Calcium-dependent Nitric Oxide Research Articles

Cellular nitric oxide synthesis is affected by disorders in the interdependent [Formula: see text] and [Formula: see text] dynamics during cystic fibrosis disease.

Calcium ([Formula: see text]), inositol trisphosphate ([Formula: see text]), and nitric oxide (NO) signaling are essential to maintain the structural integrity and physiological activity of fibroblast cells. The accumulation of excess quantity of NO for longer periods can lead to a variety of fibrotic disorders, including heart disease, penile fibrosis in Peyronie's disease (PD), and cystic fibrosis. The dynamics of these three signaling processes and their interdependence in fibroblast cells are not clearly known to date. A systems biology model is proposed using reaction-diffusion equations for calcium, [Formula: see text], and calcium-dependent NO synthesis in fibroblast cells. The finite element method (FEM) is used to examine [Formula: see text], [Formula: see text], and NO regulation and dysregulation in cells. The results throw light on the conditions that disturb the coupled [Formula: see text] and [Formula: see text] dynamics and the influence of these factors on the levels of NO concentration in the fibroblast cell. The findings suggest that changes in source inflow, buffers, and diffusion coefficient might induce an increase or reduction in nitric oxide and [Formula: see text] synthesis, resulting in fibroblast cell diseases. Furthermore, the findings provide new information regarding the size and intensity of diseases in response to changes in several factors of their dynamics, which has been linked to the development of cystic fibrosis and cancer. This knowledge could be valuable for developing novel approaches to the diagnosis of diseases and therapies for various disorders of fibroblast cells.

Loss of CFTR function is associated with reduced bitter taste receptor-stimulated nitric oxide innate immune responses in nasal epithelial cells and macrophages.

Bitter taste receptors (T2Rs) are G protein-coupled receptors identified on the tongue but expressed all over the body, including in airway cilia and macrophages, where T2Rs serve an immune role. T2R isoforms detect bitter metabolites (quinolones and acyl-homoserine lactones) secreted by gram negative bacteria, including Pseudomonas aeruginosa, a major pathogen in cystic fibrosis (CF). T2R activation by bitter bacterial products triggers calcium-dependent nitric oxide (NO) production. In airway cells, the NO increases mucociliary clearance and has direct antibacterial properties. In macrophages, the same pathway enhances phagocytosis. Because prior studies linked CF with reduced NO, we hypothesized that CF cells may have reduced T2R/NO responses, possibly contributing to reduced innate immunity in CF. Immunofluorescence, qPCR, and live cell imaging were used to measure T2R localization, calcium and NO signaling, ciliary beating, and antimicrobial responses in air-liquid interface cultures of primary human nasal epithelial cells and immortalized bronchial cell lines. Immunofluorescence and live cell imaging was used to measure T2R signaling and phagocytosis in primary human monocyte-derived macrophages. Primary nasal epithelial cells from both CF and non-CF patients exhibited similar T2R expression, localization, and calcium signals. However, CF cells exhibited reduced NO production also observed in immortalized CFBE41o- CF cells and non-CF 16HBE cells CRISPR modified with CF-causing mutations in the CF transmembrane conductance regulator (CFTR). NO was restored by VX-770/VX-809 corrector/potentiator pre-treatment, suggesting reduced NO in CF cells is due to loss of CFTR function. In nasal cells, reduced NO correlated with reduced ciliary and antibacterial responses. In primary human macrophages, inhibition of CFTR reduced NO production and phagocytosis during T2R stimulation. Together, these data suggest an intrinsic deficiency in T2R/NO signaling caused by loss of CFTR function that may contribute to intrinsic susceptibilities of CF patients to P. aeruginosa and other gram-negative bacteria that activate T2Rs.

The mitochondrial NO-synthase/guanylate cyclase/protein kinase G signaling system underpins the dual effects of nitric oxide on mitochondrial respiration and opening of the permeability transition pore.

