Influence of Nitric Oxide-Cyclic GMP and Oxidative STRESS on Amyloid-ß Peptide Induced Decrease of Na,K-ATPase Activity in Rat Hippocampal Slices.

Amyloid-ß peptide (Aß) has been shown to cause synaptic dysfunction and can render neurons vulnerable to excitotoxicity and oxidative stress. Na,K-ATPase plays an important role to maintain cell ionic equilibrium and it can be modulated by N-methyl-D-aspartate (NMDA)-nitric oxide (NO)-cyclic GMP pathway. Disruption of NO synthase (NOS) activity and reactive oxygen species (ROS) production could lead to changes in Na,K-ATPase isoforms' activities that may be detrimental to the cells. Our aim was to evaluate the signaling pathways of Aß in relation to NMDA-NOS-cyclic GMP versus oxidative stress on α1-/α2,3-Na,K-ATPase activities in rat hippocampal slices. Aß1-40 induced a concentration-dependent increase of NOS activity and increased cyclic guanosine monophosphate (cGMP), TBARS (thiobarbituric acid reactive substances), and 3-Nitrotyrosine (3-NT)-modified protein levels in rat hippocampal slices. The increase in NOS activity and cyclic GMP levels induced by Aß1-40 was completely blocked by MK-801 (inhibitor of NMDA receptor) and L-NAME (inhibitor of NOS) pre-treatment but changes in TBARS levels were only partially blocked by both compounds. The Aß treatment also decreased Na,K-ATPase activity which was reverted by N-nitro-L-arginine methyl ester hydrochloride (L-NAME) but not by MK-801 pre-treatment. The decrease in enzyme activity induced by Aß was isoform-specific since only α1-Na,K-ATPase was affected. These findings suggest that the activation of NMDA-NOS signaling cascade linked to α2,3-Na,K-ATPase activity may mediate an adaptive, neuroprotective response to Aß in rat hippocampus.

The α2 Na+/K+-ATPase isoform mediates LPS-induced neuroinflammation.

Na+/K+-ATPase is a transmembrane ion pump that is essential for the maintenance of ion gradients and regulation of multiple cellular functions. Na+/K+-ATPase has been associated with nuclear factor kappa B (NFκB) signalling, a signal associated with lipopolysaccharides (LPSs)-induced immune response in connection with activated Toll-like receptor 4 (TLR4) signalling. However, the contribution of Na+/K+-ATPase to regulating inflammatory responses remains elusive. We report that mice haploinsufficient for the astrocyte-enriched α2Na+/K+-ATPase isoform (α2+/G301R mice) have a reduced proinflammatory response to LPS, accompanied by a reduced hypothermic reaction compared to wild type litter mates. Following intraperitoneal injection of LPS, gene expressions of Tnf-α, Il-1ß, and Il-6 was reduced in the hypothalamus and hippocampus from α2+/G301R mice compared to α2+/+ littermates. The α2+/G301R mice experienced increased expression of the gene encoding an antioxidant enzyme, NRF2, in hippocampal astrocytes. Our findings indicate that α2Na+/K+-ATPase haploinsufficiency negatively modulates LPS-induced immune responses, highlighting a rational pharmacological target for reducing LPS-induced inflammation.

Intermittent fasting uncovers and rescues cognitive phenotypes in PTEN neuronal haploinsufficient mice.

Phosphatase and tensin homolog (PTEN) is an important protein with key modulatory functions in cell growth and survival. PTEN is crucial during embryogenesis and plays a key role in the central nervous system (CNS), where it directly modulates neuronal development and synaptic plasticity. Loss of PTEN signaling function is associated with cognitive deficits and synaptic plasticity impairment. Accordingly, Pten mutations have a strong link with autism spectrum disorder. In this study, neuronal Pten haploinsufficient male mice were subjected to a long-term environmental intervention - intermittent fasting (IF) - and then evaluated for alterations in exploratory, anxiety and learning and memory behaviors. Although no significant effects on spatial memory were observed, mutant mice showed impaired contextual fear memory in the passive avoidance test - an outcome that was effectively rescued by IF. In this study, we demonstrated that IF modulation, in addition to its rescue of the memory deficit, was also required to uncover behavioral phenotypes otherwise hidden in this neuronal Pten haploinsufficiency model.

