The signaling pathway of nitric oxide (NO) depends mainly on guanosine 3',5'-cyclic monophosphate (cGMP). Here we report the formation and chemical biology of a nitrated derivative of cGMP, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), in NO-mediated signal transduction. Immunocytochemistry demonstrated marked 8-nitro-cGMP production in various cultured cells in an NO-dependent manner. This finding was confirmed by HPLC plus electrochemical detection and tandem mass spectrometry. 8-Nitro-cGMP activated cGMP-dependent protein kinase and showed unique redox-active properties independent of cGMP activity. Formation of protein Cys-cGMP adducts by 8-nitro-cGMP was identified as a new post-translational modification, which we call protein S-guanylation. 8-Nitro-cGMP seems to regulate the redox-sensor signaling protein Keap1, via S-guanylation of the highly nucleophilic cysteine sulfhydryls of Keap1. This study reveals 8-nitro-cGMP to be a second messenger of NO and sheds light on new areas of the physiology and chemical biology of signal transduction by NO.

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The purpose of this work was to test whether brain-penetrating angiotensin-converting enzyme (ACE) inhibitors (e.g., perindopril), as opposed to non-brain-penetrating ACE inhibitors (e.g., enalapril and imidapril), may reduce the cognitive decline and brain injury in Alzheimer's disease (AD). We first compared the effect of perindopril, enalapril, and imidapril on cognitive impairment and brain injury in a mouse model of AD induced by intracerebroventricular (i.c.v.) injection of amyloid-β (Aβ)₁₋₄₀. Perindopril, with significant inhibition of hippocampal ACE, significantly prevented cognitive impairment in this AD mouse model. This beneficial effect was attributed to the suppression of microglia/astrocyte activation and the attenuation of oxidative stress caused by iNOS induction and extracellular superoxide dismutase down-regulation. In contrast, neither enalapril nor imidapril prevented cognitive impairment and brain injury in this AD mouse. We next examined the protective effects of perindopril on cognitive impairment in PS2APP-transgenic mice overexpressing Aβ in the brain. Perindopril, without affecting brain Aβ deposition, significantly suppressed the increase in hippocampal ACE activity and improved cognition in PS2APP-transgenic mice, being associated with the suppression of hippocampal astrocyte activation and attenuation of superoxide. Our data demonstrated that the brain-penetrating ACE inhibitor perindopril, as compared to non-brain-penetrating ACE inhibitors, protected against cognitive impairment and brain injury in experimental AD models.

Abstract-Dietary obesity is associated with type 2 diabetes and cardiovascular diseases, although the underlying mechanism is unknown. This study was undertaken to elucidate the role of angiotensin II and apoptosis signal regulating kinase-1 (ASK1) in obesity/diabetes-associated cardiovascular complications and hepatic steatosis. Mice fed a high-fat diet were treated with olmesartan, an angiotensin II type 1 receptor blocker, to elucidate the role of angiotensin II in diabetic mice. Treatment of mice fed a high-fat diet with olmesartan markedly suppressed cardiac inflammation and fibrosis, as well as vascular endothelial dysfunction and remodeling, induced by obesity/diabetes. Moreover, olmesartan suppressed the disruption of the vascular endothelial NO synthase dimer in diabetic mice. Olmesartan also significantly prevented hepatic steatosis and fibrosis in diabetic mice. These beneficial effects of olmesartan on diabetic mice were associated with the attenuation of ASK1 activation in these mice. ASK1-deficient mice and wild-type mice were compared, regarding the effects of a high-fat diet. A comparison between ASK1-deficient and wild-type mice showed that ASK1 deficiency attenuated cardiac inflammation and fibrosis, as well as vascular endothelial dysfunction and remodeling induced by obesity/diabetes. The amelioration of vascular endothelial impairment by ASK1 deficiency was attributed to the prevention of endothelial NO synthase dimer disruption. ASK1 deficiency also significantly lessened hepatic steatosis in diabetic mice. In conclusion, our work provided the evidence that ASK1 is significantly activated in diet-induced diabetic mice and contributes to cardiovascular diseases and hepatic steatosis in diabetic mice. Moreover, the beneficial effects of angiotensin II inhibition on dietary diabetic mice seem to be mediated by the inhibition of ASK1 activation. Key Words: diabetes Ⅲ obesity Ⅲ angiotensin Ⅲ ASK1 Ⅲ reactive oxygen species Ⅲ vascular endothelial function Ⅲ cardiac injury O besity, particularly dietary obesity, is associated with type 2 diabetes 1 and an increased risk of cardiovascular diseases. 2-5 However, the underlying mechanism is poorly understood. Accumulating experimental and clinical evidence indicate that the renin-angiotensin system is involved not only in hypertension but also in various cardiovascular diseases. 6 Furthermore, emerging experimental and clinical data support the notion that the renin-angiotensin system participates in the pathophysiology of obesity and type 2 diabetes, 7-13 although the underlying mechanism remains to be elucidated.Reactive oxygen species (ROS) are supposed to be involved in obesity, 14 -17 insulin resistance, diabetes, 1,18 and cardiovascular diseases. 19,20 Apoptosis signal regulating kinase-1 (ASK1), one of the mitogen-activated protein kinase kinase kinases, is markedly activated by ROS and plays a critical role in a variety of cellular responses induced by ROS, including cell apoptosis, growth, differentiation, gene expression, etc. 21-23 Previous...

