Optimization of a novel series of N-phenylindoline-5-sulfonamide-based acyl CoA:monoacylglycerol acyltransferase-2 inhibitors: Mitigation of CYP3A4 time-dependent inhibition and phototoxic liabilities.

Acyl CoA:monoacylglycerol acyltransferase-2 (MGAT2) has emerged as a potential peripheral target for the treatment of obesity and metabolic disorders. We previously identified a novel series of N-phenylindoline-5-sulfonamide derivatives exemplified by 2 as potent and orally bioavailable MGAT2 inhibitors. Despite its attractive potency, further assessment revealed that this compound exhibited time-dependent inhibition (TDI) of cytochrome P450 3A4 (CYP3A4). To remove the undesirable CYP3A4 TDI activity, structural modification was focused on the 2,4-difluoroaniline moiety on the basis of the assumption that this moiety would be involved in mechanism-based inhibition of CYP3A4 via oxidative metabolism. This led to the finding that the introduction of 4-chloro-2,6-difluoroaniline significantly improved CYP3A4 TDI risk. Further optimization resulted in the discovery of N-(4-chloro-2,6-difluorophenyl)-1-{5-[1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl]pyrimidin-2-yl}-7-(2-oxopyrrolidin-1-yl)-2,3-dihydro-1H-indole-5-sulfonamide (27c) with potent MGAT2 inhibitory activity (IC50=7.8 nM) and excellent ADME-Tox profiles including metabolic stability, oral bioavailability, and CYP3A4 TDI. In a mouse oral fat tolerance test, compound 27c effectively and dose-dependently suppressed the elevation of plasma triacylglycerol levels after oral administration at doses of 1 and 3mg/kg. We also discuss mitigation of the phototoxic liability of biaryl derivatives on the basis of the HOMO-LUMO gap hypothesis during the course of optimization efforts.

Targeted disruption of AdipoR1 and AdipoR2 causes abrogation of adiponectin binding and metabolic actions.

Adiponectin plays a central role as an antidiabetic and antiatherogenic adipokine. AdipoR1 and AdipoR2 serve as receptors for adiponectin in vitro, and their reduction in obesity seems to be correlated with reduced adiponectin sensitivity. Here we show that adenovirus-mediated expression of AdipoR1 and R2 in the liver of Lepr(-/-) mice increased AMP-activated protein kinase (AMPK) activation and peroxisome proliferator-activated receptor (PPAR)-alpha signaling pathways, respectively. Activation of AMPK reduced gluconeogenesis, whereas expression of the receptors in both cases increased fatty acid oxidation and lead to an amelioration of diabetes. Alternatively, targeted disruption of AdipoR1 resulted in the abrogation of adiponectin-induced AMPK activation, whereas that of AdipoR2 resulted in decreased activity of PPAR-alpha signaling pathways. Simultaneous disruption of both AdipoR1 and R2 abolished adiponectin binding and actions, resulting in increased tissue triglyceride content, inflammation and oxidative stress, and thus leading to insulin resistance and marked glucose intolerance. Therefore, AdipoR1 and R2 serve as the predominant receptors for adiponectin in vivo and play important roles in the regulation of glucose and lipid metabolism, inflammation and oxidative stress in vivo.

Inhibition of MGAT2 modulates fat-induced gut peptide release and fat intake in normal mice and ameliorates obesity and diabetes in ob/ob mice fed on a high-fat diet.

Monoacylglycerol O-acyltransferase 2 (MGAT2) is one of the key enzymes responsible for triglyceride (TG) re-synthesis in the small intestine. We have previously demonstrated that pharmacological inhibition of MGAT2 has beneficial effects on obesity and metabolic disorders in mice. Here, we further investigate the effects of MGAT2 inhibition on (a) fat-induced gut peptide release and fat intake in normal mice and (b) metabolic disorders in high-fat diet (HFD)-fed ob/ob mice, a model of severe obesity and type 2 diabetes mellitus, using an orally bioavailable MGAT2 inhibitor Compound B (CpdB). CpdB inhibited elevation of plasma TG in mice challenged with an oil-supplemented liquid meal. Oil challenge stimulated the secretion of two gut anorectic hormones (peptide tyrosine-tyrosine and glucagon-like peptide-1) into the bloodstream, and these responses were augmented in mice pretreated with CpdB. In a two-choice test using an HFD and a low-fat diet, CpdB selectively inhibited intake of the HFD in normal mice. Administration of CpdB to HFD-fed ob/ob mice for 5 weeks suppressed food intake and body weight gain and inhibited elevation of glycated hemoglobin. These results indicate that pharmacological MGAT2 inhibition modulates fat-induced gut peptide release and fat intake in normal mice and improves obesity and diabetes in HFD-fed ob/ob mice and thus may have potential for development into a treatment of obesity and its related metabolic diseases.