Clinical efficacy, safety and anti-inflammatory activity of two sevelamer tablet forms in patients on low-flux hemodialysis.

Sevelamer hydrochloride is an ionic exchange resin with high affinity for phosphate. This phosphate-binding agent has few serious adverse reactions with the advantage of reducing total and low density lipoprotein (LDL) cholesterol levels. However, it is controversial as to whether sevelamer hydrochloride can modulate the inflammatory response via endotoxin reduction. Therefore, a single-center, open-label, prospective and randomized study was performed to compare the clinical efficacy, safety and anti-inflammatory activity of two sevelamer hydrochloride tablet forms a branded tablet form, Renagel (Genzyme manufacturer) and its generic equivalent (EMS manufacturer). Twenty-eight chronic kidney disease volunteer patients at stage 5 (CDK 5D), on chronic low-flux hemodialysis carried out in 4-hour sessions, three times a week, were studied. The serum phosphorus, ionic calcium, total cholesterol and fractions, bicarbonate, blood pH, interleukin (IL)-6, IL-10, IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) levels were collected prior to dialysis at mid-week. The incidence of gastrointestinal adverse effects were determined at the end of the phosphate-binder washout period as well as at the end of the fourth and eighth weeks of use of both tablet forms. The same magnitude of reduction in serum phosphorus was observed with both sevelamer tablet forms. Only the Renagel group showed lower total cholesterol and lower LDL cholesterol levels at the fourth and eighth week versus baseline. No significant differences in serum cytokine levels were identified in either drug group. However, the incidence of intestinal obstipation was higher among patients who used the generic equivalent form. In conclusion, Renagel and its EMS generic equivalent tablet forms have a similar clinical efficacy in reducing phosphorus in CKD 5D patients on low-flux hemodialysis and a similar safety profile.

Influence of the dopaminergic system, CREB, and transcription factor-B on cocaine neurotoxicity

Cocaine is a widely used drug and its abuse is associated with physical, psychiatric and social problems. Abnormalities in newborns have been demonstrated to be due to the toxic effects of cocaine during fetal development. The mechanism by which cocaine causes neurological damage is complex and involves interactions of the drug with several neurotransmitter systems, such as the increase of extracellular levels of dopamine and free radicals, and modulation of transcription factors. The aim of this review was to evaluate the importance of the dopaminergic system and the participation of inflammatory signaling in cocaine neurotoxicity. Our study showed that cocaine activates the transcription factors NF-κB and CREB, which regulate genes involved in cellular death. GBR 12909 (an inhibitor of dopamine reuptake), lidocaine (a local anesthetic), and dopamine did not activate NF-κB in the same way as cocaine. However, the attenuation of NF-κB activity after the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, suggests that the activation of NF-κB by cocaine is, at least partially, due to activation of D1 receptors. NF-κB seems to have a protective role in these cells because its inhibition increased cellular death caused by cocaine. The increase in BDNF (brain-derived neurotrophic factor) mRNA can also be related to the protective role of both CREB and NF-κB transcription factors. An understanding of the mechanisms by which cocaine induces cell death in the brain will contribute to the development of new therapies for drug abusers, which can help to slow down the progress of degenerative processes.

Ouabain activates NFκB through an NMDA signaling pathway in cultured cerebellar cells.

