The critical role played by the renin–angiotensin–aldosterone system (RAAS) in the regulation of blood pressure and body fluid homeostasis has been well recognized. Angiotensin (Ang) II and aldosterone are the most powerful biologically active products of the RAAS, although there are also other bioactive Ang peptides involved in this system, including AngIII, AngIV, and Ang1 – 7. In addition to their physiological roles, AngII and aldosterone induce inflammation, cell growth, mitogenesis, apoptosis, migration, and differentiation; regulate gene expression of bioactive substances; and activate multiple intracellular signaling pathways, all of which contribute to cardiovascular tissue injury. During the last decade, both clinical and preclinical studies have demonstrated that various pharmacological interventions of the RAAS exert blood pressure–independent cardiovascular-protective effects. In this Forum Minireview entitled “New approaches to blockade of the renin–angiotensin–aldosterone system”, we will discuss the impact of RAAS inhibitors on prevention of the development of hypertension and cardiovascular diseases. Before going into details about the insights into RAAS inhibition that each of the four groups will provide, we herein briefly overview our current understanding of regulation of the circulating RAAS.
Recent randomized controlled trials showed that blockade of the renin–angiotensin system (RAS) by angiotensin-converting enzyme (ACE) inhibitors and angiotensin II–receptor blockers (ARBs) reduced cardiovascular and renal events. These drugs are widely used in the management of cardiovascular and renal diseases. Results from Randomized Controlled Trials (RCTs) so far, however, also raise several questions to be addressed. It should be noted that the residual event rate in the treatment arm in outcome studies that have employed ACE inhibitors or ARBs remains high. Such insufficient efficacy of RAS inhibition may result from the fact that neither ACE inhibitors nor ARBs completely suppress activity of RAS. Since then effort has been made to determine whether the dual blockade of RAS could provide further improvement in cardiovascular and renal outcome. This review extracts unsolved questions in the treatment with RAS inhibitors from outcome studies and discusses them from the clinical pharmacological point of view.
Since renin inhibition interferes with the first and rate-limiting steps in the renin–angiotensin system, the renin step is a very attractive target for lowering blood pressure and minimizing target-organ damage. The newly developed direct renin inhibitor aliskiren has several attractive characteristics: it definitively reduces plasma renin activity among inhibitors of the renin–angiotensin system, is remarkably specific for human renin, exhibits a long half-life in plasma comparable to that of amlodipine, and has a high affinity for renal glomeruli and vasculature. Although these characteristics suggest the clinical usefulness and safety of aliskiren, several problems remain unsolved. Why does aliskiren have beneficial effects on the heart and kidneys of patients treated with angiotensin-converting enzyme (ACE) inhibitors and/or angiotensin II type 1–receptor blockers (ARBs)? Is the blood-pressure–lowering effect of aliskiren dependent on the plasma renin activity? Does aliskiren exert a possible adverse effect via (pro)renin receptor–dependent intracellular signals? Here, we review the characteristics and usefulness of aliskiren and discuss the current issues associated with this direct renin inhibitor.
Chymase plays a crucial role in angiotensin II formation in various tissues. Angiotensin II induces gene expression of transforming growth factor (TGF)-β and matrix metalloproteinase (MMP)-9 precursors, and chymase can convert precursors of TGF-β and MMP-9 to their active forms. In cultured fibroblasts, significant increases in cell growth and TGF-β levels were observed after chymase injection; these increases were inhibited by a chymase inhibitor, but not by an angiotensin II–receptor blocker. In apolipoprotein E–deficient mice, abdominal aortic aneurysm (AAA) development depends on an increase in MMP-9 activities induced by angiotensin II infusion, but the inhibition of MMP-9 activation by a chymase inhibitor resulted in attenuation of the angiotensin II–induced AAA development. The upregulation of MMP-9 and TGF-β levels is involved in damage to various organs, but these gene expressions are not completely induced by angiotensin II alone. Therefore, chymase inhibition may be useful for attenuating MMP-9 and TGF-β levels, in addition to reducing angiotensin II formation, and this function may provide powerful organ protection. In this review, we propose the possible use of chymase inhibitors as agents to prevent organ damage.