The available data on the involvement of nitric oxide (NO) and mitochondrial calcium-dependent NO synthase (mtNOS) in the control of mitochondrial respiration and the permeability transition pore (mPTP) are contradictory. We have proposed that the mitochondrial mtNOS/guanylate cyclase/protein kinase G signaling system (mtNOS-SS) is also implicated in the control of respiration and mPTP, providing the interplay between NO and mtNOS-SS, which, in turn, may result in inconsistent effects of NO. Therefore, using rat liver mitochondria, we applied specific inhibitors of the enzymes of this signaling system to evaluate its role in the control of respiration and mPTP opening. Steady-state respiration was supported by pyruvate, glutamate, or succinate in the presence of hexokinase, glucose, and ADP. When applied at low concentrations, l-arginine (to 500µm) and NO donors (to 50µm) activated the respiration and increased the threshold concentrations of calcium and d,l-palmitoylcarnitine required for the dissipation of the mitochondrial membrane potential and pore opening. Both effects were eliminated by the inhibitors of NO synthase, guanylate cyclase, and kinase G, which denotes the involvement of mtNOS-SS in the activation of respiration and deceleration of mPTP opening. At high concentrations, l-arginine and NO donors inhibited the respiration and promoted pore opening, indicating that adverse effects induced by an NO excess dominate over the protection provided by mtNOS-SS. Thus, these results demonstrate the opposite impact of NO and mtNOS-SS on the respiration and mPTP control, which can explain the dual effects of NO.

How chronic administration of benzodiazepines leads to unexplained chronic illnesses: A hypothesis

It is thought that an ill defined biochemical cascade may lead to protracted withdrawal symptoms subsequent to discontinuance of routine use of benzodiazepine class drugs and establish chronic illness in some patients. In this review, published findings are presented that support the novel concept that withdrawal from benzodiazepine class drugs can trigger elevated and sustained levels of a potent oxidant called peroxynitrite via potentiation of the L-type voltage-gated calcium channels, and in the later stages of withdrawal, via excessive N-methyl-D-aspartate receptor activity, as well. Potentiation of L-type voltage-gated calcium channels and excessive N-methyl-D-aspartate receptor activity both result in calcium influx into the cell that triggers nitric oxide synthesis. In pathophysiological conditions, such increased nitric oxide synthesis leads to peroxynitrite formation.The downstream effects of peroxynitrite formation that may occur during withdrawal ultimately lead to further peroxynitrite production in a system of overlapping vicious cycles collectively referred to as the NO/ONOO(−) cycle. Once triggered, the elements of the NO/ONOO(−) cycle perpetuate pathophysiology, perhaps including reduced GABAA receptor functioning, that may explain protracted withdrawal associated symptoms while the vicious cycle nature of the NO/ONOO(−) cycle may explain how withdrawal becomes a chronic state.Suboptimal levels of tetrahydrobiopterin may be one risk factor for the development of the protracted withdrawal syndrome as this will lead to partial nitric oxide uncoupling and resultant peroxynitrite formation. Nitric oxide uncoupling results in superoxide production as calcium-dependent nitric oxide synthases attempt to produce nitric oxide in response to L-type voltage-gated calcium channel-mediated calcium influx that is known to occur during withdrawal. The combination of nitric oxide and superoxide produced, as when partial uncoupling occurs, react together in a very rapid, diffusion limited reaction to form peroxynitrite and thereby trigger the NO/ONOO(−) cycle.The NO/ONOO(−) cycle may explain the nature of the protracted withdrawal syndrome and the related constellation of symptoms that are also common in other illnesses characterized as NO/ONOO(−) disorders such as myalgic encephalomyelitis/chronic fatigue syndrome and fibromyalgia.

Production, amplification and systemic propagation of redox messengers in plants? The phloem can do it all!