Effects of tanshinone VI on phosphorylation of ERK and Akt in isolated cardiomyocytes and cardiac fibroblasts.

Effects of tanshinone VI, a diterpene from Tan-Shen, on humoral factor-induced phosphorylation of ERK and Akt during hypertrophy of cardiomyocytes and fibrosis of cardiac fibroblasts isolated from neonatal rats were examined. Treatment of cultured cardiomyocytes with 10 nM insulin-like growth factor-1 (IGF-1) or 10 nM endothelin-1 resulted in an increase in leucine incorporation into acid-insoluble fraction. Treatment of cultured cardiac fibroblasts with 10 nM IGF-1 or 10 nM angiotensin II increased incorporation of proline. IGF-1 increased phosphorylated extracellular signal-regulated kinase (pERK) and protein kinase B (pAkt) of cardiomyocytes, whereas endothelin-1 increased pERK, but not pAkt. Treatment of cardiac fibroblasts with 10 nM IGF-1 or 10 nM angiotensin II also increased pERK, whereas pAkt was increased by treatment with IGF-1 alone. When the cardiomyocytes were incubated in the presence of 10 microM tanshinone VI, IGF-1- and endothelin-1-induced increases in pERK, but not pAkt, were partially attenuated. Treatment of cardiac fibroblasts with 10 microM tanshinone VI also attenuated IGF-1-induced increases in pERK and pAkt. Tanshinone VI also partially attenuated angiotensin II-induced increase in proline incorporation into cardiac fibroblasts. PD98059, an inhibitor for phosphorylation of extracellular signal-regulated kinase (ERK), but not wortmannin, that for protein kinase B phosphorylation, attenuated an increase in leucine incorporation into cardiomyocytes in the presence of either IGF-1 or endothelin-1. These results suggest that tanshinone VI is a possible agent that can attenuate the humoral factor-induced hypertrophy of cardiomyocytes and fibrosis of cardiac fibroblasts via an attenuation of ERK phosphorylation in these cells.

Insulin receptor substrate 2 plays a crucial role in beta cells and the hypothalamus.

We previously demonstrated that insulin receptor substrate 2 (Irs2) KO mice develop diabetes associated with hepatic insulin resistance, lack of compensatory beta cell hyperplasia, and leptin resistance. To more precisely determine the roles of Irs2 in beta cells and the hypothalamus, we generated beta cell-specific Irs2 KO and hypothalamus-specific Irs2 knockdown (betaHT-IRS2) mice. Expression of Irs2 mRNA was reduced by approximately 90% in pancreatic islets and was markedly reduced in the arcuate nucleus of the hypothalamus. By contrast, Irs2 expression in liver, muscle, and adipose tissue of betaHT-IRS2 mice was indistinguishable from that of control mice. The betaHT-IRS2 mice displayed obesity and leptin resistance. At 4 weeks of age, the betaHT-IRS2 mice showed normal insulin sensitivity, but at 8 and 12 weeks, they were insulin resistant with progressive obesity. Despite their normal insulin sensitivity at 8 weeks with caloric restriction, the betaHT-IRS2 mice exhibited glucose intolerance and impaired glucose-induced insulin secretion. beta Cell mass and beta cell proliferation in the betaHT-IRS2 mice were reduced significantly at 8 and 12 weeks but not at 10 days. Insulin secretion, normalized by cell number per islet, was significantly increased at high glucose concentrations in the betaHT-IRS2 mice. We conclude that, in beta cells and the hypothalamus, Irs2 is crucially involved in the regulation of beta cell mass and leptin sensitivity.