Na,K-ATPase, an ion pump, has been shown to interact with other proteins in signaling complexes in cardiac myocytes, renal and glial cells, and several other cell types. Our previous in vivo studies indicated that intrahippocampal administration of ouabain (OUA), an inhibitor of Na,K-ATPase, induces NFκB activation, leading to an increase in mRNA levels of target genes of this transcription factor in the rat hippocampus. The present work investigated whether OUA can regulate NF-κB in primary cultured rat cerebellar cells. Cells were treated with different concentrations of OUA (1, 10 or 100 µM) for different periods of time (1, 2 and 4 h). OUA induced a time- and concentration-dependent activation of NFκB (peak of activation: 10 µM, 2 h), involving both p50/p65 and p50/p50 NFκB dimers. OUA (10 µM, 2 h) induced upregulation of tumor necrosis factor α (Tnf-α), interleukin-1ß (Il-1ß), and brain derived neurotrophic factor (Bdnf) mRNA levels. Both NFκB activation and gene expression activation induced by OUA (10 µM) were abolished when cells were pre-treated for 20 min with MK-801 (N-Methyl-D-Aspartate (NMDA) receptor antagonist), manumycin A (farnesyltransferase inhibitor), PP-1(Src-family tyrosine kinase inhibitor) and PD98059 (mitogen-activated protein kinase (MAPK) inhibitor). OUA (10 µM) alone or in the presence of MK-801, PP-1, PD98059 did not cause cell death or DNA fragmentation. These findings suggest that OUA activates NFκB by NMDA-Src-Ras-like protein through MAPK pathways in cultured cerebellar cells. This pathway may mediate an adaptive response in the central nervous system.

Curcumin requires tumor necrosis factor α signaling to alleviate cognitive impairment elicited by lipopolysaccharide.

A decline in cognitive ability is a typical feature of the normal aging process, and of neurodegenerative disorders such as Alzheimer's, Parkinson's and Huntington's diseases. Although their etiologies differ, all of these disorders involve local activation of innate immune pathways and associated inflammatory cytokines. However, clinical trials of anti-inflammatory agents in neurodegenerative disorders have been disappointing, and it is therefore necessary to better understand the complex roles of the inflammatory process in neurological dysfunction. The dietary phytochemical curcumin can exert anti-inflammatory, antioxidant and neuroprotective actions. Here we provide evidence that curcumin ameliorates cognitive deficits associated with activation of the innate immune response by mechanisms requiring functional tumor necrosis factor α receptor 2 (TNFR2) signaling. In vivo, the ability of curcumin to counteract hippocampus-dependent spatial memory deficits, to stimulate neuroprotective mechanisms such as upregulation of BDNF, to decrease glutaminase levels, and to modulate N-methyl-D-aspartate receptor levels was absent in mice lacking functional TNFRs. Curcumin treatment protected cultured neurons against glutamate-induced excitotoxicity by a mechanism requiring TNFR2 activation. Our results suggest the possibility that therapeutic approaches against cognitive decline designed to selectively enhance TNFR2 signaling are likely to be more beneficial than the use of anti-inflammatory drugs per se.

Effect of activation of canonical Wnt signaling by the Wnt-3a protein on the susceptibility of PC12 cells to oxidative and apoptotic insults

Wnt proteins are involved in tissue development and their signaling pathways play an important role during embryogenesis. Wnt signaling can promote cell survival, which is beneficial for neurons, but could also lead to tumor development in different tissues. The present study investigated the effects of a Wnt protein on the susceptibility of a neural tumor cell line (PC12 cells) to the cytotoxic compounds ferrous sulfate (10 mM), staurosporine (100 and 500 nM), 3-nitropropionic acid (5 mM), and amyloid β-peptide (Aβ25-35; 50 µM). Cells (1 x 10(6) cells/mL) were treated with the Wnt-3a recombinant peptide (200 ng/mL) for 24 h before exposure to toxic insults. The Wnt-3a protein partially protected PC12 cells, with a 6-15 percent increase in cell viability in the presence of toxic agents, similar to the effect measured using the MTT and lactate dehydrogenase cell viability assays. The Wnt-3a protein increased protein expression of β-catenin by 52 percent compared to control. These findings suggest that Wnt signaling can protect neural cells against apoptosis induced by toxic agents, which are relevant to the pathogenesis of Alzheimer’s and Huntington’s diseases.

Effect of activation of canonical Wnt signaling by the Wnt-3a protein on the susceptibility of PC12 cells to oxidative and apoptotic insults.

