Glucose metabolism in glycolysis and in mitochondria is pivotal to glucose-induced insulin secretion from pancreatic beta cells. One or more factors derived from glycolysis other than pyruvate appear to be required for the generation of mitochondrial signals that lead to insulin secretion. The electrons of the glycolysis-derived reduced form of nicotinamide adenine dinucleotide (NADH) are transferred to mitochondria through the NADH shuttle system. By abolishing the NADH shuttle function, glucose-induced increases in NADH autofluorescence, mitochondrial membrane potential, and adenosine triphosphate content were reduced and glucose-induced insulin secretion was abrogated. The NADH shuttle evidently couples glycolysis with activation of mitochondrial energy metabolism to trigger insulin secretion.

Abstract-The klotho gene, originally identified by insertional mutagenesis in mice, suppresses the expression of multiple aging-associated phenotypes. This gene is predominantly expressed in the kidney. Recent studies have shown that expression of renal klotho gene is regulated in animal models of metabolic diseases and in humans with chronic renal failure. However, little is known about the mechanisms and the physiological relevance of the regulation of the expression of the klotho gene in the kidney in some diseased conditions. In the present study, we first investigated the role of angiotensin II in the regulation of renal klotho gene expression. Long-term infusion of angiotensin II downregulated renal klotho gene expression at both the mRNA and protein levels. This angiotensin II-induced renal klotho downregulation was an angiotensin type 1 receptor-dependent but pressor-independent event. Adenovirus harboring mouse klotho gene (ad-klotho, 3.3ϫ10 10 plaque forming units) was also intravenously administered immediately before starting angiotensin II infusion in some rats. This resulted in a robust induction of Klotho protein in the liver at day 4, which was still detectable 14 days after the gene transfer. T he klotho gene, identified by insertional mutagenesis in mice, is a suppressor of the expression of multiple aging phenotypes similar to age-related diseases in humans, such as arteriosclerosis, osteoporosis, infertility, pulmonary emphysema, and short lifespan. 1 Interestingly, expression of klotho mRNA in the kidney can be only faintly detected in the prenatal rat, and it is markedly augmented after 4 days of age. 2 Although the klotho gene has a role in phenotypic alterations in various organs, expression of klotho mRNA is predominantly observed in the kidney, 1 suggesting that the Klotho protein or its metabolites may function as humoral factors. Recent studies have shown that expression of renal klotho gene is regulated in animals 2 and in humans 3 in some diseased conditions. At present, however, the mechanism regulating klotho gene expression is poorly understood.In the present study, we have investigated the role of angiotensin (Ang) II in the regulation of renal klotho gene expression. In addition, to clarify the possible physiological role of the klotho gene in the Ang II-infused rats, exogenous klotho gene was delivered into Ang II-infused rats, and functional and histological changes in the kidney were analyzed. Methods Animal ModelsThe experiments were performed in accordance with the guidelines and practices established by the Animal Center for Biomedical Research, University of Tokyo, Faculty of Medicine. The rat Ang II hypertension model was induced in male Sprague-Dawley rats (Nippon Bio-Supply Center, Tokyo, Japan) by the continuous infusion of [Val 5 ]-Ang II (Sigma) at a dose of 0.7 mg/kg per day via an osmotic minipump (Alza) as described previously. 4 In some experiments, the selective angiotensin type 1 receptor antagonist losartan (25 mg/kg per day; a gift from Merck, Rahway, NJ) ...