Peroxisome proliferator-activated receptor (PPAR)alpha activation increases adiponectin receptors and reduces obesity-related inflammation in adipose tissue: comparison of activation of PPARalpha, PPARgamma, and their combination.

We examined the effects of activation of peroxisome proliferator-activated receptor (PPAR)alpha, PPARgamma, and both of them in combination in obese diabetic KKAy mice and investigated the mechanisms by which they improve insulin sensitivity. PPARalpha activation by its agonist, Wy-14,643, as well as PPARgamma activation by its agonist, rosiglitazone, markedly improved insulin sensitivity. Interestingly, dual activation of PPARalpha and -gamma by a combination of Wy-14,643 and rosiglitazone showed increased efficacy. Adipocyte size in Wy-14,643-treated KKAy mice was much smaller than that of vehicle- or rosiglitazone-treated mice, suggesting that activation of PPARalpha prevents adipocyte hypertrophy. Moreover, Wy-14,643 treatment reduced inflammation and the expression of macrophage-specific genes in white adipose tissue (WAT). Importantly, Wy-14,643 treatment upregulated expression of the adiponectin receptor (AdipoR)-1 and AdipoR2 in WAT, which was decreased in WAT of KKAy mice compared with that in nondiabetic control mice. Furthermore, Wy-14,643 directly increased expression of AdipoRs and decreased monocyte chemoattractant protein-1 expression in adipocytes and macrophages. Rosiglitazone increased serum adiponectin concentrations and the ratio of high molecular weight multimers of adiponectin to total adiponectin. A combination of rosiglitazone and Wy-14,643 increased both serum adiponectin concentrations and AdipoR expression in WAT. These data suggest that PPARalpha activation prevents inflammation in WAT and that dual activation of PPARalpha and -gamma enhances the action of adiponectin by increasing both adiponectin and AdipoRs, which can result in the amelioration of obesity-induced insulin resistance.

Pharmacological Inhibition of Monoacylglycerol O-Acyltransferase 2 Improves Hyperlipidemia, Obesity, and Diabetes by Change in Intestinal Fat Utilization.

Monoacylglycerol O-acyltransferase 2 (MGAT2) catalyzes the synthesis of diacylglycerol (DG), a triacylglycerol precursor and potential peripheral target for novel anti-obesity therapeutics. High-throughput screening identified lead compounds with MGAT2 inhibitory activity. Through structural modification, a potent, selective, and orally bioavailable MGAT2 inhibitor, compound A (compA), was discovered. CompA dose-dependently inhibited postprandial increases in plasma triglyceride (TG) levels. Metabolic flux analysis revealed that compA inhibited triglyceride/diacylglycerol resynthesis in the small intestine and increased free fatty acid and acyl-carnitine with shorter acyl chains than originally labelled fatty acid. CompA decreased high-fat diet (HFD) intake in C57BL/6J mice. MGAT2-null mice showed a similar phenotype as compA-treated mice and compA did not suppress a food intake in MGAT2 KO mice, indicating that the anorectic effects were dependent on MGAT2 inhibition. Chronic administration of compA significantly prevented body weight gain and fat accumulation in mice fed HFD. MGAT2 inhibition by CompA under severe diabetes ameliorated hyperglycemia and fatty liver in HFD-streptozotocin (STZ)-treated mice. Homeostatic model assessments (HOMA-IR) revealed that compA treatment significantly improved insulin sensitivity. The proximal half of the small intestine displayed weight gain following compA treatment. A similar phenomenon has been observed in Roux-en-Y gastric bypass-treated animals and some studies have reported that this intestinal remodeling is essential to the anti-diabetic effects of bariatric surgery. These results clearly demonstrated that MGAT2 inhibition improved dyslipidemia, obesity, and diabetes, suggesting that compA is an effective therapeutic for obesity-related metabolic disorders.

Production and autocrine/paracrine effects of endogenous insulin-like growth factor-1 in rat cardiac fibroblasts.