Wnt proteins are involved in tissue development and their signaling pathways play an important role during embryogenesis. Wnt signaling can promote cell survival, which is beneficial for neurons, but could also lead to tumor development in different tissues. The present study investigated the effects of a Wnt protein on the susceptibility of a neural tumor cell line (PC12 cells) to the cytotoxic compounds ferrous sulfate (10 mM), staurosporine (100 and 500 nM), 3-nitropropionic acid (5 mM), and amyloid ß-peptide (Aß25-35; 50 µM). Cells (1 x 10(6) cells/mL) were treated with the Wnt-3a recombinant peptide (200 ng/mL) for 24 h before exposure to toxic insults. The Wnt-3a protein partially protected PC12 cells, with a 6-15% increase in cell viability in the presence of toxic agents, similar to the effect measured using the MTT and lactate dehydrogenase cell viability assays. The Wnt-3a protein increased protein expression of ß-catenin by 52% compared to control. These findings suggest that Wnt signaling can protect neural cells against apoptosis induced by toxic agents, which are relevant to the pathogenesis of Alzheimer's and Huntington's diseases.

Influence of N-methyl-D-aspartate receptors on ouabain activation of nuclear factor-κB in the rat hippocampus.

It has been shown that ouabain (OUA) can activate the Na,K-ATPase complex and mediate intracellular signaling in the central nervous system (CNS). Inflammatory stimulus increases glutamatergic transmission, especially at N-methyl-D-aspartate (NMDA) receptors, which are usually coupled to the activation of nitric oxide synthase (NOS). Nuclear factor-κB (NF-κB) activation modulates the expression of genes involved in development, plasticity, and inflammation. The present work investigated the effects of OUA on NF-κB binding activity in rat hippocampus and the influence of this OUA-Na,K-ATPase signaling cascade in NMDA-mediated NF-κB activation. The findings presented here are the first report indicating that intrahippocampal administration of OUA, in a concentration that did not alter Na,K-ATPase or NOS activity, induced an activation of NF-κB, leading to increases in brain-derived neurotrophic factor (Bdnf), inducible NOS (iNos), tumor necrosis factor-α (Tnf-α), and B-cell leukemia/lymphoma 2 (Bcl2) mRNA levels. This response was not linked to any significant signs of neurodegeneration as showed via Fluoro-Jade B and Nissl stain. Intrahippocampal administration of NMDA induced NF-κB activation and increased NOS and α(2/3) -Na,K-ATPase activities. NMDA treatment further increased OUA-induced NF-κB activation, which was partially blocked by MK-801, an antagonist of NMDA receptor. These results suggest that OUA-induced NF-κB activation is at least in part dependent on Na,K-ATPase modulatory action of NMDA receptor in hippocampus. The interaction of these signaling pathways could be associated with biological mechanisms that may underlie the basal homeostatic state linked to the inflammatory signaling cascade in the brain.

Stress-induced neuroinflammation: mechanisms and new pharmacological targets

Stress is triggered by numerous unexpected environmental, social or pathological stimuli occurring during the life of animals, including humans, which determine changes in all of their systems. Although acute stress is essential for survival, chronic, long-lasting stress can be detrimental. In this review, we present data supporting the hypothesis that stress-related events are characterized by modifications of oxidative/nitrosative pathways in the brain in response to the activation of inflammatory mediators. Recent findings indicate a key role for nitric oxide (NO) and an excess of pro-oxidants in various brain areas as responsible for both neuronal functional impairment and structural damage. Similarly, cyclooxygenase-2 (COX-2), another known source of oxidants, may account for stress-induced brain damage. Interestingly, some of the COX-2-derived mediators, such as the prostaglandin 15d-PGJ2 and its peroxisome proliferator-activated nuclear receptor PPARγ, are activated in the brain in response to stress, constituting a possible endogenous anti-inflammatory mechanism of defense against excessive inflammation. The stress-induced activation of both biochemical pathways depends on the activation of the N-methyl-D-aspartate (NMDA) glutamate receptor and on the activation of the transcription factor nuclear factor kappa B (NFκB). In the case of inducible NO synthase (iNOS), release of the cytokine TNF-α also accounts for its expression. Different pharmacological strategies directed towards different sites in iNOS or COX-2 pathways have been shown to be neuroprotective in stress-induced brain damage: NMDA receptor blockers, inhibitors of TNF-α activation and release, inhibitors of NFκB, specific inhibitors of iNOS and COX-2 activities and PPARγ agonists. This article reviews recent contributions to this area addressing possible new pharmacological targets for the treatment of stress-induced neuropsychiatric disorders.