The role of angiotensin II in mediating hypertension and renal diseases is well documented, and inhibition of the renin–angiotensin–aldosterone system elicits antihypertensive and renoprotective effects. There is increasing evidence implicating aldosterone, in addition to angiotensin II, in the pathogenesis of hypertension and renal diseases. Beneficial effects of mineralocorticoid receptor (MR) blockers against these diseases have been reported and are independent of the effects exerted by renin–angiotensin system (RAS) inhibitors. MR blockers are increasingly being used, not only for primary aldosteronism but also for other resistant hypertensive patients whose blood pressure is insufficiently controlled by RAS inhibitors. In these settings, MR blockers have shown impressive results. In addition, anti-proteinuric effects of MR blockers have been observed in hypertensive patients treated with RAS inhibitors, but without significant effects on blood pressure. Interestingly, these effects of MR blockers are not always dependent on plasma aldosterone levels. These data suggest that MR blockers provide a potential therapeutic approach for patients with hypertension and renal impairment who are being treated with RAS inhibitors.
This study was conducted to determine the rate of P-glycoprotein (P-gp)–mediated efflux of digoxin analogues and metabolites and to assess the effects of macrolide antibiotics on this efflux. Bidirectional transport studies were conducted using our Caco-2 sub clone with high P-gp expression (CLEFF9). HPLC methods were employed to measure drug transport. All digoxin metabolites were P-gp substrates, although digoxin had the greatest efflux ratio. Erythromycin had no effect on the transport of digoxin, maintaining a basolateral to apical efflux ratio of 14.8, although it did reduce the efflux ratio of dihydrodigoxin and digoxigenin by 34% and 43%, respectively. Azithromycin also had little effect on the transport of digoxin or any of its metabolites. In contrast, clarithromycin and roxithromycin almost eliminated basolateral targeted efflux. Using paclitaxel as a known P-gp substrate, erythromycin demonstrated only partial P-gp inhibitory capacity, maintaining an efflux ratio over 100. In contrast, clarithromycin and roxithromycin were 10-fold greater P-gp inhibitors. Clarithromycin and roxithromycin are likely to exhibit drug interactions with digoxin via inhibition of efflux mechanisms. Azithromycin appears to have little influence on P-gp–mediated digoxin absorption or excretion and would be the safest macrolide to use concurrently with oral digoxin.
Diabetic cardiomyopathy and nephropathy induce endoplasmic reticulum stress (ERS) and ERS-initiated apoptosis. The primary function of 14-3-3 protein is to inhibit apoptosis, but the roles of this protein in protecting against cardiac ERS and apoptosis in the diabetic heart are largely unknown. In this study, we investigated the in vivo role of 14-3-3 protein in diabetic ERS and apoptosis using streptozotocin (STZ)-induced transgenic mice that showed cardiac-specific expression of a dominant negative (DN) 14-3-3η protein mutant. The expression levels of cardiac glucose-regulated protein (GRP) 78, inositol-requiring enzyme (Ire) 1α, and tumor necrosis factor receptor (TNFR)-associated factor (TRAF) 2 protein were significantly increased in the diabetic DN 14-3-3η mice compared with the diabetic wild-type. Moreover, cardiac apoptosis and the expression of CCAAT / enhancer binding protein homology protein (CHOP), caspase-12, and cleaved caspase-12 protein were significantly increased in the diabetic DN 14-3-3η mice. In conclusion, partial depletion of 14-3-3 protein in the diabetic heart exacerbates cardiac ERS and activates ERS-induced apoptosis pathways, at least in part, through the regulation of CHOP and caspase-12 via the Ire1α/TRAF2 pathway. The enhancement of 14-3-3 protein expression can be used as a novel protective therapy against ERS and ERS-initiated apoptosis in the diabetic heart.
Cardiac ischemia–reperfusion injury is evoked by reactive oxygen species (ROS). We previously reported that sulfaphenazole (SPZ) attenuated cardiac ROS levels and ischemia–reperfusion injury in rats. SPZ has distinct two actions: a) elimination of ROS and b) inhibition of cytochrome P450 (CYP) that is responsible for ROS production. The aim of this study is to determine which action contributes to the attenuation of cardiac ischemia–reperfusion injury using SPZ and its derivatives [acetyl-SPZ (Ac-SPZ) and dichloro-SPZ (2Cl-SPZ)]. Administration of 2Cl-SPZ or SPZ prior to ischemia significantly reduced myocardial infarct size, myocardial lipid peroxides, and ROS levels. In addition, they inhibited rat cardiac CYP activity. However, Ac-SPZ neither reduced infarct size nor inhibited cardiac CYP activity. The three compounds had similar effects on ROS scavenging activity in that they scarcely scavenged hydrogen peroxide and superoxide anions but reduced hydroxyl radicals with the same efficacy. The serum concentration of each compound was almost the same until 24 h after reperfusion. Collectively, our findings indicate that the suppressive effects of SPZ and 2Cl-SPZ on ischemia–reperfusion injury are associated with the reduction of ROS levels, which is primarily due to a decrease in ROS production via inhibition of cardiac CYP, not via ROS scavenging activity.
