Dipyridamole with Low-Dose Aspirin Augments the Infarct Size-Limiting Effects of Simvastatin

Purpose

Statins protect against ischemia-reperfusion injury and limit myocardial infarct size (IS). This effect is dependent on increased generation of adenosine by ecto-5′ nucleotidase and downstream activation of cyclooxygenase-2 (COX2). Dipyridamole (DIP) augments the IS-limiting effects of statins by blocking the cellular reuptake of adenosine; whereas aspirin (ASA) attenuates the effect by inhibiting COX2. We studied the effect of acute administration of DIP, ASA and their combination on the IS-limiting effect of simvastatin (SIM).

Methods

Rats received oral SIM (10 mg/kg/d) or vehicle for 3 days. Rats underwent 30 min of coronary artery occlusion and 4 h reperfusion. After 5 min of ischemia rats received i.v. DIP (5 mg/kg), ASA (20 mg/kg or 2 mg/kg) or DIP+ASA (2 mg/kg) or vehicle alone. Ischemia area at risk (AR) was assessed by blue dye and IS by TTC. Myocardial samples were analyzed for the activation of Akt, ERK 1/2, endothelial nitric oxide synthase (eNOS), and cyclic-AMP-response-element-binding-protein (CREB).

Results

SIM limited IS. High- or low-dose ASA alone had no effect on IS. DIP alone or with low-dose ASA significantly reduced IS. Low-dose ASA did not attenuate the SIM effect, whereas high-dose ASA completely blocked the effect. The combination of DIP+low-dose ASA+SIM resulted in the smallest IS. Both SIM and DIP+low-dose ASA augmented Akt phosphorylation and their effect was additive. Both SIM and DIP+low-dose ASA augmented eNOS, ERK 1/2 and CREB phosphorylation.

Conclusions

During acute myocardial ischemia, DIP alone or with low-dose ASA limits IS and does not attenuate the IS-limiting effect of SIM as high-dose ASA.     

Metformin suppresses hepatic gluconeogenesis and lowers fasting blood glucose levels through reactive nitrogen species in mice

Aims/hypothesis

Metformin, the major target of which is liver, is commonly used to treat type 2 diabetes. Although metformin activates AMP-activated protein kinase (AMPK) in hepatocytes, the mechanism of activation is still not well known. To investigate AMPK activation by metformin in liver, we examined the role of reactive nitrogen species (RNS) in suppression of hepatic gluconeogenesis.

Methods

To determine RNS, we performed fluorescence examination and immunocytochemical staining in mouse hepatocytes. Since metformin is a mild mitochondrial complex I inhibitor, we compared its effects on suppression of gluconeogenesis, AMPK activation and generation of the RNS peroxynitrite (ONOO^?) with those of rotenone, a representative complex I inhibitor. To determine whether endogenous nitric oxide production is required for ONOO^? generation and metformin action, we used mice lacking endothelial nitric oxide synthase (eNOS).

Results

Metformin and rotenone significantly decreased gluconeogenesis and increased phosphorylation of AMPK in wild-type mouse hepatocytes. However, unlike rotenone, metformin did not increase the AMP/ATP ratio. It did, however, increase ONOO^? generation, whereas rotenone did not. Exposure of eNOS-deficient hepatocytes to metformin did not suppress gluconeogenesis, activate AMPK or increase ONOO^? generation. Furthermore, metformin lowered fasting blood glucose levels in wild-type diabetic mice, but not in eNOS-deficient diabetic mice.

Conclusions/interpretation

Activation of AMPK by metformin is dependent on ONOO^?. For metformin action in liver, intra-hepatocellular eNOS is required.     

Nitric oxide and prostaglandin as mediators in the pathogenesis of hyperkinetic circulatory state in a model of endotoxemia-induced portal hypertension

Aims

To evaluate the participation of nitric oxide (NO) and prostaglandin (PGI₂) on hyperdynamic state in endotoxemia-induced portal hypertension (EIP) induced by chronic endotoxemia.