Inflammation is a hallmark of many diseases, such as atherosclerosis, chronic obstructive pulmonary disease, arthritis, infectious diseases, and cancer. Although steroids and cyclooxygenase inhibitors are effective antiinflammatory therapeutical agents, they may cause serious side effects. Therefore, developing unique antiinflammatory agents without significant adverse effects is urgently needed. Vinpocetine, a derivative of the alkaloid vincamine, has long been used for cerebrovascular disorders and cognitive impairment. Its role in inhibiting inflammation, however, remains unexplored. Here, we show that vinpocetine acts as an antiinflammatory agent in vitro and in vivo. In particular, vinpocetine inhibits TNF-α-induced NF-κB activation and the subsequent induction of proinflammatory mediators in multiple cell types, including vascular smooth muscle cells, endothelial cells, macrophages, and epithelial cells. We also show that vinpocetine inhibits monocyte adhesion and chemotaxis, which are critical processes during inflammation. Moreover, vinpocetine potently inhibits TNF-α-or LPS-induced up-regulation of proinflammatory mediators, including TNF-α, IL-1β, and macrophage inflammatory protein-2, and decreases interstitial infiltration of polymorphonuclear leukocytes in a mouse model of TNF-α-or LPSinduced lung inflammation. Interestingly, vinpocetine inhibits NF-κB-dependent inflammatory responses by directly targeting IKK, independent of its well-known inhibitory effects on phosphodiesterase and Ca 2+ regulation. These studies thus identify vinpocetine as a unique antiinflammatory agent that may be repositioned for the treatment of many inflammatory diseases.vinpocetine | inflammation | NF-κB | IKK

Abstract-Angiotensin II (Ang II) and nitric oxide (NO) signaling pathways mutually regulate each other by multiple mechanisms. Ang II regulates the expression of NO synthase and NO production, whereas NO downregulates the Ang II type I (AT1) receptor. A ngiotensin II (Ang II), the primary effector of the renin-angiotensin system (RAS), is a multifunctional hormone that plays an important role in vascular function. The local RAS, acting in both autocrine and paracrine fashions, is also functionally operative and important in the vasculature. 1,2 The role of the RAS, particularly of Ang II, is of great interest in cardiovascular physiology and pathology because of the beneficial effects of Ang II-converting enzyme (ACE) inhibitors and Ang II receptor blockers in cardiovascular diseases (hypertension, atherosclerosis, heart failure, and stroke). [3][4][5][6] Nitric oxide (NO) as an endogenous endothelium-derived relaxing factor has also been extensively studied. NO plays critical roles in the maintenance of vascular homeostasis. Reduction of NO production due to endothelial dysfunction is the result of many cardiovascular risk factors.In the vasculature, Ang II and NO interact with each other (albeit indirectly) to influence each other's functions. The interaction between Ang II and NO occurs in both the endothelial cell (EC) and vascular smooth muscle cell (VSMC). Vascular smooth muscle constricts in response to Ang II and dilates in response to NO. In addition to vascular tone, these 2 molecules antagonize each other in many vascular functions, such as cell growth, apoptosis, and inflammation. Ang II Signaling PathwaysThe actions of Ang II are primarily mediated by 2 receptors, Ang II type 1 (AT1) and type 2 (AT2). The AT1 receptor is widely present in many organs, such as the heart, kidneys, adrenal glands, and brain. The vast majority of well-known physiological and pathophysiological effects of Ang II have been shown to occur via the AT1 receptor. In the vasculature, the AT1 receptor is mainly expressed in VSMCs, where it mediates the vasoconstrictor, proliferative, and inflammatory actions of Ang II. There are AT1 receptors in the endothelium and in monocytes/macrophages as well, 7,8 which are pathophysiologically important because Ang II induces the oxidized LDL receptor in the endothelium and stimulates macrophages to express tumor necrosis factor-␣. 8,9 The AT2 receptor is highly and ubiquitously expressed in fetal tissue, and its expression is dramatically reduced after birth. 10,11 The fact that AT2 receptor expression is much higher in fetal compared with normal adult tissues has led to speculation as to its possible role in cell growth, development, and differentiation. AT2 receptor-mediated signaling pathways and function are not very well understood but in general appear to antagonize the effects of the AT1 receptor.Functions of the AT1 receptor, a G protein-coupled receptor, have been best characterized in VSMCs. The AT1 receptor coupled to Gq leads to phospholipase C (PLC) activation and, in turn, ...