Rapid long-distance signalling is an emerging topic in plant research, and is particularly associated with responses to biotic and abiotic stress. Systemic acquired resistance (SAR) to pathogen attack is dependent on nitric oxide (NO) and reactive oxygen species (ROS) such as hydrogen peroxide (H2 O2 ). By comparison, systemic wound responses (SWRs) and systemic acquired acclimation (SAA) to abiotic stress encounters are triggered by rapid waves of H2 O2 , calcium and electrical signalling. Efforts have been made to decipher the relationship between redox messengers, calcium and other known systemic defence signals. Less is known about possible routes of signal transduction throughout the entire plant. Previously, the phloem has been suggested to be a transport conduit for mobile signals inducing SAR, SWR and SAA. This review highlights the role of the phloem in systemic redox signalling by NO and ROS. A not yet identified calcium-dependent NO source and S-nitrosoglutathione reductase are candidate regulators of NO homeostasis in the phloem, whereas ROS concentrations are controlled by NADPH oxidases and the H2 O2 -scavenging enzyme ascorbate peroxidase. Possible amplification mechanisms in phloem-mediated systemic redox signalling are discussed.

Resveratrol lowers blood pressure in spontaneously hypertensive rats via calcium-dependent endothelial NO production

ABSTRACTObjective: Resveratrol, a polyphenol of natural compounds, has beneficial cardiovascular effects, many of which are mediated by nitric oxide (NO). Resveratrol increases intracellular calcium and activates AMP-activated protein kinase (AMPK), all of which could increase NO production. We hypothesized that resveratrol via a calcium-dependent NO production lowers blood pressure (BP) in spontaneously hypertensive rats (SHR). Methods: Acetylcholine (Ach)-induced endothelium-dependent relaxations in rat aortas were examined by organ chamber. Blood pressures were determined by radiotelemetry methods. Results: Incubation of isolated aortas from SHR with resveratrol dramatically improved vasorelaxation induced by Ach. Preincubation of aortas with endothelial NO synthase (eNOS) inhibitor or calcium chelant blunted the effects of resveratrol on Ach-induced relaxation, as wells as NO production and eNOS phosphorylation. In animal studies, administration of resveratrol significantly lowered systemic BP in SHR. Conclusion: Resveratrol increases endothelial NO production to improve endothelial dysfunction and lowers BP in hypertensive rats, which depends on calcium-eNOS activation.

Photoluminescence of Bioceramic Materials (PLB) as a Complementary and Alternative Therapy for Diabetes

The rapid rise of diabetes in Asia and Africa is surpassing that of western countries. Diabetes incurs a significant financial burden on patients and national economies. Consequently, factors such as a miserable life-long treatment of hypoglycemic therapy and the possibility of drug intolerance necessitate a search for non-pharmacological alternatives to reduce the requirement of anti-diabetic drugs. Photoluminescence refers to materials that absorb light energy and then release that energy in the form of light. In this study, Photoluminescence of Bioceramic Materials (PLB) was applied to control hyperglycemia and glycosuria in diabetes from the bench to the clinical bedside examination. The PLB treatments resulted in a tendency to promote glucose diffusion into C2C12 cell line and show a significant decrease in glycosuria in STZ (streptozotocin) induced diabetic rats. The possible mechanisms of the PLB effects on hyperglycemia also correlate with our previous publication and include molecular diffusion, calcium dependent nitric oxide, suppression of oxidative stress and autonomous nervous system regulation. In the future, PLB may have the role of clinical applications on ameliorating hyperglycemia and improving diabetes-related complications.

Enhancement of Transdermal Delivery of Indomethacin and Tamoxifen by Far-Infrared Ray-Emitting Ceramic Material (BIOCERAMIC): A Pilot Study

BIOCERAMIC have been found to modulate various biological effects. Our earlier published research on various cell lines demonstrated that BIOCERAMIC promoted microcirculation, upregulated calcium-dependent nitric oxide and calmodulin, and exerted an antioxidant effect by increasing hydrogen peroxide scavenging ability. The development of pain relief systems requires most possible minimum doses and methods for effective local control of pain, so as to protect liver and renal function. There is also clinical necessary to develop targeted delivery of estrogen inhibitor in the breast using a local drug release system, to protect the breast from the increased cancer risk associated with the use of estrogen therapy. We compared the viscosity of BIOCERAMIC irradiated water and control water, and found that BIOCERAMIC might weaken the hydrogen bonds. Such breaks are caused by the loss of hydrogen bond covalence resulting from electron rearrangement. The purposes of this study were thus to investigated a transdermal drug delivery model using Franz cell apparatus for Indomethacin and Tamoxifen. The results showed that BIOCERAMIC enhanced the diffusion and permeability of the drugs. Therefore, we suggest that BIOCERAMIC might enhance the penetration performed by hydrogen bond weakening due to physical induction, and may facilitate local drug delivery in transdermal systems.