Methods

The portal pressure (PP) and mean arterial pressure (MAP) were recorded, in vivo before and after administration of l-NAME (NOS inhibitor) and indomethacin (specific blocker of COX). The vasoactive responses to acetylcholine of thoracic rat aortic rings were studied in vitro before and after nitric oxide and cyclooxygenase blockade using multichannel organ bath. The mRNA expression for isoforms of (cyclooxygenase) COX and nitric oxide synthase (NOS) were analyzed using RT-PCR.

Results

Administration of both l-NAME and indomethacin in EIP rabbits significantly reduced (p < 0.05) the PP and reversed the MAP to normal as compared to sham-operated (SO) rabbits. There was impaired vasodilatory response to acetylcholine in EIP rabbits. l-NAME caused a significant reduction in acetylcholine-induced vasorelaxation in SO rabbits than EIP due to preexisting hyperemia in EIP. Indomethacin partially restored vasoresponsiveness to acetylcholine in EIP group. The mRNA expression of eNOS (endothelial NOS) and COX-1 (constitutive COX) were significantly higher in SO than EIP rabbits. iNOS (inducible NOS) and COX-2 (inducible COX) mRNA expression was seen only in EIP rabbits.

Conclusions

A significant component of acetylcholine-mediated vasorelaxation in EIP model is modulated by eNOS. There was increased production of contractile prostaglandin in EIP rabbits. iNOS and COX-2 play an important role in the hemodynamic abnormalities of PHT. This novel model of PHT produced by chronic splanchnic endotoxemia in rabbit, mimics impaired vasodilation and vasoreactivity akin to other models of PHT.     

L-citrulline Prevents Alveolar and Vascular Derangement in a Rat Model of Moderate Hyperoxia-induced Lung Injury

Background

Moderate normobaric hyperoxia causes alveolar and vascular lung derangement in the newborn rat. Endogenous nitric oxide (NO), which promotes lung growth, is produced from the metabolism of l-arginine to l-citrulline in endothelial cells. We investigated whether administering l-citrulline by raising the serum levels of l-arginine and enhancing NO endogenous synthesis attenuates moderate hyperoxia-induced lung injury.

Methods

Newborn rats were exposed to FiO₂?=?0.6 or room air for 14?days to induce lung derangement and then were administered l-citrulline or a vehicle (sham). Lung histopathology was studied with morphometric features. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected for analysis. Lung vascular endothelial growth factor (VEGF), nitric oxide synthase (eNOS), and matrix metalloproteinase 2 (MMP2) gene and protein expressions were assessed.

Results

Serum l-arginine rose in the L-citr?+?hyperoxia group (p?=?0.05), as well as the Von Willebrand factor stained vessels count (p?=?0.0008). Lung VEGF immune staining, localized on endothelial cells, was weaker in the sections under hyperoxia than the l-citr?+?hyperoxia and room air groups. This pattern was comparable with the VEGF gene and protein expression profiles. Mean alveolar size increased in the untreated hyperoxia and sham-treated groups compared with the groups reared in room air or treated with l-citrulline under exposure to hyperoxia (p?=?0.0001). Lung VEGF and eNOS increased in the l-citrulline-treated rats, though this treatment did not change MMP2 gene expression but regulated the MMP2 active protein, which rose in BALF (p?=?0.003).

Conclusions

We conclude that administering l-citrulline proved effective in improving alveolar and vascular growth in a model of oxygen-induced pulmonary damage, suggesting better lung growth and matrix regulation than in untreated groups.     

Methylglyoxal impairs endothelial insulin sensitivity both in vitro and in vivo

Aims/hypothesis

Insulin exerts a direct action on vascular cells, thereby affecting the outcome and progression of diabetic vascular complications. However, the mechanism through which insulin signalling is impaired in the endothelium of diabetic individuals remains unclear. In this work, we have evaluated the role of the AGE precursor methylglyoxal (MGO) in generating endothelial insulin resistance both in cells and in animal models.

Methods

Time course experiments were performed on mouse aortic endothelial cells (MAECs) incubated with 500 μmol/l MGO. The glyoxalase-1 inhibitor S-p-bromobenzylglutathione-cyclopentyl-diester (SpBrBzGSHCp2) was used to increase the endogenous levels of MGO. For the in vivo study, an MGO solution was administrated i.p. to C57BL/6 mice for 7 weeks.