Abstract-In this study, we investigated the regulation and physiological role of heme oxygenase-1 (HO-1) in the kidney of rats with hypertension. Rats were continuously administered either angiotensin II (Ang II) or norepinephrine with an osmotic minipump for up to 7 days. Ang II infusion decreased the glomerular filtration rate (GFR) as determined through creatinine clearance (3.2Ϯ0. Key Words: hypertension Ⅲ angiotensin II Ⅲ proteinuria Ⅲ oxidative stress Ⅲ kidney H ypertension induces structural and functional alterations in the kidney, eventually leading to end-stage renal disease. The control of blood pressure retards the progression of renal failure and reduces the morbidity and mortality rates associated with hypertensive vascular disease. 1,2 Although several clinical trials have shown a similar slowing of the progression with the use of either ACE inhibitors or other antihypertensive drugs, 3 the superiority of ACE inhibitors has been demonstrated in some studies. Compared with other classes of antihypertensive drugs, ACE inhibitors were more effective than other agents in preservation of the glomerular filtration rate 4 and in their antiproteinuric effects 5,6 in essential hypertension. The extra effectiveness of ACE inhibitors may derive from their ability to preferentially dilate the efferent arterioles 7 and their antiproliferative effects. 8 Heme oxygenase (HO) is a rate-limiting enzyme of heme catabolism that has 2 isoforms: HO-1, an inducible form, 9,10 and HO-2, a constitutive form. Induced HO-1 is thought to act as an antioxidative and anti-inflammatory defense mechanism through the degradation of cellular heme (pro-oxidant) and increase in biliverdin (antioxidant 11 ). The carbon monoxide (CO) that is produced also has physiological functions, such as vascular relaxation 12 and inhibition of platelet aggregation, 13 through the activation of soluble guanylyl cyclase. The recent findings that HO-1 gene transfer ameliorated oxidative tissue injury 14 and that oxidant-induced cellular injury was increased in HO-1 knockout mice 15 and in human HO-1 deficiency 16 provide further direct evidence that HO-1 acts favorably against oxidative stress. In the kidney, both HO-1 and HO-2 are present in the tubular epithelial cells, 17,18 suggesting that the HO system also plays a role in the kidney. In fact, HO-1 induction exerts a protective effect on renal function in animal models of rhabdomyolysis, 19 cisplatin nephrotoxicity, 20 and nephrotoxic nephritis. 21 In previous reports, we demonstrated that HO-1 expression was regulated by Ang II in vascular smooth muscle in both pressordependent 22 and pressor-independent 23 manners.Renal damage occurs via pressor-dependent and -independent mechanisms. The present study was designed to examine both such mechanisms in the angiotensin II (Ang II)

Clinical studies have demonstrated that some antihypertensive agents provide renoprotection independent of BP lowering. Recent in vitro and in vivo studies evaluated the mechanisms involved in this protection. First, the in vitro effects of several angiotensin II type 1 receptor blockers (ARB), calcium channel blockers (CCB), and ␤ blockers (BB) on various mediators were compared: Formation of pentosidine (an advanced glycation end product), hydroxyl radical-induced formation of o-tyrosine, and transition metals-induced oxidation of ascorbic acid (the Fenton reaction). All of the six tested ARB but neither the six CCB nor the nine BB inhibited pentosidine formation. ARB, as well as BB but not CCB, inhibited hydroxyl radicals-mediated o-tyrosine formation. ARB but neither BB nor CCB inhibited efficiently transition metals-catalyzed oxidation of ascorbic acid. Second, the in vivo consequences for the kidney of these various in vitro effects were evaluated. Hypertensive, type 2 diabetic rats with nephropathy, SHR/NDmcr-cp, were given for 20 wk either olmesartan (ARB) or nifedipine (CCB), or atenolol (BB). Despite similar BP reduction, only ARB significantly reduced proteinuria and prevented glomerular and tubulointerstitial damage (mesangial activation, podocyte injury, tubulointerstitial injury, and inflammatory cell infiltration). It is interesting that only ARB prevented abnormal iron deposition in the interstitium, corrected chronic hypoxia, reduced expressions of heme oxygenase and p47phox (a subunit of NADPHoxidase), and inhibited pentosidine formation (which correlates well with proteinuria). These observations