The activity of pyruvate dehydrogenase (PDH) is reduced in diabetic patients. Phosphorylation of the PDH E1α subunit by PDH kinase contributes to the suppression of PDH activity. PDH requires thiamine as a coenzyme. We investigated the exact mechanism of diabetes-induced PDH inhibition, and the effect of thiamine in both in vivo and in vitro experiments. Treatment of rats with thiamine significantly, although partially, recovered streptozotocin (STZ)-induced reductions in mitochondrial PDH activity. Nevertheless, we found that PDH E1α phosphorylation in the thiamine-treated STZ group was perfectly diminished to the same level as that in the control group. STZ treatment significantly caused enhancements of the expression of O-glycosylated protein in the rat hearts, which was decreased by thiamine repletion. Next, the rat cardiac fibroblasts (RCFs) were cultured in the presence of high glucose levels. Thiamine dramatically recovered high glucose–induced PDH inhibition. High glucose loads did not alter the phosphorylated PDH E1α. PDH inhibition in RCFs was not accompanied by an increase in the PDH E1α phosphorylation. The O-glycosylated protein was markedly increased in RCFs exposed to high glucose, which was inhibited by thiamine. These results suggest that thiamine ameliorates diabetes-induced PDH inhibition by suppressing the increased expression of the O-glycosylated protein. The O-glycosylation of PDH E1α may be involved in the regulation of the PDH activity.
Hypoxia-induced gene expression frequently occurs in malignant solid tumors because they often have hypoxic areas in which circulation is compromised due to structurally disorganized blood vessels. Hypoxia-response elements (HREs) are responsible for activating gene transcription in response to hypoxia. In this study, we constructed a hypoxia-response plasmid vector producing short hairpin RNA (shRNA) against B-cell leukemia/lymphoma-2 (bcl-2), an anti-apoptotic factor. The hypoxia-response promoter was made by inserting tandem repeats of HREs upstream of cytomegalovirus (CMV) promoter (HRE-CMV). HRE-CMV shbcl-2 vector consisted of bcl-2 shRNA under the control of HRE-CMV promoter. In hypoxic mouse rectum carcinoma cells (colon-26), the production of bcl-2 shRNA driven by HRE-CMV promoter was approximately 2-fold greater than that driven by CMV promoter. A single intratumoral (i.t.) injection of 40 μg HRE-CMV shbcl-2 to colon-26 tumor–bearing mice caused apoptotic cell death, and repetitive treatment with HRE-CMV shbcl-2 (40 μg/mouse, i.t.) also significantly suppressed the growth of colon-26 tumor cells implanted in mice. Apoptotic and anti-tumor effects were not observed in tumor-bearing mice treated with CMV shbcl-2. These results reveal the ability of HRE-CMV shbcl-2 vector to suppress the expression of bcl-2 in hypoxic tumor cells and suggest the usefulness of our constructed hypoxia-response plasmid vector to treat malignant tumors. [Supplementary Figures: available only at http://dx.doi.org/10.1254/jphs.10054FP]
Serotonin 2C receptor (5-HT2CR) mRNA receives editing at 5 nucleotide positions (sites A – E) located in the sequence encoding the second intracellular loop of 5-HT2CR. 5-HT2CR mRNA without editing and with editing at sites AB, ABD, ABC, ABCD, and C are translated to 6 isoforms of 5-HT2CR: INI(non-edited), VNI(AB), VNV(ABD), VSI(ABC), VSV(ABCD), and ISI(C), respectively. In this study, we investigated electrophysiologically the ability of these isoforms to couple with the G protein / phospholipase C (PLC) system using Xenopus oocytes injected with edited 5-HT2CR RNAs and muscarinic M1 receptor (M1R) RNA. The efficacy with which 5-HT stimulated each isoform was calculated by comparing 5-HT–induced current with 100 μM acetylcholine–induced M1R current. Stimulation with 5-HT of INI(non-edited), VNI(AB), VNV(ABD), VSI(ABC), VSV(ABCD), and ISI(C) expressed in Xenopus oocytes showed concentration-dependent responses with EC50 values of 8.6, 17.2, 76,5, 22.0, 91.2, and 20.3 nM, respectively. No significant difference in the ability of 5-HT to induce currents among the oocytes expressing these isoforms was detected, but in the oocytes expressing VSI(ABC) or VSV(ABCD), 5-HT had a significantly reduced ability to induce currents. These results suggest that editing at site C together with sites A and B and/or D markedly reduces 5-HT2CR function by generating isoforms with reduced ability to activate PLC.