Results

MGO prevented the insulin-dependent activation of the IRS1/protein kinase Akt/endothelial nitric oxide synthase (eNOS) pathway, thereby blunting nitric oxide (NO) production, while extracellular signal-regulated kinase (ERK1/2) activation and endothelin-1 (ET-1) release were increased by MGO in MAECs. Similar results were obtained in MAECs treated with SpBrBzGSHCp2. In MGO- and SpBrBzGSHCp2-exposed cells, inhibition of ERK1/2 decreased IRS1 phosphorylation on S616 and rescued insulin-dependent Akt activation and NO generation, indicating that MGO inhibition of the IRS1/Akt/eNOS pathway is mediated, at least in part, by ERK1/2. Chronic administration of MGO to C57BL/6 mice impaired whole-body insulin sensitivity and induced endothelial insulin resistance.

Conclusions/interpretation

MGO impairs the action of insulin on the endothelium both in vitro and in vivo, at least in part through an ERK1/2-mediated mechanism. These findings may be instrumental in developing novel strategies for preserving endothelial function in diabetes.     

Noninvasive assessment of subclinical atherosclerosis in normotensive gravidae with gestational diabetes

Aim

Carotid artery intima-media thickness (CIMT), hyperhomocysteinemia, microalbuminuria, and nitric oxide reflect subclinical atherosclerosis and predict the risk of future cardiovascular events. We aimed to evaluate the presence of subclinical atherosclerosis and endothelial dysfunction in normotensive patients with gestational diabetes mellitus (GDM) noninvasively.

Patients and methods

We enrolled 41 normotensive patients with GDM and 44 healthy gravidae in the study. Serum homocysteine and nitric oxide levels, urinary albumin excretion (microalbuminuria), and CIMT were evaluated along with lipid parameters and anthropometric measurements.

Results

Patients with GDM had significantly higher levels of serum homocysteine, urinary albumin excretion, and increased CIMT (p?Conclusion Independent of elevated blood pressure, subclinical atherosclerosis and endothelial dysfunction exist in normotensive patients with GDM. Further studies with a large number of participants are required to clarify these data.     

Nitrergic innervation and relaxant response of rectal circular smooth muscle

PURPOSE: This study was designed to investigate whether nitric oxide mediates inhibitory innervation in human rectal circular smooth muscle. METHODS: Tissue was obtained from the midrectum of patients undergoing anterior resection for carcinoma. Adjacent strips of circular muscle were dissected and mounted in superfusion organ baths for isometric tension recording and initially loaded with 1 g of weight. Strips were continuously bathed with standard Krebs solution (37°C, bubbled with 97 percent O ₂ /3 percent CO ₂ ) containing 3×10 ^?6 M guanethidine and 3×10 ^?6 M atropine sulfate to block adrenergic and muscarinic cholinergic neurotransmission. After equilibration, strips had no intrinsic tone, and reproducible and stable tension was, therefore, induced by the addition of 3×10 ^?6 M histamine for five-minute “test” periods, during which electrical field stimulation (EFS) and additional drugs were applied. RESULTS: EFS elicited frequency-dependent, neurogenic (tetrodotoxin-sensitive) relaxations of precontracted strips. Addition of N-ω-nitro- l -arginine, a powerful competitive inhibitor of nitric oxide synthase, reduced the relaxant response to EFS in a dose-dependent fashion, an effect reversed by addition of 3×10 ^?4 M l -arginine but not by D-arginine. Addition of exogenous nitric oxide (sodium nitroprusside) mimicked the relaxant response induced by EFS. CONCLUSIONS: Human rectal circular smooth muscle receives an intrinsic inhibitory innervation mediated by nitric oxide. The presence of a residual response following blockade of the enzyme nitric oxide synthase suggests the involvement of additional neurotransmitters.     

Effect of Biliary Cirrhosis on Neurogenic Relaxation of Rat Gastric Fundus and Anococcygeus Muscle: Role of Nitric Oxide Pathway

Background

Cirrhosis, associated with a host of hemodynamic abnormalities, could affect the gastrointestinal (GI) tract motility. On the other hand, the nonadrenergic noncholinergic (NANC) neurotransmission has been shown to play a pivotal role in GI tract motility and has been linked with release of nitric oxide (NO) on electrical stimulation. In this study, we investigated the effect of biliary cirrhosis on the neurogenic relaxation of rat gastric fundus and anococcygeus muscle and also the possible role of nitric oxide system in this manner.