confirm unique renoprotective properties of ARB, independent of BP lowering but related to decreased oxidative stress (hydroxyl radicals scavenging and inhibition of the Fenton reaction), correction of chronic hypoxia, and inhibition of advanced glycation end product formation and of abnormal iron deposition. These benefits of ARB may contribute to the renoprotection observed beyond BP lowering. S everal clinical studies, mainly but not only in diabetic patients, have provided evidence that some antihypertensive agents that inhibit the renin-angiotensin system (RAS), namely angiotensin II type 1 receptor blockers (ARB) and angiotensin-converting enzyme inhibitors (ACEI), are renoprotective (1-4). Recently, the renoprotection provided by these drugs seems at least partly independent of BP lowering and related perhaps to the inhibition of the RAS (5-8). ARB and ACEI thus now are part of the standard treatment of patients with diabetic nephropathy, regardless of the presence of systemic hypertension. A similar renoprotective effect has been claimed for nifedipine or other calcium channel blockers (CCB) acting independent of the RAS (9,10), but results of clinical studies remain disputed (5).The nature of the renoprotection provided by some antihypertensive agents independent of BP lowering and inhibition of the RAS thus has become a topic of major interest. We previously demonstrated in vitro that ARB an...

cAMP plays crucial roles in cardiac remodeling and the progression of heart failure. Recently, we found that expression of cAMP hydrolyzing phosphodiesterase 3A (PDE3A) was significantly reduced in human failing hearts, accompanied by up-regulation of inducible cAMP early repressor (ICER) expression. Angiotensin II (Ang II) and the ␤-adrenergic receptor agonist isoproterenol (ISO) also induced persistent PDE3A down-regulation and concomitant ICER up-regulation in vitro, which is important in Ang II-and ISO-induced cardiomyocyte apoptosis. We hypothesized that interactions between PDE3A and ICER may constitute an autoregulatory positive feedback loop (PDE3A-ICER feedback loop), and this loop would cause persistent PDE3A down-regulation and ICER up-regulation. Here, we demonstrate that ICER induction repressed PDE3A gene transcription. PDE3A down-regulation activated cAMP͞PKA signaling, leading to ICER up-regulation via PKAdependent stabilization of ICER. With respect to Ang II, the initiation of the PDE3A-ICER feedback loop depends on activation of Ang II type 1 receptor (AT1R), classical PKC(s), and CREB (cAMP response element binding protein). We further show that the PDE3A-ICER feedback loop is essential for Ang II-induced cardiomyocyte apoptosis. ISO and PDE3 inhibitors also induced the PDE3A-ICER feedback loop and subsequent cardiomyocyte apoptosis, highlighting the importance of this PDE3A-ICER feedback loop and cAMP signaling in cardiomyocyte apoptosis. Our findings may provide a therapeutic paradigm to prevent cardiomyocyte apoptosis and the progression of heart failure by inhibiting the PDE3A-ICER feedback loop.angiotension II ͉ ␤-andrenergic receptor ͉ PKC ͉ heart failure C ardiac remodeling including dysregulated myocyte apoptosis contributes to the development and progression of myocardial remodeling and the transition from cardiac hypertrophy to chronic heart failure (1-3). Stimulation of the renin-angiotensin and ␤-adrenergic receptor (␤-AR) systems are broadly involved in contraction, growth control, and cell death (3, 4). Both systems are subject to counterregulatory balances under normal physiological circumstances, which are overridden by chronic activation in heart failure. For example, chronic exposure to angiotensin II (Ang II) and ISO promote cardiac dysfunction resulting from cardiac remodeling including hypertrophy and apoptosis (4-7). Many clinical trials have indicated that angiotensinconverting enzyme inhibitors and ␤-AR blockers significantly improve the survival rates of heart failure patients by decreasing cardiac remodeling (8). However, the exact mechanisms of myocyte apoptosis, especially those mediated by angiotensin II, remain unclear.cAMP signaling plays important roles in both physiologic and pathologic regulation of cardiac performance (9). cAMP is one of the most well characterized signaling molecules in ␤-AR signaling, but its contribution to Ang II signaling in cardiomyocytes is not fully understood. Clinical and experimental studies indicate that acute stimulation of ␤-AR...