Therapeutic carbamazepine (CBZ) and valproic acid (VPA) monitoring in children using saliva as a biologic fluid / Monitoramento terapêutico de carbamazepina e ácido valproico em saliva de crianças

OBJECTIVE: The aim of the study was to analyze retrospectively carbamazepine (CBZ) and valproic acid (VPA) salivary data collected from epileptic children during a 3-year period. METHODS: Saliva samples stimulated by citric acid were assayed by FPIA method. One hundred and three patients (aged 1-14 years) were in CBZ or VPA monotherapy or in CBZ-VPA combined therapy. RESULTS: VPA salivary levels were linearly related with daily dose, but a non-linear relationship was found for CBZ, in patients under monotherapy. VPA did not alter saliva CBZ concentration. Conversely, CBZ reduced VPA salivary levels. Non-responsive children displayed higher VPA concentrations. CBZ levels in uncontrolled patients showed non-significant difference in relation with controlled subjects even though their daily doses were higher. CONCLUSION: Citric acid stimulated saliva is reliable enough to perform therapeutic drug monitoring. Saliva drug levels in non-responsive patients would be explained according to the generalized efflux transporter overexpression hypothesis.

OBJETIVO: O objetivo deste estudo foi avaliar retrospectivamente por 3 anos a partir de dados salivares, as terapias com carbamacepina (CBZ) e ácido valproico (VPA) em pacientes pediátricos. MÉTODOS: Foram avaliadas amostras de saliva estimuladas com ácido cítrico por método FPIA em 103 pacientes (idades 1-14 anos) em monoterapia com CBZ ou VPA ou terapia combinada CBZ-VPA. RESULTADOS: Níveis salivares de VPA se relacionaram linearmente com a dose diária, e a relação não linear foi encontrada em pacientes com CBZ. VPA não alterou as concentrações salivares de CBZ, porém a CBZ reduziu os níveis salivares de VPA em pacientes com terapia combinada. Pacientes refratários apresentaram altas concentrações de VPA. Os níveis de CBZ em pacientes não controlados não apresentaram diferenças significativas em relação aos pacientes controlados quando as doses diárias foram mais elevadas. CONCLUSÃO: Saliva estimulada com ácido cítrico é adequada para o monitoramento terapêutico. Níveis da droga na saliva em pacientes que não responderam ao tratamento pode ser explicado pelo transporte de efluxo generalizado.

Stress-induced neuroinflammation: mechanisms and new pharmacological targets.

Stress is triggered by numerous unexpected environmental, social or pathological stimuli occurring during the life of animals, including humans, which determine changes in all of their systems. Although acute stress is essential for survival, chronic, long-lasting stress can be detrimental. In this review, we present data supporting the hypothesis that stress-related events are characterized by modifications of oxidative/nitrosative pathways in the brain in response to the activation of inflammatory mediators. Recent findings indicate a key role for nitric oxide (NO) and an excess of pro-oxidants in various brain areas as responsible for both neuronal functional impairment and structural damage. Similarly, cyclooxygenase-2 (COX-2), another known source of oxidants, may account for stress-induced brain damage. Interestingly, some of the COX-2-derived mediators, such as the prostaglandin 15d-PGJ2 and its peroxisome proliferator-activated nuclear receptor PPARgamma, are activated in the brain in response to stress, constituting a possible endogenous anti-inflammatory mechanism of defense against excessive inflammation. The stress-induced activation of both biochemical pathways depends on the activation of the N-methyl-D-aspartate (NMDA) glutamate receptor and on the activation of the transcription factor nuclear factor kappa B (NFkappaB). In the case of inducible NO synthase (iNOS), release of the cytokine TNF-alpha also accounts for its expression. Different pharmacological strategies directed towards different sites in iNOS or COX-2 pathways have been shown to be neuroprotective in stress-induced brain damage: NMDA receptor blockers, inhibitors of TNF-alpha activation and release, inhibitors of NFkappaB, specific inhibitors of iNOS and COX-2 activities and PPARgamma agonists. This article reviews recent contributions to this area addressing possible new pharmacological targets for the treatment of stress-induced neuropsychiatric disorders.