Methods

Isolated gastric fundus and anococcygeus strips of sham-operated and biliary cirrhotic (4 weeks after bile duct ligation) rats were mounted under tension in a standard organ bath. Electrical stimulation was applied to obtain NANC-mediated relaxations in precontracted gastric fundus and anococcygeus muscle. The neurogenic relaxations were examined in the presence of different doses of NO synthase inhibitor, N ^w-Nitro-l-Arginine Methyl Ester (l-NAME). The concentration-dependent relaxant responses to the NO donor sodium nitroprusside were also evaluated.

Results

The neurogenic relaxation of both gastric fundus and anococcygeus muscle was significantly (P < 0.001) increased in cirrhotic animals. l-NAME (0.03–1,000 µM) inhibited relaxations in both groups in a dose-dependent manner (P < 0.001), but cirrhotic groups were more resistant to the inhibitory effects of l-NAME (P < 0.01). Sodium nitroprusside-mediated relaxations were similar in two groups.

Conclusions

This study for the first time demonstrated that cirrhosis increases the NO-mediated neurogenic relaxation of both rat gastric fundus and anococcygeus muscle, suggesting a crucial role for the neurogenic NO in the pathophysiology of disturbed GI motility in cirrhosis.     

Inhibition of calcium-dependent nitric oxide synthase causes ileitis and leukocytosis in guinea pigs

As nitric oxide reduces gut epithelial permeability, we designed a study to determine if chronic nitric oxide synthase inhibition predisposes the gut to inflammation. Nitric oxide synthase (NOS) inhibitors were administered in the drinking waterad libitum, for seven days: aminoguanidine (10 µg/ml), a selective inhibitor of the inducible form of nitric oxide synthase; andN ^G-nitro-l-arginine methyl ester (l-NAME, 1, 10, and 100 µg/ml), which inhibits both the constitutive and inducible forms. Control animals drank tap water only or water withd-NAME, the inactive enantiomer. After one week, circulating leukocyte count and tissue myeloperoxidase activity were measured.l-NAME (100 µg/ml), but notd-NAME or aminoguanidine, caused a twofold increase in a circulating leukocyte numbers. This increase in leukocyte numbers was time- and dose-dependent, but the differential count was unaltered. Tissue myeloperoxidase (MPO) activity as an index of granulocyte infiltration was comparable in all groups in the stomach, jejunum, colon, liver, lung, kidney, heart, and skeletal muscle. However, ileal MPO activity was elevated threefold in thel-NAME- (100 µg/ml) treated group (P<0.05). Results in thed-NAME and aminoguanidine groups were similar to controls.l-NAME administration resulted in a reduction in NOS activity ([¹⁴C]citrulline formation) in the ileum but not jejunum, whereas cGMP levels were elevated in both ileum and jejunum. We conclude that chronic inhibition of the constitutive form of nitric oxide synthase predisposes the ileum to inflammation and leads to a progressive leukocytosis.     

Inhibition of atrial fibrillation by low-level vagus nerve stimulation: the role of the nitric oxide signaling pathway

Purpose

We examined the role of the phosphatidylinositol-3 kinase (PI3K)/nitric oxide (NO) signaling pathway in low-level vagus nerve stimulation (LLVNS)-mediated inhibition of atrial fibrillation (AF).

Methods

In 17 pentobarbital anesthetized dogs, bilateral thoracotomies allowed the attachment of electrode catheters to the superior and inferior pulmonary veins and atrial appendages. Rapid atrial pacing (RAP) was maintained for 6 h. Each hour, programmed stimulation was used to determine the window of vulnerability (WOV), a measure of AF inducibility, at all sites. During the last 3 h, RAP was overlapped with right LLVNS (50 % below that which slows the sinus rate). In group 1 (n?=?7), LLVNS was the only intervention, whereas in groups 2 (n?=?6) and 3 (n?=?4), the NO synthase inhibitor N ^G-nitro-l-arginine methyl ester (l-NAME) and the PI3K inhibitor wortmannin, respectively, were injected in the right-sided ganglionated plexi (GP) during the last 3 h. The duration of acetylcholine-induced AF was determined at baseline and at 6 h. Voltage–sinus rate curves were constructed to assess GP function.