Insulin-like growth factor (IGF)-1 appears to play an important role in cardiac hypertrophy or remodeling. However, the role of endogenous IGF-1 in the growth of cardiac myocytes and fibroblasts remains unclear. This study investigated the major site of the production of cardiac IGF-1 and the local effects of endogenous IGF-1 secreted from cardiac cells. A significant expression of IGF-1 mRNA was found in cultured neonatal and adult rat cardiac fibroblasts, but not in myocytes. In addition, an in vivo examination by in situ hybridization histochemical analyses demonstrated the IGF-1 transcripts in the interstitial fibrotic tissue of the ventricle. Time-dependent secretion of IGF-1 protein was also observed in cultured cardiac fibroblasts. An antibody against IGF-1 decreased collagen synthesis in cardiac fibroblasts under basal conditions. Fibroblast-conditioned medium, as well as exogenous IGF-1, increased protein synthesis in cardiac myocytes, and this increase was inhibited by antibodies against IGF-1 and IGF-1 receptor, IGF binding protein-3, and IGF-1 receptor antagonist. These observations suggest that IGF-1 is produced and released mainly from cardiac fibroblasts and that endogenous IGF-1 promotes collagen synthesis by cardiac fibroblasts and hypertrophy of myocytes as an autocrine and a paracrine factor. Cardiac IGF-1 may function as an endogenous modulator of cardiac hypertrophy or remodeling.

Retrograde degeneration of the corticospinal tract associated with pontine infarction.

We report a 40-year-old man with myotonic dystrophy who survived for 5 years after pontine infarction. Serial MRI detected abnormal T2 elongation of the corticospinal tract at the cerebral peduncle 4 years after the infarction. An autopsy confirmed the existence of retrograde degeneration extending from the pons to the internal capsule, evidence that retrograde degeneration in the corticospinal tract occurs above the pontine level. MRI was suggested to be useful for detection of the degenerative process.

Discovery of a Novel Series of N-Phenylindoline-5-sulfonamide Derivatives as Potent, Selective, and Orally Bioavailable Acyl CoA:Monoacylglycerol Acyltransferase-2 Inhibitors.

Acyl CoA:monoacylglycerol acyltransferase-2 (MGAT2) has attracted interest as a novel target for the treatment of obesity and metabolic diseases. Starting from N-phenylbenzenesulfonamide derivative 1 with moderate potency for MGAT2 inhibition, we explored an effective location of the hydrophobic group at the 1-position to enhance MGAT2 inhibitory activity. Shifting the hydrophobic group to the adjacent position followed by introduction of a bicyclic central core to restrict the substituent orientation produced N-phenylindoline-5-sulfonamide derivative 10b, which displayed much improved potency, with an IC50 value of 1.0 nM. This compound also exhibited excellent selectivity (greater than 30,000-fold) against related acyltransferases (MGAT3, DGAT1, DGAT2, and ACAT1). Subsequent optimization efforts were directed toward improving pharmacokinetic profiles, which resulted in the identification of 5-[(2,4-difluorophenyl)sulfamoyl]-7-(2-oxopyrrolidin-1-yl)-N-[4-(trifluoromethyl)phenyl]-2,3-dihydro-1H-indole-1-carboxamide (24d) endowed with potent MGAT2 inhibitory activity (IC50 = 3.4 nM) and high oral bioavailability (F = 52%, mouse). In a mouse oral fat tolerance test, oral administration of this compound effectively suppressed the elevation of plasma triacylglycerol levels.

Gene expression, secretion, and autocrine action of C-type natriuretic peptide in cultured adult rat cardiac fibroblasts.