Amyloid beta-peptide activates nuclear factor-kappaB through an N-methyl-D-aspartate signaling pathway in cultured cerebellar cells.

Amyloid beta-peptide (A beta) likely causes functional alterations in neurons well prior to their death. Nuclear factor-kappaB (NF-kappaB), a transcription factor that is known to play important roles in cell survival and apoptosis, has been shown to be modulated by A beta in neurons and glia, but the mechanism is unknown. Because A beta has also been shown to enhance activation of N-methyl-D-aspartate (NMDA) receptors, we investigated the role of NMDA receptor-mediated intracellular signaling pathways in A beta-induced NF-kappaB activation in primary cultured rat cerebellar cells. Cells were treated with different concentrations of A beta1-40 (1 or 2 microM) for different periods (6, 12, or 24 hr). MK-801 (NMDA antagonist), manumycin A and FTase inhibitor 1 (farnesyltransferase inhibitors), PP1 (Src-family tyrosine kinase inhibitor), PD98059 [mitogen-activated protein kinase (MAPK) inhibitor], and LY294002 [phosphatidylinositol 3-kinase (PI3-k) inhibitor] were added 20 min before A beta treatment of the cells. A beta induced a time- and concentration-dependent activation of NF-kappaB (1 microM, 12 hr); both p50/p65 and p50/p50 NF-kappaB dimers were involved. This activation was abolished by MK-801 and attenuated by manumycin A, FTase inhibitor 1, PP1, PD98059, and LY294002. A beta at 1 microM increased the expression of inhibitory protein I kappaB, brain-derived neurotrophic factor, inducible nitric oxide synthase, tumor necrosis factor-alpha, and interleukin-1 beta as shown by RT-PCR assays. Collectively, these findings suggest that A beta activates NF-kappaB by an NMDA-Src-Ras-like protein through MAPK and PI3-k pathways in cultured cerebellar cells. This pathway may mediate an adaptive, neuroprotective response to A beta.

Neutrophil function and metabolism in individuals with diabetes mellitus

Neutrophils act as first-line-of-defense cells and the reduction of their functional activity contributes to the high susceptibilityto and severity of infections in diabetes mellitus. Clinical investigations in diabetic patients and experimental studies in diabetic rats and mice clearly demonstrated consistent defects of neutrophil chemotactic, phagocytic and microbicidal activities. Other alterations that have been reported to occur during inflammation in diabetes mellitus include: decreased microvascular responses to inflammatory mediators such as histamine and bradykinin, reduced protein leakage and edema formation, reduced mast cell degranulation, impairment of neutrophil adhesionto the endothelium and migration to the site of inflammation, production of reactive oxygen species and reduced release of cytokines and prostaglandin by neutrophils, increased leukocyte apoptosis, and reduction in lymph node retention capacity. Since neutrophil function requires energy, metabolic changes (i.e., glycolytic and glutaminolytic pathways) may be involved in the reduction of neutrophil function observed in diabetic states. Metabolic routes by which hyperglycemia is linked to neutrophil dysfunction include the advanced protein glycosylation reaction, the polyol pathway, oxygen-free radical formation, the nitric oxide-cyclic guanosine-3'-5'monophosphate pathway, and the glycolytic and glutaminolytic pathways. Lowering of blood glucose levels by insulin treatment of diabetic patients or experimental animals has been reported to have significant correlation with improvement of neutrophil functional activity. Therefore, changes might be primarily linked to a continuing insulin deficiency or to secondary hyperglycemia occurring in the diabetic individual. Accordingly, effective control with insulin treatment is likely to be relevant during infection in diabetic patients.