Results

LLVNS significantly decreased the acetylcholine-induced AF duration by 8.2?±?0.9 min (p?<?0.0001). Both l-NAME and wortmannin abrogated this effect. The cumulative WOV (the sum of the individual WOVs) decreased toward baseline with LLVNS (p?<?0.0001). l-NAME and wortmannin blunted this effect during the fifth (l-NAME only, p?<?0.05) and the sixth hour (l-NAME and wortmannin, p?<?0.05). LLVNS suppressed the ability of GP stimulation to slow the sinus rate, whereas l-NAME and wortmannin abolished this effect.

Conclusion

The anti-arrhythmic effects of LLVNS involve the PI3K/NO signaling pathway.     

Inhibition of neuronal nitric oxide synthase activity does not alter vasopressin secretion in septic rats

Background/Purpose

During the early phase of sepsis, hypotension is accompanied by increase of plasma vasopressin hormone (AVP) levels, which decline during the late phase. This hypotension is due in part to increase of nitric oxide (NO) synthesis by nitric oxide synthase (NOS) enzyme. Neuronal isoform of this enzyme (nNOS) is present in vasopressinergics neurons of hypothalamus, but its role in vasopressin secretion during sepsis is unknown.

Methods

We evaluated the role of nNOS in NO production and vasopressin secretion during sepsis. Wistar rats received 7-nitroindazole (50 mg/kg, i.p.), an inhibitor of nNOS activity, or vehicle and were submitted to septic stimulus by cecal ligation and puncture (CLP). At the time points 0, 4, 6, 18 and 24 h after sepsis induction the animals were decapitated and neurohypophysis and hypothalamus were removed for analysis of vasopressin content and NOS activity, respectively. Hematocrit, serum sodium, osmolality, proteins and plasmatic AVP were quantified.

Results

Mortality was not affected by 7-nitroindazole (7-NI). Sodium and plasma proteins levels decreased after CLP and the treatment anticipated the protein loss, and delayed serum sodium decrease. Septic animals treated with 7-NI showed decrease of osmolality 4 h after CLP. Nitric oxide synthase activity in hypothalamus increased at 4 and 24 h after CLP and was reduced with 7-NI. Neurohypophysis content of AVP diminished after CLP and 7-NI did not alter this parameter. Plasma AVP levels increased at 6 h and decreased 18 and 24 h after CLP. Treatment with 7-NI did not alter plasma vasopressin levels.

Conclusion

We concluded that nNOS does not have a substantial role in vasopressin secretion during experimental sepsis.

     

Inhibition of matrix metalloproteinases and inducible nitric oxide synthase by andrographolide in human osteoarthritic chondrocytes

Objective

The aim of this study was to investigate the effects of andrographolide on matrix metalloproteinases (MMP) 1, 3, and 13 and inducible nitric oxide synthase (iNOS) in human articular chondrocytes from osteoarthritic cartilage.

Methods

Passaged chondrocytes were pretreated with or without andrographolide for 2 h, followed by coincubation with interleukin-1 beta (IL-1β) 1 ng/ml for 24 h. Expression levels of MMP-1, 3, and 13, tissue inhibitor of metalloproteinase-1 (TIMP-1), and iNOS were evaluated using real-time-quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blotting. Nitric oxide (NO) was analyzed using the Griess reaction assay. Involvement of nuclear factor kappa B (NF-κB) was assessed by Western blotting, transient transfection, and luciferase reporter assay.

Results

Andrographolide tested in these in vitro studies was found be an effective antiarthritic agent, as evidenced by potent inhibition of MMP-1, 3, and 13 and iNOS expression, as well as upregulation of TIMP-1 in IL-1β-stimulated human articular chondrocytes (p < 0.05). The mechanism of andrographolide’s inhibitory effects was mediated by attenuating the activation of NF-κB in human chondrocytes in the presence of IL-1β.

Conclusions

Andrographolide was a potent inhibitor of the production of inflammatory and catabolic mediators by chondrocytes, suggesting that this natural compound may merit consideration as a therapeutic agent for treating and preventing osteoarthritis.     

