actin [8]. Recently, the pathogenesis of interstitial fibrosis has been less a specific gene (Fsp1) [9,10] was cloned, which triggers well understood. The mechanisms that contribute to the transformation of tubular epithelial cells into these two types of matrix accumulation in the kidney myofibroblasts [11] in response to cytokines, such as are in part common, but to some extent also different. TGF-b. Furthermore, it has been shown that AII In this brief editorial, we focus on some new directly induced the expression of TGF-b in cultured approaches to interstitial fibrosis that have recently renal tubular cells and in fibroblasts [12]. Taken helped to elucidate its pathogenesis. together, the activation of AT1 receptors in glomeruli, tubular epithelial cells and interstitial fibroblasts might be involved in the progression of interstitial fibrosis. New roles of the renin–angiotensin system in the In the rat remnant kidney model, an AT1 receptor development of interstitial fibrosis antagonist, candesartan cilexetil (TCV-116), was superior in preventing interstitial fibrosis and glomRecent reports suggest that angiotensin II (AII ) plays erulosclerosis compared to angiotensin-converting important roles in the development and amelioration enzyme inhibitor (ACI) [13]. The beneficial effects of of interstitial fibrosis through different types of AII AT1 receptor antagonist were initially thought to be receptors. AII not only contributes to glomerular capilmediated mainly by the blockade of AT1 receptors. lary hypertension and an impairment of glomerular In contrast to the AT1 receptor, much less is known permselectivity, but influences also directly some funcabout the functional properties of AT2 receptor. Since tions of interstitial fibroblasts and tubular epithelial AT2 receptor expression is high in embryonic tissues cells [3,4]. To date, molecular cloning and pharmacoand declines dramatically after birth [14], it was postulogical studies have defined two major classes of AII lated that the AT2 receptor played a role in cell growth receptors, i.e. AT1 and AT2 receptors. Most known and differentiation. In contrast, recent reports sugeffects of AII are mediated by AT1 receptors [5,6 ]. gested that AT2 receptors mediate antiproliferative effects and participate in cell apoptosis [5,6 ]. Since AII Correspondence and offprint requests to: Masafumi Fukagawa MD exhibits growth promoting properties via AT1 receptor PhD, Division of Nephrology and Clinical Research Center, Tokyo in some cell types, AT1 and AT2 receptors might Teishin Hospital, 2-14-23 Fujimi, Chiyoda-ku, Tokyo 102-8798, Japan. mediate counterbalancing signals [15]. A similar recip

The nonhydroyzable GTP analog guanosine 5′-(3-O-thio)triphosphatase (GTPγS) was previously shown to enhance Ca2+-dependent contraction and phosphorylation of 20-kDa myosin light chain (MLC20) in skinned smooth muscle preparations. In β-escin-permeabilized cultured aortic smooth muscle cells, GTPγS enhances Ca2+-induced phosphorylation of MLC20 in a concentration-dependent manner, which is inhibited by the nonhydrolyzable GDP analog guanosine 5′-O-(β-thio)diphosphate (GDPβS). Down-regulation of protein kinase C by pretreatment with phorbol-12,13-dibutyrate does not affect enhancement by GTPγS of Ca2+-dependent MLC20 phosphorylation, indicating that protein kinase C is not involved in GTPγS-induced enhancement of MLC20 phosphorylation. Western blot analysis reveals that the smallmolecular-weight G protein rho p21 is abundant in aortic smooth muscle cells. Pretreatment of permeabilized cells with the botulinum exoenzyme C3, which specifically ADP-ribosylates and inactivates the rho family proteins, completely abolishes GTPγS-induced enhancement of Ca2+-dependent MLC20 phosphorylation. Under the same condition C3 completely ADP-ribosylates cellular proteins with a relative molecular weight of 21 kDa in permeabilized cells. C3 pretreatment of cells does not potentiate thiophosphorylation of MCL20 but decreases the rate of dephosphorylation of MLC20. These data suggest that rho p21 is involved in the GTPγS-induced Ca2+ sensitization of MLC20 phosphorylation in aortic smooth muscle cells, and that this effect of rho p21 is due to inhibition of protein phosphatase activity toward MLC20.