C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, is known to be synthesized in the central nervous system and vascular endothelial cells, in contrast to atrial natriuretic peptide and brain natriuretic peptide. However, there have been no studies concerning CNP production in cultured cardiac cells. Here, we examined the production and the local effect of CNP in cultured ventricular cells. Under serum-free conditions, adult rat cardiac fibroblasts secreted immunoreactive CNP time dependently. TGF-beta1, basic fibroblast growth factor, and endothelin-1 significantly stimulated CNP secretion. Northern blot analysis detected significant expressions of CNP and its specific receptor (guanylyl cyclase-B) mRNA in cardiac fibroblasts. CNP stimulated intracellular cGMP production in fibroblasts more intensely than atrial and brain natriuretic peptides. CNP inhibited both DNA and collagen syntheses of cardiac fibroblasts, and these inhibitory effects by CNP were stronger than by atrial and brain natriuretic peptides. The inhibition by CNP of DNA and collagen syntheses was reproduced by a cGMP analog, 8-bromo cGMP. The present findings demonstrate that CNP is synthesized in and secreted from cardiac fibroblasts and suggest that CNP has a suppressive effect on fibroblast proliferation and extracellular matrix production, probably via the guanylyl cyclase-B-mediated cGMP-dependent process. CNP produced by cardiac fibroblasts may play a role as an autocrine regulator against excessive cardiac fibrosis.

Utilization behavior as a white matter disconnection syndrome.

We report a case of utilization behavior that was examined neuropathologically. A 72-year-old right-handed male patient, who was admitted with a complaint of transient loss of consciousness, displayed utilization behavior several times. He used daily objects that were placed in front of him, such as a teacup and a toothbrush, without instructions to do so. If the examiner asked the patient not to use the objects, the patient did not use them. MRI revealed acute infarction of the left superior frontal gyrus, where decreased blood flow was revealed by SPECT. The patient died of an acute worsening of dilated cardiomyopathy. Neuropathological examination demonstrated an acute phase infarction of the subcortical white matter of the left superior frontal lobe, which correlated well with neuroradiological findings. Utilization behavior has been thought a "frontal lobe symptom". However, we propose that utilization behavior might be considered a white matter disconnection syndrome.

Effects of tanshinone VI on insulin-like growth factor-1-induced hypertrophy of isolated cardiomyocytes from neonatal rats.

The effects of tanshinone VI (Tan), a diterpene extracted from Salvia miltiorrhiza, on insulin-like growth factor-1 (IGF-1)-induced hypertrophy of cardiomyocytes were examined. Cultured cardiomyocytes were isolated from neonatal rat hearts. The incorporation of [(3)H]-leucine into the trichloroacetic acid (TCA)-insoluble fraction was measured as a marker of protein synthesis, which revealed cardiomyocyte hypertrophy. Various concentrations of IGF-1, ranging from 0.1 nM to 10 nM, increased [(3)H]-leucine incorporation into the TCA-insoluble fraction of cardiomyocytes in a dose-dependent manner. IGF-1 induced an increase in phosphorylated extracellular signal-regulated kinase 1/2 (ERK), but did not change ERK protein content in cardiomyocytes. When the cells were incubated in the presence of Tan (0.1 muM to 10 muM), [(3)H]-leucine incorporation into IGF-1-untreated cells was unaltered. When the cells were incubated with 10 muM Tan, IGF-1-induced increases in [(3)H]-leucine incorporation into the TCA-insoluble fraction and phosphorylated ERK were attenuated. These results suggest that Tan is a possible agent for the suppression of IGF-1-induced hypertrophy of cardiomyocytes via an attenuation of ERK activation.

Clinical symptoms of bilateral anterior cerebral artery territory infarction.

Bilateral anterior cerebral artery (ACA) territory infarction is rare and its associated symptoms are still not well understood. We evaluated the clinical symptoms of four patients with bilateral ACA infarction. The common clinical features were various primitive reflexes and parkinsonian symptoms including akinesia, rigidity and hypophonia. Frontal release signs were present in all patients with ACA infarction even without direct involvement of the prefrontal cortex. Enhanced glabellar tap response, previously not reported in patients with ACA infarction, was the most consistent finding, and electrophysiological studies confirmed hyperexcitability of the late component of the blink reflex. The bilateral lesions in the deep white matter may be associated with the observed symptoms, reflecting functional disconnection of the medial prefrontal-subcortical circuitry.

Cortical propagation of Creutzfeldt-Jakob disease with codon 180 mutation.