Caulophine is a new fluorenone alkaloid isolated from the radix of Caulophyllum robustum MAXIM and identified as 3-(2-(dimethylamino) ethyl)-4,5-dihydroxy-1,6-dimethoxy-9H-fluoren-9-one. Due to its new chemical structure, the pharmacological activities of caulophine are not well characterized. The present study evaluated the protective effect and the primary mechanisms of caulophine on cardiomyocyte injury. Viability of cardiomyocytes was assayed with the MTT method, and cell apoptosis was detected by flow cytometry. Myocardial infarction was produced by ligating the coronary artery, and myocardial ischemia was induced by isoproterenol in rats. Myocardial infarction size was estimated with p-nitro-blue tetrazolium staining. Lactate dehydrogenase (LDH), creatine kinase (CK), superoxide dismutase (SOD), malondialdehyde (MDA), and free fatty acid (FFA) were spectrophotometrically determined. Histopathological and ultrastructural changes of ischemic myocardium were observed. The results showed that pretreatment with caulophine increased the viability of H2O2- and adriamycin-injured cardiomyocytes; decreased CK, LDH, and MDA; increased SOD; and inhibited H2O2-induced cellular apoptosis. Caulophine reduced myocardial infarct size and serum CK, LDH, FFA, and MDA; raised serum SOD; and improved histopathological and ultrastructural changes of ischemic myocardium. The results demonstrate that caulophine has the ability to protect cardiomyocytes from oxidative and ischemic injury through an antioxidative mechanism that provides a basis for further study and development of caulophine as a promising agent for treating coronary heart disease.
Recent evidence indicates that strychnine-sensitive glycine receptors are located in upper brain regions including the hippocampus. Because of excitatory effects of glycine via facilitation of NMDA-receptor function, however, the net effects of increased extracellular glycine on neuronal excitability in either physiological or pathophysiological conditions are mostly unclear. Here, we addressed the potential neuroprotective effect of either exogenous application of glycine and taurine, which are both strychnine-sensitive glycine-receptor agonists, or an endogenous increase of glycine via blockade of glycine transporter 1 (GlyT1) by assessing their ability to facilitate the functional recovery of field excitatory postsynaptic potentials (fEPSPs) after termination of brief oxygen/glucose deprivation (OGD) in the CA1 region in mouse hippocampal slices. Glycine and taurine promoted restoration of the fEPSPs after reperfusion, but this was never observed in the presence of strychnine. Interestingly, glycine and taurine appeared to generate neuroprotective effects only at their optimum concentration range. By contrast, blockade of GlyT1 by N-[3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)propyl]sarcosine or sarcosine did not elicit significant neuroprotection. These results suggest that activation of strychnine-sensitive glycine receptors potentially produces neuroprotection against metabolic stress such as OGD. However, GlyT1 inhibition is unlikely to elicit a sufficient increase in the extracellular level of glycine to generate neuroprotection.