Astrocyte-induced cortical vasodilation is mediated by D-serine and endothelial nitric oxide synthase

Astrocytes play a critical role in neurovascular coupling by providing a physical linkage from synapses to arterioles and releasing vaso-active gliotransmitters. We identified a gliotransmitter pathway by which astrocytes influence arteriole lumen diameter. Astrocytes synthesize and release NMDA receptor coagonist, D-serine, in response to neurotransmitter input. Mouse cortical slice astrocyte activation by metabotropic glutamate receptors or photolysis of caged Ca²⁺ produced dilation of penetrating arterioles in a manner attenuated by scavenging D-serine with D-amino acid oxidase, deleting the enzyme responsible for D-serine synthesis (serine racemase) or blocking NMDA receptor glycine coagonist sites with 5,7-dichlorokynurenic acid. We also found that dilatory responses were dramatically reduced by inhibition or elimination of endothelial nitric oxide synthase and that the vasodilatory effect of endothelial nitric oxide synthase is likely mediated by suppressing levels of the vasoconstrictor arachidonic acid metabolite, 20-hydroxy arachidonic acid. Our results provide evidence that D-serine coactivation of NMDA receptors and endothelial nitric oxide synthase is involved in astrocyte-mediated neurovascular coupling.     

Enhancement of the endothelial NO synthase attenuates experimental diastolic heart failure

Background

Diastolic heart failure is a rising problem with a high incidence and similar mortality and morbidity compared to patients with systolic heart failure. Nevertheless, the underlying pathophysiology is still debated.

Aim

We investigated the effect of pharmacological enhancement of endothelial nitric oxide synthase (eNOS) on experimental diastolic heart failure (DHF).

Methods

DHF was induced in 60 DAHL salt-sensitive rats by salt diet in 8-week-old animals. 30 were treated with the eNOS enhancer AVE3085 (DHFeNOS) and 30 with placebo (DHF). Rats with normal salt intake served as controls.

Results and conclusion

Diastolic dysfunction with increased diastolic stiffness constant and increased left ventricular (LV) pressure was analyzed by invasive pressure–volume loop measurements in the DHF group compared to controls. Cardiac hypertrophy as indicated by LV mass measurements by echocardiography, and increased cardiac collagen content as measured by immunohistochemistry were associated with an increased activation state of calcineurin, AKT, ERK½, but not JNK and p38 kinases. Titin isoforms were not altered in this model of DHF. Treatment with AVE3085 significantly increased eNOS mRNA and protein levels in the cardiac tissue and decreases NAD(P)H oxidase subunits p22phox and gp91phox. Diastolic dysfunction was attenuated and cardiac hypertrophy and fibrosis were improved in comparison with untreated DHF animals. This was associated with a normalized activation state of calcineurin, AKT and ERK½. Therefore, we suggest that targeting the NO system might yield a future therapeutic aim for the treatment of DHF.     

Counteracting neuronal nitric oxide synthase proteasomal degradation improves glucose transport in insulin-resistant skeletal muscle from Zucker fa/fa rats

Aims/hypothesis

Insulin-mediated glucose transport and utilisation are decreased in skeletal muscle from type 2 diabetic and glucose-intolerant individuals because of alterations in insulin receptor signalling, GLUT4 translocation to the plasma membrane and microvascular blood flow. Catalytic activity of the muscle-specific isoform of neuronal nitric oxide synthase (nNOS) also participates in the regulation of glucose transport and appears to be decreased in a relevant animal model of drastic insulin resistance, the obese Zucker fa/fa rat. Our objective was to determine the molecular mechanisms involved in this defect.

Methods

Isolated rat muscles and primary cultures of myocytes were used for western blot analysis of protein expression, immunohistochemistry, glucose uptake measurements and GLUT4 translocation assays.

Results

nNOS expression was reduced in skeletal muscle from fa/fa rats. This was caused by increased ubiquitination of the enzyme and subsequent degradation by the ubiquitin proteasome pathway. The degradation occurred through a greater interaction of nNOS with the chaperone heat-shock protein 70 and the co-chaperone, carboxyl terminus of Hsc70-interacting protein (CHIP). In addition, an alteration in nNOS sarcolemmal localisation was observed. We confirmed the implication of nNOS breakdown in defective insulin-induced glucose transport by demonstrating that blockade of proteasomal degradation or overexpression of nNOS improved basal and/or insulin-stimulated glucose uptake and GLUT4 translocation in primary cultures of insulin-resistant myocytes.