Repetitive cyclic stretch (60 cycles/min) of rat aortic smooth muscle cells dramatically enhances the effect of angiotensin II (AII) on mRNA expression of the vasorelaxant, parathyroid hormone-related peptide (PTHrP). Thus, combined stimulation of rat aortic smooth muscle cells by cyclic stretch and low concentrations of AII, but not either alone, induces a synergistic, marked increase in the PTHrP mRNA level, in a manner dependent on the strength of stretch. This response is accompanied by a synergistic increase in secretion of PTHrP from smooth muscle cells. Removal of extracellular Ca2+ or addition of Ca2+ channel blockers, including Gd3+ and nitrendipine, does not considerably reduce the combined effects of stretch and AII, indicating that this response is not dependent on stretch-induced Ca2+ influx across the plasma membrane. The combined effect of stretch and AII on PTHrP mRNA expression is strongly attenuated by the protein kinase C (PKC) inhibitor staurosporine or by down-regulation of PKC, suggesting that PKC plays an important role in the synergistic response. However, stretch neither elicits activation of phospholipase C or PKC by itself, nor does it enhance AII-induced activation of these enzymes. These results indicate that in vascular smooth muscle cells mechanical stretch acts together with the vasoconstrictor AII to regulate the expression of the vasodilator PTHrP and suggest the role of PTHrP as a local modulator of myogenic tone.

In dispersed rabbit adrenocortical glomerulosa cells, a non-peptide angiotensin II (AT 1 ) receptor antagonist, CV-11974 (10 −10 − 10 −5 M), competitively inhibited angiotensin II- or angiotensin III-stimulated aldosterone production, whereas PD123177, an angiotensin AT 2 receptor antagonist, did not. CV-11974 inhibited aldosterone production induced by 4 mM K + but not by 12 mM K + . CV-11974 had no effect on adrenocorticotropic hormone-stimulated aldosterone or corticosterone production, but inhibited angiotensin II-stimulated corticosterone production. In the rat, TCV-116, the prodrug of CV-11974, (0.1 and 1 mg/kg, p.o.) markedly reduced the elevation of both plasma aldosterone concentration and blood pressure induced by i.v. infusion of angiotensin II. In spontaneously hypertensive rats, TCV-116 at daily p.o. doses of 0.1, 1 and 10 mg/kg for 2 weeks caused a dose-dependent reduction of blood pressure and plasma aldosterone concentration without affecting plasma corticosterone. Thus, TCV-116 inhibited the induction of aldosterone production by not only exogenpus but also endogenous angiotensin II.

The angiotensin II (AII) antagonistic action of CV-11974 (2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl] benzimidazole-7-carboxylic acid) was investigated in an AII-receptor binding assay using rabbit aortic membranes and an AII-induced contraction assay using rabbit aortic strips. A single class of [125I]AII-(Sar1,Ile8) binding sites was found in the membranes with a dissociation constant (Kd) of 0.15 nM and a receptor concentration (Bmax) of 86.9 fmol/mg protein. CV-11974 markedly reduced Kd without affecting Bmax. The specific binding of [125I]AII-(Sar1,Ile8) in this preparation was inhibited completely by CV-11974 [the inhibition constant (Ki) = 0.64 nM], DuP 753 [an angiotensin II type I (AT1) receptor-selective antagonist] (Ki = 51 nM) and EXP3174 (an active metabolite of DuP 753) (Ki = 6.8 nM), but was not affected by PD123177 (an AT2 receptor-selective antagonist). These results suggest that the single binding site in rabbit aortic membranes is an AT1 receptor subtype. The affinity of CV-11974 to these AT1 receptors was approximately 80 and 10 times higher than that of DuP 753 and EXP3174, respectively. CV-11974 showed no appreciable affinity for the AT2 receptors found in bovine cerebellum. In the in vitro functional study, CV-11974 markedly reduced the AII-induced maximal contractile response of rabbit aortic strips (pD'2 = 9.97). In contrast, Compound 7-H, which lacks the carboxyl group at the benzimidazole ring of CV-11974, inhibited the contraction in a competitive manner. The inhibition by CV-11974 was long lasting. These results suggest that CV-11974 is a potent and long-acting AT1 receptor-selective, competitive antagonist. The carboxyl group at the benzimidazole ring plays an important role in the interaction between CV-11974 and the AT1 receptor.