Distant effects of nitric oxide inhalation in lavage‐induced lung injury in anaesthetised pigs

Inhalation of nitric oxide (INO) exerts both local and distant effects. INO in healthy pigs causes down-regulation of endogenous nitric oxide (NO) production and vasoconstriction in lung regions not reached by INO, especially in hypoxic regions, which augments hypoxic pulmonary vasoconstriction. In contrast, in pigs with endotoxemia-induced lung injury, INO causes increased NO production in lung regions not reached by INO. The aim of this study was to investigate whether INO exerts distant effects in surfactant-depleted lungs. Twelve pigs were anaesthetised, and the left lower lobe (LLL) was separately ventilated. Lavage injury was induced in all lung regions, except the LLL. In six pigs, 40 ppm INO was given to the LLL (INO group), and the effects on endogenous NO production and blood flow in the lavage-injured lung regions were studied. Six pigs served as a control group. NO concentration in exhaled air (ENO), NO synthase (NOS) activity and cyclic guanosine monophosphate (cGMP) in lung tissue, and regional pulmonary blood flow were measured. The calcium (Ca(2+) )-dependent NOS activity was lower (P < 0.05) in the lavage-injured lung regions in the INO group than in the control group. There were no measurable differences between the groups for Ca(2+) -independent NOS activity, cGMP, ENO, or regional pulmonary blood flow. Regional INO did not increase endogenous NO production in lavage-injured lung regions not directly reached by INO, but instead down-regulated the constitutive calcium-dependent nitric oxide synthase activity, indicating that NO may inhibit its own synthesis.

NMDA receptor involvement in antidepressant-like effect of pioglitazone in the forced swimming test in mice

Previously, we showed that pioglitazone exerts its antidepressant-like effect through peroxisome proliferator-activated receptor gamma receptors and demonstrated the possible involvement of calcium-dependent nitric oxide synthase inhibitors. Based upon the in vitro results, pioglitazone reduces N-methyl-D-aspartate (NMDA)-mediated calcium currents in hippocampal neurons. In this study, we evaluated the involvement of the NMDA receptor (NMDAR) on the antidepressant-like effect of pioglitazone in the forced swimming test (FST) in mice. After the assessment of locomotor activity in the open-field test, mice were forced to swim individually and the immobility time of the last 4 min was evaluated. Pioglitazone was administered orally with doses of 5, 10, and 20 mg/kg 4 h before FST. To assess the involvement of NMDARs in the possible antidepressant-like effect of pioglitazone, a selective glutamate receptor agonist, NMDA (75 mg/kg, intraperitoneally [i.p.] or 20 ng/mouse, intracerebroventricularly [i.c.v.]), was administered before pioglitazone (20 mg/kg). To further determine a possible role of NMDARs in this effect, a noncompetitive antagonist of the NMDA, MK-801 (0.05 mg/kg, i.p. or 100 ng/mouse, i.c.v.), was coadministered with pioglitazone (10 mg/kg) 4 h prior to FST. Pioglitazone (20 mg/kg) administered 4 h prior to FST significantly reduced the immobility time. Coadministration of the noneffective doses of pioglitazone and MK-801 revealed an antidepressant-like effect in FST. Moreover, NMDA significantly reversed the antidepressant-like effect of pioglitazone administered 4 h prior to FST. The antidepressant-like effect of pioglitazone in the FST is mediated partly through NMDAR signaling. This study provides a new approach for the treatment of depression.