Conclusions/interpretation

Recovery of nNOS in insulin-resistant muscles should be considered a potential new approach to address insulin resistance.     

Distribution of nitric oxide synthase containing neurons in the rectal myenteric plexus and anal canal

PURPOSE: Following the demonstration that a novel neurotransmitter, nitric oxide (NO), is released during neurogenic relaxation of the internal anal sphincter in vitro,it has been suggested that NO could mediate the rectoanal inhibitory reflex in vivo.The aim of this study was to establish whether the distribution of NO-producing nerves in the anorectum is consistent with this proposed role. METHODS: NO is synthesized in neurons which contain the enzyme nitric oxide synthase and their presence in the anorectum was determined in tissue obtained from nine abdominoperineal and three anterior resection specimens in patients undergoing surgery for rectal carcinoma. Cryostat sections were stained for nitric oxide synthase immunoreactivity, pan-neuronal/axonal immunoreactivity, and NADPH diaphorase activity. RESULTS: Nitric oxide synthase immunoreactivity is present in a subpopulation of neurons in rectal myenteric ganglia which also contain NADPH diaphorase activity. Use of the latter histochemical technique enabled the structure and distribution of nitric oxide synthase containing neurons to be determined in whole-mount preparations. Individual neurons have Dogiel type 1 morphology and are present throughout the rectal myenteric plexus. In the distal rectum, positively stained axons enter shunt fascicles which descend into the anal canal, where they ramify into and throughout the internal anal sphincter. Within the sphincter, positively stained nerves lie in close proximity to smooth muscle cells. CONCLUSION: These results are consistent with the hypothesis that NO is the neurotransmitter that mediates the rectoanal inhibitory reflex.     

Abnormal modulation of cell protective systems in response to ischemic/reperfusion injury is important in the development of mouse sickle cell hepatopathy

Background

Sickle cell disease, a genetic red cell disorder inherited in an autosomal recessive manner, occurs throughout the world. Hepatic dysfunction and liver damage may be present in sickle cell disease, but the pathogenesis of these conditions is only partially understood.

Design and Methods

Transgenic mice with sickle cell disease (SAD mice) and wild-type mice were exposed to an ischemic/reperfusion stress. The following parameters were evaluated: hematologic profile, transaminase and bilirubin levels, liver histopathology, and mRNA levels of nuclear factor-κB p65, endothelial nitric oxide synthase, inducible nitric oxide synthase, heme oxygenase-1 and phosphodiesterase-1, -2, -3, and -4 genes in hepatocytes obtained by laser-capture microdissection. Immunoblotting was used to analyze the expression of the following proteins: nuclear factor-κB p65 and phospho-nuclear factor-κB p65, heme oxygenase-1, biliverdin reductase, heat shock protein-70, heat shock protein-27 and peroxiredoxin-6. A subgroup of SAD mice was treated with the phosphodiesterase-4 inhibitor rolipram (30 mg/Kg/day by gavage) during the ischemic/reperfusion protocol.

Results

In SAD mice the ischemic/reperfusion stress induced liver damage compatible with sickle cell disease hepatopathy, which was associated with: (i) lack of hypoxia-induced nuclear factor-κB p65 activation; (ii) imbalance in the endothelial/inducible nitric oxide synthase response to ischemic/reperfusion stress; (iii) lack of hypoxia-induced increased expression of heme oxygenase-1/biliverdin reductase paralleled by a compensatory increased expression of heat shock proteins 70 and 27 and peroxiredoxin-6; and (iv) up-regulation of the phosphodiesterase-1, -2, -3, and -4 genes. In SAD mice the phosphodiesterase-4 inhibitor rolipram attenuated the ischemic/reperfusion-related microcirculatory dysfunction, reduced the inflammatory cell infiltration and induced the heme oxygenase-1/biliverdin reductase cytoprotective systems.