We examined the effects of transforming growth factor β1 (TGFβ1) on cellular functions in human lung cancer cell line A549. Treatment of A549 cells with 1 ng/ml TGFβ1 for more than 3 days altered their morphology from an epithelial cobblestone-like appearance to a fibroblast-like one, reduced the expression of E-cadherin mRNA and protein, and induced the formation of F-actin fibers. These hallmarks indicate that TGFβ1 induced the epithelial–mesenchymal transition in A549 cells. Migration of TGFβ1-treated A549 cells, which was quantified by the wound-healing assay, was markedly accelerated by 3 μM ATPγS, a non-hydrolyzable ATP analogue. ATPγS-induced migration of TGFβ1-treated A549 cells was reversed by the P2 antagonist suramin. In contrast, migration of control A549 cells was not altered by ATPγS. TGFβ1-treated A549 cells showed an augmentation of ATP-induced Ca2+ transients, thapsigargin-induced Ca2+ transients, and store-operated Ca2+ entry compared with those in control cells. Basal level of the extracellular ATP concentration was significantly lower in TGFβ1-treated A549 cells than in control cells. We conclude from these results that TGFβ1 augments ATP-induced Ca2+ mobilization, which leads to the acceleration of migration, in A549 cells but, it markedly reduces endogenous ATP release. This implies that the actions of ATP would become a novel therapeutic target for inhibiting cancer cell migration.
Hyperlipidemia is a major risk factor for cardiovascular diseases. In this study, we investigated the potential effects of cordycepin (3′-deoxyadenosine), a bioactive component of the fungus Cordyceps militaris, on hyperlipidemia. We found that in male Syrian golden hamsters fed a high-fat diet (HFD), daily administration of cordycepin effectively reduced the accumulation of serum total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-c) and suppressed HFD-associated increases in relative retroperitoneal fat. It also increased the levels of phospho–AMP-activated protein kinase (AMPK) and phospho–acetyl-CoA carboxylase (phospho-ACC) in liver and retroperitoneal adipose tissues. In HepG2 cells, cordycepin stimulated robust concentration- and time-dependent AMPK activation that correlated with the activation of ACC and the suppression of lipid biosynthesis. However, pretreatment with compound C, a specific inhibitor of AMPK, substantially abolished the effects of cordycepin on AMPK activation and lipid biosynthesis inhibition. These results indicate that cordycepin prevents hyperlipidemia via activation of AMPK. Experiments on abnormal metabolic mice indicated that cordycepin can also improve insulin sensitivity effectively.
Parishin C, a major component of Gastrodia elata BLUME (GE), was purified from GE. Because GE modulates the serotonergic system and the 5-HT1A receptor is an important therapeutic target of schizophrenia, we examined whether parishin C affects phencyclidine-induced abnormal behaviors in mice. Phencyclidine-induced abnormal behaviors were significantly ameliorated by parishin C. These effects were reversed by WAY 100635, a 5HT1A–receptor antagonist. Consistently, parishin C showed high affinity at 5-HT1A receptor as well as a 5-HT1A–agonist activity in a 8-OH-DPAT–stimulated [35S]GTP-γS binding assay. Our results suggest that the antipsychotic effects of parishin C require activation of 5-HT1A receptors.
Our previous results suggested that ruscogenin inhibited tumor necrosis factor α (TNF-α)–induced leukocyte adhesion, which correlated with its suppression of intercellular adhesion molecule-1 (ICAM-1) expression in endothelial cells. In the present studies, we further examined its effects on the main signaling pathways involved in upregulation of ICAM-1 induced by TNF-α in human umbilical vein endothelial cells (HUVECs). The results showed that ruscogenin significantly suppressed p65 phosphorylation, IκB-α phosphorylation and degradation, and inhibited IκB kinase α (IKKα) and IKKβ activation induced by TNF-α. However, it exerted weak effects on TNF-α–induced phosphorylations of p38, JNK, ERK1/2, and Akt. Overall, our results indicated that downregulation of ICAM-1 expression by ruscogenin in HUVECs might be mediated by nuclear factor-κB (NF-κB), but not by mitogen-activated protein kinase (MAPK) and Akt signaling pathways.
A chemoattractant lectin from the dorsal spines of the redfin velvetfish, Hypodytes rubripinnis, was isolated using a combination of affinity chromatography techniques. The glycoprotein, with a molecular mass of 110 kDa, is named Karatoxin. Karatoxin caused agglutination of rabbit erythrocytes. This agglutination was effectively inhibited by D-mannose. In addition, Karatoxin exhibited not only mitogenic activity in the presence of murine splenocytes, but also chemotactic activity in the presence of guinea-pig neutrophils and macrophages. Thus, Karatoxin appears to be a novel chemoattractant lectin. These results suggest that the redfin velvetfish Hypodytes rubripinnis may be a novel source of biologically active substances.