A patient with Creutzfeldt-Jakob disease (CJD) with prion protein (PrP) gene codon 180 mutation (CJD 180) experienced cognitive decline over the 1.5-year period before her death. Serial magnetic resonance imaging (MRI) studies tracked stepwise propagation of cortical abnormal swelling and T2 elongations. On postmortem examination, the cortical areas affected by CJD for relatively short periods were associated with mild spongiform changes with the number of neurons being largely preserved. The residual neurons in these areas exhibited vacuole-like dilatation of their cell body. In contrast, the atrophic cortical areas affected by CJD for long periods exhibited predominant gemistocytic astrocytosis with severe neuronal loss. The present report depicts the unique cortical propagation of CJD 180 with corresponding radiological and pathological findings. Axonal transport through corticocortical connections might underlie the disease's propagation. MRI appeared to be useful for discriminating between different pathological states and tracking the progression of CJD 180.

Adiponectin inhibits the growth and peritoneal metastasis of gastric cancer through its specific membrane receptors AdipoR1 and AdipoR2.

Adiponectin, a circulating peptide hormone produced in adipose tissue, has been shown to be reduced in the plasma of patients with cancer, suggesting that this adipokine may be mechanically involved in the pathogenesis of adiposity-related carcinogenesis. In this study, we examined the expression of adiponectin receptors (AdipoR1 and AdipoR2) and assessed the function of adiponectin in gastric cancer. All of the six gastric cancer cell lines significantly expressed mRNA and protein of both receptors with variable levels. Addition of 30 microg/mL adiponectin potently induced apoptosis and inhibited the proliferation of AZ521 and HCG27. Down-regulation of either AdipoR1 or AdipoR2 by specific siRNA significantly suppressed the growth inhibitory effects of adiponectin in both cell lines. Moreover, a local injection of adiponectin markedly inhibited the growth of AZ521 inoculated subcutaneously in nude mice. Similarly, the continuous intraperitoneal infusion of adiponectin effectively suppressed the development of peritoneal metastasis of AZ521. Adiponectin negatively regulates the progression of gastric cancer cells possibly through both AdipoR1 and AdipoR2. Although adiponectin was already reported to have antiangiogenic effects, our results suggest that the antitumor effect of adiponectin was, at least partially, dependent on the direct effects on tumor cells.

Disturbance-free high-speed sinusoidal phase-modulating laser diode interferometer.

A surface profiler that incorporates a feedback controller to eliminate external disturbances is proposed and demonstrated. Its overall performance is dependent on the frequency response of the feedback loop. The frequency of the modulating signal strongly influences the response of the feedback controller. When we used the integrating-bucket method, the CCD camera had to be operated at a low-frequency video rate. Our technique uses a CCD camera equipped with an electronic shutter. The shutter function enables us to apply high-speed sinusoidal phase modulation to the conventional integrating-bucket method under the standard video rate.

Beneficial effects of sampatrilat, a novel vasopeptidase inhibitor, on cardiac remodeling and function of rats with chronic heart failure following left coronary artery ligation.

Sampatrilat is a novel vasopeptidase inhibitor that may offer a greater benefit than traditional angiotensin-converting enzyme (ACE) inhibitors in the treatment of chronic heart failure (CHF). The present study was undertaken to determine whether sampatrilat improves hemodynamic function and cardiac remodeling through a direct action on the failing heart in rats with CHF following left coronary artery ligation (CAL). Sampatrilat (30 mg/kg a day) was administered orally to the animals from the 1st to 6th week after the operation. Sampatrilat reduced the mortality of the rats with CAL (20 versus 57% for untreated rats). Treatment with sampatrilat for 5 weeks suppressed tissue ACE and neutral endopeptidase (NEP) activities. Sampatrilat did not affect the arterial blood pressure, whereas it attenuated the CAL-induced increases in the left ventricular end-diastolic pressure, heart weight, and collagen content of the viable left ventricle. To assess the direct effects of sampatrilat on collagen synthesis, we measured the incorporation of [(3)H]proline into cultured cardiac fibroblasts. Sampatrilat at concentrations that inhibited NEP activity in vitro augmented the atrial natriuretic peptide-induced decrease in [(3)H]proline incorporation by the cells. In addition, sampatrilat prevented the angiotensin I-induced increase in [(3)H]proline incorporation, whereas captopril did not. The results suggest that long-term treatment with sampatrilat regresses cardiac remodeling in rats with CAL, which is associated with improvement of hemodynamic function. The mechanism by which sampatrilat improved cardiac remodeling may be attributable to the direct inhibition of cardiac fibrosis, possibly acting through the cardiac natriuretic peptide system.