Abstract-In response to biological and mechanical injury, or in vitro culturing, vascular smooth muscle cells (VSMCs) undergo phenotypic modulation from a differentiated "contractile" phenotype to a dedifferentiated "synthetic" one. This results in the capacity to proliferate, migrate, and produce extracellular matrix proteins, thus contributing to neointimal formation. Cyclic nucleotide phosphodiesterases (PDEs), by hydrolyzing cAMP or cGMP, are critical in the homeostasis of cyclic nucleotides that regulate VSMC growth. Here, we demonstrate that PDE1A, a Ca 2ϩ -calmodulin-stimulated PDE preferentially hydrolyzing cGMP, is predominantly cytoplasmic in medial "contractile" VSMCs but is nuclear in neointimal "synthetic" VSMCs. Using primary VSMCs, we show that cytoplasmic and nuclear PDE1A were associated with a contractile marker (SM-calponin) and a growth marker (Ki-67), respectively. This suggests that cytoplasmic PDE1A is associated with the "contractile" phenotype, whereas nuclear PDE1A is with the "synthetic" phenotype. To determine the role of nuclear PDE1A, we examined the effects loss-of-PDE1A function on subcultured VSMC growth and survival using PDE1A RNA interference and pharmacological inhibition. Reducing PDE1A function significantly attenuated VSMC growth by decreasing proliferation via G 1 arrest and inducing apoptosis. Inhibiting PDE1A also led to intracellular cGMP elevation, p27Kip1 upregulation, cyclin D1 downregulation, and p53 activation. We further demonstrated that in subcultured VSMCs redifferentiated by growth on collagen gels, cytoplasmic PDE1A regulates myosin light chain phosphorylation with little effect on apoptosis, whereas inhibiting nuclear PDE1A has the opposite effects. These suggest that nuclear PDE1A is important in VSMC growth and survival and may contribute to the neointima formation in atherosclerosis and restenosis. (Circ Res. 2006;98:777-784.) Key Words: PDE Ⅲ smooth muscle cell Ⅲ growth Ⅲ apoptosis Ⅲ vascular injury V ascular smooth muscle cells (VSMCs) in response to injury and hormonal stimuli exhibit phenotypic plasticity, changing from a differentiated (quiescent, contractile) phenotype to a dedifferentiated (active, synthetic) one. 1 This process was originally defined as "phenotypic modulation." 2 Under normal conditions, VSMCs residing in the media of vessels are quiescent with a very low turnover rate. 3,4 Quiescent VSMCs are fully differentiated cells that possess the "contractile" phenotype and function principally to maintain vascular tone. If the vessel is injured or cells are placed in tissue culture, VSMCs respond by changing from the "contractile" to the "synthetic" phenotype. 4 Synthetic VSMCs contribute to neointima formation by downregulating contractile proteins and acquiring the capacity to proliferate, migrate, and produce extracellular matrix proteins. 5 Therefore, phenotypic modulation of VSMCs plays a key role in the pathogenesis of cardiovascular disorders such as atherosclerosis, postangioplasty restenosis, bypass vein graft failure, and ca...