Differences in the In Vitro Antiplatelet Effect of Dexibuprofen, Ibuprofen, and Flurbiprofen in Human Blood

In this study, we compared the in vitro pharmacodynamic profile of dexibuprofen, ibuprofen, and flurbiprofen to identify possible differences in antiplatelet activity. In whole blood samples from healthy volunteers, we measured platelet aggregation induced by adenosine diphosphate, collagen and arachidonic acid, platelet thromboxane B(2) (TxB(2)), lipopolysaccharide-induced prostaglandin E(2), leukocyte 6-keto-prostaglandin F(1α) (PGF(1α)), and nitric oxide induced by both constitutive and inducible pathways before and after incubation with increasing concentrations of acetylsalicylic acid, dexibuprofen, ibuprofen, or flurbiprofen. The concentration that inhibited (IC(50)) or increased each variable by 50% was calculated. All 3 drugs inhibited platelet aggregation in a dose-dependent manner, TxB(2), prostaglandin E(2), and 6-keto-PGF(1α), and increased calcium-induced nitric oxide production. Dexibuprofen showed greater antiplatelet potency than ibuprofen and flurbiprofen, and its profile was similar to that of aspirin. For example, IC(50) values for arachidonic acid-induced platelet aggregation were 0.85 ± 0.06 μM for dexibuprofen, 14.76 ± 1.22 μM for ibuprofen, 6.39 ± 0.51 μM for flurbiprofen, and 0.38 ± 0.03 μM for aspirin. All drugs inhibited both thromboxane and prostacyclin synthesis, but the IC(50) anti-TxB(2)/IC(50) anti-6-keto-PGF(1α) ratio was 0.21 ± 0.03 for dexibuprofen, 1.05 ± 0.08 for ibuprofen, 0.79 ± 0.11 for flurbiprofen, and 0.46 ± 0.06 for aspirin. All drugs increased calcium-dependent nitric oxide production. The aryl propionic acid derivative dexibuprofen was the most potent antiplatelet drug, and its pharmacodynamic profile is similar to aspirin.

Resolvin E1 protects the rat heart against reperfusion injury

The purpose of the present study was to assess whether resolvin E1 (RvE1), an anti-inflammatory mediator derived from eicosapentaenoic acid, would limit myocardial infarct size in the rat. The H9c2 cell line was used to assess whether RvE1 has direct protective effects on cardiomyocytes. In in vivo experiments, Male Sprague-Dawley rats underwent 30 min of ischemia/4 h of reperfusion. Before reperfusion, rats received intravenous RvE1 (0, 0.03, 0.1, or 0.3mg/kg). In in vitro experiments, H9c2 cells were incubated with RvE1 (0, 1, 10, 100, or 1000 nM). Cells were subjected to 18 h of incubation under normoxic conditions, 16 h of hypoxia, or 16 h of hypoxia and 2 h of reoxygenation. In vivo, RvE1 dose dependently reduced infarct size (30.7 +/- 1.7% of the area at risk in the control group and 29.1 +/- 1.6%, 14.7 +/- 1.3%, and 9.0 +/- 0.6% in the 0.03, 0.1, and 0.3 mg/kg groups, respectively, P < 0.001). In vitro, RvE1 increased viability and decreased apoptosis in a dose-dependent fashion in cells exposed to hypoxia or hypoxia/reoxygenation. A maximal effect was achieved at a concentration of 100 nM. RvE1 augmented phosphoinositide 3-kinase activity, attenuated caspase-3 activity, and augmented calcium-dependent nitric oxide synthase activity in cells exposed to hypoxia or hypoxia/reoxygenation. RvE1 increased Akt, ERK1/2, and endothelial nitric oxide synthase phosphorylation and attenuated the levels of activated caspase-3 and phosphorylated p38 levels. AG-1478, an EGF receptor tyrosine kinase inhibitor, blocked the protective effect of RvE1 both in vivo and in vitro and attenuated the RvE1-induced increase in Akt and ERK1/2 phosphorylation. In conclusion, RvE1, an anti-inflammatory mediator derived from eicosapentaenoic acid, has a direct protective effect on cardiomyocytes against ischemia-reperfusion injury and limits infarct size when administered intravenously before reperfusion.