Expression of inducible nitric oxide synthase is increased in patients with heart failure due to ischemic disease

The objective of the present study was to determine the relationship between nitric oxide synthases (NOS) and heart failure in cardiac tissue from patients with and without cardiac decompensation. Right atrial tissue was excised from patients with coronary artery disease (CAD) and left ventricular ejection fraction (LVEF) <35 percent (N = 10), and from patients with CAD and LVEF >60 percent (N = 10) during cardiac surgery. NOS activity was measured by the conversion of L-[H ]-arginine to L-[H ]-citrulline. Gene expression was quantified by the competitive reverse transcription-polymerase chain reaction. Both endothelial NOS (eNOS) activity and expression were significantly reduced in failing hearts compared to non-failing hearts: 0.36 ± 0.18 vs 1.51 ± 0.31 pmol mg-1 min-1 (P < 0.0001) and 0.37 ± 0.08 vs 0.78 ± 0.09 relative cDNA absorbance at 320 nm (P < 0.0001), respectively. In contrast, inducible NOS (iNOS) activity and expression were significantly higher in failing hearts than in non-failing hearts: 4.00 ± 0.90 vs 1.54 ± 0.65 pmol mg-1 min-1 (P < 0.0001) and 2.19 ± 0.27 vs 1.43 ± 0.13 cDNA absorbance at 320 nm (P < 0.0001), respectively. We conclude that heart failure down-regulates both eNOS activity and expression in cardiac tissue from patients with LVEF <35 percent. In contrast, iNOS activity and expression are increased in failing hearts and may represent an alternative mechanism for nitric oxide production in heart failure due to ischemic disease.

Expression of inducible nitric oxide synthase is increased in patients with heart failure due to ischemic disease.

The objective of the present study was to determine the relationship between nitric oxide synthases (NOS) and heart failure in cardiac tissue from patients with and without cardiac decompensation. Right atrial tissue was excised from patients with coronary artery disease (CAD) and left ventricular ejection fraction (LVEF) <35% (N = 10), and from patients with CAD and LVEF >60% (N = 10) during cardiac surgery. NOS activity was measured by the conversion of L-[H(3)]-arginine to L-[H(3)]-citrulline. Gene expression was quantified by the competitive reverse transcription-polymerase chain reaction. Both endothelial NOS (eNOS) activity and expression were significantly reduced in failing hearts compared to non-failing hearts: 0.36 +/- 0.18 vs 1.51 +/- 0.31 pmol mg-1 min-1 (P < 0.0001) and 0.37 +/- 0.08 vs 0.78 +/- 0.09 relative cDNA absorbance at 320 nm (P < 0.0001), respectively. In contrast, inducible NOS (iNOS) activity and expression were significantly higher in failing hearts than in non-failing hearts: 4.00 +/- 0.90 vs 1.54 +/- 0.65 pmol mg-1 min-1 (P < 0.0001) and 2.19 +/- 0.27 vs 1.43 +/- 0.13 cDNA absorbance at 320 nm (P < 0.0001), respectively. We conclude that heart failure down-regulates both eNOS activity and expression in cardiac tissue from patients with LVEF <35%. In contrast, iNOS activity and expression are increased in failing hearts and may represent an alternative mechanism for nitric oxide production in heart failure due to ischemic disease.

Influence of age on nitric oxide modulatory action on Na(+), K(+)-ATPase activity through cyclic GMP pathway in proximal rat trachea.

Age-related changes in the modulatory action of nitric oxide (NO) on cyclic GMP levels and Na(+),K(+)-ATPase activity in the proximal rat trachea were investigated using sodium nitroprusside, 8-bromo-cyclic GMP and okadaic acid. At 24 months, both control activities of Na(+), K(+)-ATPase and Mg(2+)-ATPase were decreased when compared to the segments from 4- and 12-month-old animals. However, cyclic GMP levels were similar among the three ages. Sodium nitroprusside (100 microM) induced stimulation of Na(+),K(+)-ATPase activity in segments from both 4- and 12-month-old animals, but not 24-month-old animals. The effect was specific for Na(+),K(+)-ATPase since Mg(2+)-ATPase activity was unaffected. Sodium nitroprusside induced an increase in nitrates/nitrites and cyclic GMP levels in proximal segments at 4, 12 and 24 months. The 8-bromo-cyclic GMP (100 microM) induced a similar specific stimulation of Na(+),K(+)-ATPase activity in segments from 4- and 12- but not 24-month-old animals. Okadaic acid (1 microM), a phosphatase-1 inhibitor, increased proximal Na(+), K(+)-ATPase but not Mg(2+)-ATPase activity in tissues from 4-, 12- and 24-month-old animals. Our results suggest that aging affects cyclic GMP pathway in proximal rat trachea by an action at the level of the cyclic GMP-dependent protein kinase.