Chronic injection of an anti-c-KIT receptor tyrosine kinase monoclonal antibody (ACK2) results in the disruption of the normal motility patterns of young BALB/c mice intestine. This effect is accompanied by a drastic decrease in the number of intestinal c-kit-expressing (c-kit+) cells when studied immunohistochemically with the fluorescence-labelled antibody. In order to clarify the mechanism underlying the ACK2 action and the physiological roles of intestinal c-kit+ cells, we studied the excitability of intestinal c-kit+ cells in primary culture by use of the nystatin perforated-patch-clamp technique. Under voltage-clamp at -40 mV, the majority of c-kit+ cells tested (59/70) elicited rhythmic current waves with an amplitude and frequency of 263 +/- 24 pA and 2.30 +/- 0.25 cycles/min (mean +/- SEM), respectively. Intracellular perfusion of the c-kit+ cells with ethylenebis (okonitrilo) tetraacetate (EGTA) as well as a nominally Ca(2+)-free external solution or low holding voltage (< -60 mV) prevented the rhythmic current. The reversal potential of the rhythmic current was close to the equilibrium potential for Cl-(ECl). Moreover the rhythmic current was depressed by a Cl- channel blocker, 4-acetoamido-4-isothiocyanat-ostilbene-2,2'-disulphoni c acid (SITS). The smooth muscle cells freshly dissociated from the same intestinal specimen revealed a Ca(2+)-activated K+ current, as has been described in a variety of smooth muscle cells. Cultured smooth muscle cells from the ileum preparation lacked neither the Ca(2+)-activated K+ nor rhythmic Cl- currents. Smooth muscle cells freshly dissociated from the same ileum preparation and those in culture showed no immunoreactivity with the labelled ACK2, which was consistent with our previous in situ study. Results provided direct evidence that the intestinal c-kit+ cells, but not the smooth muscle cells, possess a rhythmic Cl- current oscillation, suggesting their participation in pacemaker activity for the peristaltic gut movement.

Aims/hypothesis The effect of renin inhibition on type 2 diabetes is still unclear. The present study was undertaken to examine the efficacy of aliskiren, a direct renin inhibitor, on cardiovascular injuries, glucose intolerance and pancreatic injury in a mouse model of type 2 diabetes. Methods Groups of db/db mice, with obesity and type 2 diabetes, were treated with aliskiren (3, 6, 12 and 25 mg kg −1 day −1 ) or hydralazine (80 mg kg −1 day −1 ) for 6 weeks, and the protective effects were extensively compared among groups.Results All sub-pressor and hypotensive doses of aliskiren significantly attenuated cardiac fibrosis, macrophage infiltration and coronary remodelling, and improved vascular endothelial function in db/db mice. These protective effects of aliskiren were attributed to the attenuation of cardiac p22 phox -related NADPH oxidase-induced superoxide and the restoration of vascular endothelial nitric oxide synthase (eNOS) production. Aliskiren at the highest dose (25 mg kg), but not at lower doses, partially reduced glucose intolerance in db/db mice. Furthermore, the highest dose of aliskiren significantly attenuated the decreases in pancreatic islet insulin content and beta cell mass, and prevented pancreatic islet fibrosis in db/db mice, being associated with the reduction of 8-hydroxy-2′-deoxyguanosine-positive cells and Nox2 (also known as Cybb) expression in pancreatic islets by aliskiren. Conclusions/interpretation Our work provides the first evidence that direct renin inhibition with aliskiren protects against cardiovascular complications and pancreatic injury, through the attenuation of oxidative stress. Thus, we propose that aliskiren may be a promising therapeutic agent for type 2 diabetes.