Effects of tanshinone VI on the hypertrophy of cardiac myocytes and fibrosis of cardiac fibroblasts of neonatal rats.

The possible effects of tanshinone VI (tsh), a diterpene from the root of Tan-Shen (Salvia miltiorrhiza, Bunge (Labiatae)) on hypertrophy and fibrosis in cultured neonatal rat cardiac myocytes and fibroblasts were examined. Tsh had no significant effect on protein synthesis, which was evaluated by [3H]-leucine incorporation into the acid insoluble fraction in the cells, in the absence of stimulatory factors in cardiac myocytes. The amount of protein produced in cardiac myocytes was increased by 10(-8) M endothelin-1 (ET-1), 10(-6) M phenylephrine (PE), or 10(-8) M insulin-like growth factor-1 (IGF-1), suggesting that hypertrophy of cardiac myocytes in vitro was induced by these factors. The ET-1-, PE-, or IGF-1-induced increase in protein synthesis was attenuated by treatment with 10(-5) M tsh. Treatment with 10(-5) M tsh significantly decreased the synthesis of collagen by cardiac fibroblasts, which was evaluated by [3H]-proline incorpolation into acid-insoluble fraction of the fiblobrasts, in the absence of stimulatory factors for the production. Fetal bovine serum (FBS) or IGF-1 increased collagen synthesis in a concentration-dependent manner. The increase at 5% FBS or 10(-8) M IGF-1 was inhibited by 10(-5) M tsh. Fibroblast-conditioned medium (FB-CM) increased protein synthesis in cardiac myocytes in a concentration-dependent manner (10; - 100 %). Tsh attenuated the FB-CM-induced increase in protein synthesis by cardiac myocytes. These results show that tsh may attenuate the humoral factor-induced hypertrophy of cardiac myocytes and fibrosis of cardiac fibroblasts. The findings suggest that tsh may improve the development of cardiac remodeling under pathophysiological conditions. Abbreviations. ANP:atrial natriuretic peptide DMEM:Dulbecco-modified Eagle's medium ET-1:endothelin-1 FB-CM:fibroblast-conditioned medium FBS:fetal bovine serum IGF-1:insulin-like growth factor-1 PE:phenylephrine tsh:tanshinone VI

Direct inhibition of neutral endopeptidase in vasopeptidase inhibitor-mediated amelioration of cardiac remodeling in rats with chronic heart failure.

Vasopeptidase inhibitors possess dual inhibitory actions on neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE) and have beneficial effects on cardiac remodeling. However, the contribution of NEP inhibition to their effects is not yet fully understood. To address the role of cardiac NEP inhibition in the anti-remodeling effects of a vasopeptidase inhibitor, we examined the effects of omapatrilat on the development of cardiac remodeling in rats with left coronary artery ligation (CAL) and those on collagen synthesis in cultured fibroblast cells. In vivo treatment with omapatrilat (30 mg/kg/day for 5 weeks) inhibited cardiac NEP activity in rats with CAL, which was associated with a suppression of both cardiac hypertrophy and collagen deposition. In cultured cardiac fibroblasts, omapatrilat (10(-7) to approximately 10(-5) M) inhibited NEP activity and augmented the ANP-induced decrease in [3H]-proline incorporation. ONO-BB, an active metabolite of the NEP selective inhibitor ONO-9902, also augmented the ANP-induced response, whereas captopril, an ACE inhibitor, did not. The angiotensin I-induced increase in [3H]-proline incorporation was prevented by omapatrilat and captopril, but not by ONO-BB. The results suggest that vasopeptidase inhibitor suppressed cardiac remodeling in the setting of chronic heart failure, possibly acting through the direct inhibition of cardiac NEP. Vasopeptidase inhibitors may have therapeutic advantages over the classical ACE and NEP inhibitors alone with respect to the regression of cardiac fibrosis.