Participation of the mouse implanting trophoblast in nitric oxide production during pregnancy.

While considerable progress has been made in elucidating nitric oxide (NO) regulatory mechanisms in the later stages of gestation, much less is known about its synthesis and role during embryo implantation. Thus, to evaluate the participation of the trophoblast in the production of NO during this phase, this study focused on NADPH-diaphorase activity and the distribution of NO synthase isoforms (NOS) using immunohistochemistry in pre- and postimplantation mouse embryos in situ and in vitro, as well as on NO production itself, measured as total nitrite, in trophoblast culture supernatants (Griess reaction). No NADPH-diaphorase activity was found in preimplanting embryos except after culturing for at least 48 h, when a few trophoblastic giant cells were positive. Conversely, postimplantation trophoblast cells either lodged into the implantation chamber (in situ) or after culturing (in vitro) showed intense NADPH-diaphorase activity. Also in the postimplantation trophoblast, the endothelial and inducible NOS (eNOS and iNOS) isoforms were immunodetected, under both in situ and in vitro conditions, although in different patterns. Extracts of ectoplacental cone also revealed bands of 135 and 130 kDa on SDS-PAGE that reacted with anti-eNOS and anti-iNOS, respectively, on Western blot. Analysis of the culture supernatant demonstrated that the nitrite concentration was 1) proportional to the number of cultured trophoblast cells, 2) almost completely abolished in the presence of N(omega)-nitro-L-arginine methyl ester, and 3) increased 2-fold in cultures stimulated with gamma-interferon. These results strongly suggest the production of NO from constitutive and inducible isoforms of NOS by the implanting mouse trophoblast. They also emphasize the possibility of the participation of these cells in vasodilatation and angiogenesis, and in cytotoxic mechanisms involved in the intense phagocytosis of injured maternal cells, which occur during the implantation process.

Nitric oxide modulates Na+, K+-ATPase activity through cyclic GMP pathway in proximal rat trachea.

The present work demonstrated that nitric oxide (NO) modulates Na+, K+-ATPase activity in the proximal rat trachea. Sodium nitroprusside induced concentration-dependent (10-100 microM) stimulation in proximal trachea Na+, K+-ATPase activity. The effect was specific for Na+, K+-ATPase since Mg-ATPase activity was unaffected. This NO-donor changed neither Na+, K+-ATPase nor Mg-ATPase activity in the distal segment. The modulatory action on Na+, K+-ATPase induced by sodium nitroprusside was linked to an increase in nitrates/nitrites and cyclic GMP levels in proximal segments. Modulation of proximal Na+, K+-ATPase activity by sodium nitroprusside was mimicked by S-nitroso-N-acetylpenicillamine (100 microM) and 8-bromo-cyclic GMP (100 microM). Both sodium nitroprusside and 8-bromo-cyclic GMP effects on Na+, K+-ATPase activity of proximal segments of trachea were blocked by 2 microM of KT 5823 (a cyclic GMP-dependent protein kinase inhibitor), but not by 0.5 microM of KT 5720 (a cyclic AMP-dependent protein kinase inhibitor). Both kinase inhibitors decreased proximal Na+, K+-ATPase activity, but did not change Mg-ATPase activity. Okadaic acid (1 microM), a phosphatase-1 inhibitor, increased proximal Na+, K+-ATPase but not Mg-ATPase activity. The effect of okadaic acid was non-additive with that of 8-bromo-cGMP on Na+, K+-ATPase activity. Our results suggest that NO modulates proximal rat trachea Na+, K+-ATPase activity through cyclic GMP and cyclic GMP-dependent protein kinase.