Objective-This work was undertaken to investigate comparative effect of AT1 receptor blocker (ARB), 3-hydroxy-3-methylglutaryl (HMG) coenzymeA (CoA) reductase inhibitor (statin), and their combination on vascular injury of salt-sensitive hypertension. Methods and Results-Salt-loaded Dahl salt-sensitive hypertensive rats (DS rats) were treated with (1) vehicle, (2) hydralazine (5 mg/kg/d), (3) olmesartan (0.5 mg/kg/d), (4) pravastatin (100 mg/kg/d), and (5) combined olmesartan and pravastatin for 4 weeks. Olmesartan or pravastatin significantly and comparably improved vascular endotheliumdependent relaxation to acetylcholine, coronary arterial remodeling, and eNOS activity of DS rats. Olmesartan prevented vascular eNOS dimer disruption or the downregulation of dihydrofolate reductase (DHFR) more than pravastatin, whereas Akt phosphorylation was enhanced by pravastatin but not olmesartan, indicating differential pleiotropic effects between olmesartan and pravastatin. Add-on pravastatin significantly enhanced the improvement of vascular endothelial dysfunction and remodeling by olmesartan in DS rats. Moreover, pravastatin enhanced the increase in eNOS activity by olmesartan, being associated with additive effects of pravastatin on phosphorylation of Akt and eNOS. Conclusions-Olmesartan and pravastatin exerted beneficial vascular effects in salt-sensitive hypertension, via differential pleiotropic effects. Pravastatin enhanced vascular protective effects of olmesartan. Thus, the combination of ARB with statin may be the potential therapeutic strategy for vascular diseases of salt-sensitive hypertension. Key Words: eNOS dimers Ⅲ DHFR Ⅲ oxidative stress Ⅲ vascular injury Ⅲ combined ARB and statin A ccumulating evidence indicates that renin-angiotensin system (RAS) plays a crucial role in the pathophysiology of cardiovascular diseases in hypertension, and that RAS blockers, including angiotensin-converting enzyme inhibitors and AT1 receptor blockers (ARB), are the useful therapeutic agents for hypertensive cardiovascular diseases. 1 As hypertension is often accompanied by dyslipidemia in the same patients, their treatment frequently involves the combination of RAS blockers with 3-hydroxy-3-methylglutaryl coenzymeA (HMG-CoA) reductase inhibitors (statins), potent inhibitors of cholesterol biosynthesis. Clinical evidence show that statins improve endothelial dysfunction and reduce the incidence of atherosclerotic events, 2-5 and these vascular protective effects by statins are at least partially attributed to their pleiotropic vascular effects beyond lowering of plasma cholesterol. 4 -6 However, the difference in vascular pleiotropic effects between RAS blockers and statins remains to be fully understood. Moreover, the significance and the advantage of their combination therapy in hypertension, particularly salt-sensitive hypertension, are not defined.Clinically, salt-sensitive hypertensive patients are more prone to cardiovascular diseases than their salt-insensitive counterparts. 7,8 Therefore, it is a key clinical is...

Abstract-The effect of pioglitazone, a peroxisome proliferator-activated receptor ␥ agonist, on hypertensive cardiovascular injury is unknown. We examined the effect of pioglitazone on hypertensive cardiovascular injury and the significance of combination of pioglitazone with angiotensin type 1 receptor blocker. Stroke-prone spontaneously hypertensive rats (SHRSP) were orally given pioglitazone, candesartan, or combined pioglitazone and candesartan for 4 weeks to compare their effects on cardiovasucular injury. Pioglitazone, without lowering blood pressure, significantly suppressed cardiac inflammation and fibrosis and reduced vascular endothelial dysfunction, and these beneficial effects were associated with the reduction of superoxide by inhibition of cardiovascular NADPH oxidase. Thus, pioglitazone protects against hypertensive cardiovascular injury, by inhibiting reactive oxygen species (ROS). Combination of pioglitazone and candesartan suppressed cardiac hypertrophy, inflammation, and interstitial fibrosis of SHRSP to a greater extent than either monotherapy, and reduced vascular endothelial dysfunction of SHRSP more than either monotherapy. Furthermore, more beneficial effects of their combination on cardiovascular injury were associated with more reduction of NADPH oxidase-mediated cardiovascular ROS. To elucidate the underlying molecular mechanism, we examined cardiovascular NADPH oxidase subunits. Pioglitazone monotherapy significantly attenuated cardiovascular p22 phox and Rac1 in SHRSP, whereas pioglitazone combined with candesartan more attenuated p22 phox and significantly reduced Nox1. Thus, additive suppression of cardiovascular NADPH oxidase by the combination was attributed to its additive attenuation of p22 phox and Nox1 protein levels. In conclusion, we showed that pioglitazone protected against hypertensive cardiovascular damage, and the combination of pioglitazone and candesartan exerted more beneficial effects on hypertensive cardiovascular injury by more suppressing ROS. (Hypertension. 2008;51:296-301.) Key Words: cardiac remodeling Ⅲ endothelium Ⅲ hypertension Ⅲ reactive oxygen species Ⅲ inflammation Ⅲ peroxisome proliferator-activated receptor ␥ Ⅲ AT1 receptor H ypertension is frequently accompanied by type 2 diabetes in the same patients, and hence, many hypertensive patients are subjected to the combination therapy of antihypertensive drug and antidiabetic drug. Pioglitazone is a peroxisome proliferator-activated receptor ␥ (PPAR-␥) agonist 1 and is a useful therapeutic drug for type 2 diabetes, by improving insulin resistance. A growing body of experimental 2-5 and clinical 6 -9 evidence indicate that pioglitazone has beneficial pleiotropic effects on cardiovascular diseases, independently of the improvement of insulin resistance and glycemic control. However, the potential pleiotropic effects of pioglitazone on cardiovascular diseases in hypertension are not clear.Among antihypertensive drugs, an angiotensin type 1 (AT 1 ) receptor blocker (ARB) has been established to exert favorabl...