Conclusions

In SAD mice, sickle cell hepatopathy is associated with perturbed nuclear factor-κB p65 signaling with an imbalance of endothelial/inducible nitric oxide synthase levels, lack of heme oxygenase-1/biliverdin reductase expression and up-regulation of two novel cytoprotective systems: heat shock protein-27 and peroxiredoxin-6.     

l-arginine and endogenous nitric oxide protect the gastric mucosa from endothelin-1-induced gastric ulcers in rats

We have reported that endothelin-1 induces gastric ulcer characterized by a potent long-lasting vasoconstriction of the regional microvasculature. Nitric oxide synthesized froml-arginine has been shown to regulated gastric mucosal blood flow, and inhibition of its synthesis has been shown to delay the healing of gastric ulcers. We examined the effect of exogenousl-arginine and the inhibition of nitric oxide synthesis on the development of endothelin-1-induced gastric ulcers. In rats anesthetized with urethane, a continuous intravenous infusion ofl-ord-arginine (10 mg·kg^?1·min^?1) was followed, 15 min later, by a submucosal injection of endothelin-1 (200 pmol/kg) in the anterior wall of the gastric body. In another group, rats were intravenously pretreated with N^ω-nitro-l-arginine-methyl ester (1–10mg/kg), a nitric oxide synthesis inhibitor, and then injected with endothelin-1 (40 pmol/kg). Twenty-four h later,l-arginine, but notd-arginine, had significantly reduced the extent and the severity of the endothelin-1-induced ulcer (mucosal wall damage, 18.11 ± 4.79% and 88.14 ±7.06%, respectively; mean ± SD,P<0.001), and the nitric oxide synthesis inhibitor (10mg/kg) had increased the endothelin-1-induced mucosal damage (ulcer length, 3.8 ± 1.2 mm and 1.1 ± 0.2 mm, respectively,P<0.01). Continuous gastric mucosal blood flow measurements showed thatl-arginine antagonized the endothelin-1-induced vasoconstriction.l-arginine protected the gastric mucosa from the ulcerogenic action of endothelin-1 and antagonized its vasoconstrictive action. The inhibition of endogenous nitric oxide potentiated the ulcerogenic effect of endothelin-1 on rat gastric mucosa.     

Glucagon-like Peptide-1 Receptor Agonist Liraglutide Inhibits Endothelin-1 in Endothelial Cell by Repressing Nuclear Factor-Kappa B Activation

Purpose

The increase in endothelin-1 (ET-1) and the decrease in endothelial nitric oxide synthase (eNOS) both induce vasoconstriction and lead to molecular changes associated with diabetes mellitus and atherosclerosis. Glucagon-like peptide-1 (GLP-1) activation stimulates insulin secretion and may prevent atherosclerosis by increasing eNOS synthesis. However, there is paucity of information on the effect of GLP-1 activation on ET-1 expression. This study was conducted to address this issue.

Methods and Results

Human umbilical vein endothelial cells (HUVECs) were incubated with different concentrations of liraglutide, a GLP-1 agonist, and the expression of ET-1 and eNOS and activity of NF-κB were measured. Liraglutide, in a concentration-dependent manner, was observed to promote eNOS expression and to inhibit ET-1 expression both at mRNA and protein levels. Liraglutide also inhibited NF-κB phosphorylation and its translocation from cytoplasm to the nucleus. To ascertain the role of NF-κB activation in the altered expression of ET-1 and eNOS, we treated HUVECs with phorbol 12-myristate 13-acetate (PMA). PMA activated NF-κB and reversed the effects of liraglutide on eNOS and ET-1 expression. The effects of PMA on eNOS and ET-1 expression were reproduced in experiments wherein cells were treated with TNF-α. Further, we measured the generation of IL-6, apowerful pro-inflammatory molecule released by endothelial cells, as a measure of cellular function. PMA increased IL-6 generation, and this effect was blocked by liraglutide.

Conclusions

Our observations suggest liraglutide suppresses ET-1 expression by inhibiting the phosphorylation of NF-κB. This mechanism may underlie the potential anti-atherosclerotic effects of GLP-1 agonists. Of note, these effects of liraglutide were seen in an in vitro setting wherein cellular glucose concentrations were elevated.