Molecular, biochemical characterization and localization of neuronal nitric oxide synthase in human neutrophil

Nitric oxide (NO) is a potent free radical with diverse roles in regulating several important functions of neutrophils (PMNs), including chemotaxis, adhesion, aggregation, apoptosis, bacterial killing, however the expression status, biochemical characterization and localization study remains the least defined in human PMNs. The present study was attempted to characterize expression of NOS isoforms and NO generation potential in healthy human PMNs. Real time PCR using NOS primers have identified neuronal NOS (nNOS) as higher copy number (45 fold, *p&lt;0.001) than inducible NOS (iNOS). Identification of nNOS PDZ domain (Exon‐2, N‐terminal) was done by RT‐PCR using exon‐2 specific primer and Western blotting using N‐terminal specific antibody. Localization of nNOS was explored by electron and confocal microscopy. Biochemical study involving NOS activity (L‐Arginine to L‐Citruline conversion) exhibited higher calcium dependent activity (0.21±0.05 pmol/30 min./107cells) than calcium independent (0.039±.007 pmol/30 min/107) which positively correlated with higher expression of nNOS. Immunocytochemical studies exhibited distribution of nNOS isoform primarily in the cytosol, granules, mitochondria and nucleus. The results thus suggest constitutive expression of PDZ domain containing nNOS localized mainly to cytoplasm contributes to calcium dependent activity and NO generation in human PMNs.

Additive Effect of TAK-491, a New Angiotensin Receptor Blocker, and Pioglitazone, in Reducing Myocardial Infarct Size

We assessed the effects of TAK-491 (a newly designed potent and selective ARB) alone and in combination with pioglitazone (PIO) on myocardial infarct size (IS). Rats received TAK-491 (0.3, 1, 3, or 10 mgkg(-1)d(-1)), PIO (1.0 or 2.5 mgkg(-1)d(-1)), or PIO 2.5 mgkg(-1)d(-1) with TAK-491 (1 or 3 mgkg(-1)d(-1)) for 4 days. On day 5 rats underwent 30-minute coronary artery occlusion and 4-hour reperfusion. Area at risk (AR) was assessed by blue dye and IS by TTC. Left ventricular (LV) dimensions and function was assessed by echocardiography 35 days after infarction. TAK (1.0-10 mgkg(-1)d(-1)), PIO (1.0 to 2.5 mgkg(-1)d(-1)), PIO2.5+TAK1.0, and PIO2.5+TAK3.0 significantly reduced IS. IS was the smallest in the TAK 10.0, followed by PIO+TAK 3.0. The protective effects of TAK and PIO were additive, as IS was smaller in the PIO2.5+TAK1.0 than in PIO 2.5 alone (p = 0.008) or TAK1.0 alone (p = 0.002); and in PIO2.5+TAK3.0 than in PIO alone (p < 0.001) or TAK3.0 alone (p < 0.001). TAK, PIO and their combination tended to attenuate LV remodeling and improved LV function 35 days after infarction; however, the differences among individual groups were not statistically significant. Both TAK-491 and PIO increased calcium-dependent nitric oxide synthase activity, whereas only PIO increased COX2 expression and activity. Both PIO and TAK-491 increased Akt, ERK 1/2 and eNOS phosphorylation and inhibited BAX activation. TAK-491 and PIO independently limited myocardial IS in a dose-dependent fashion; and the effects were additive. The mechanism of protection and the role of TAK-491 in this clinical setting should be further investigated.

Immunoglobulin G from Breast Cancer Patients in Stage I Stimulates Muscarinic Acetylcholine Receptors in MCF7 Cells and Induces Proliferation. Participation of Nitric Oxide Synthase-Derived Nitric Oxide

Muscarinic acetylcholine receptors (mAChR) belong to the G-protein-coupled receptor family and are extensively expressed in most cells in mammals. We had reported the expression of mAChR in murine and human breast tumors. The presence of antibodies in the sera of patients with different tumors directed against self-proteins has been recently described. In this work, we investigated the presence of autoantibodies against mAChR in the sera of breast cancer patients in stage I (T1N0Mx-IgG). IgG purification was performed by affinity chromatography in protein G-agarose. We also studied the ability of these antibodies to modulate the proliferation of MCF-7 breast tumor cells by the MTS colorimetric assay. The ability of T1N0Mx-IgG to stimulate muscarinic signaling pathway via nitric oxide synthase was tested by Griess reaction. We demonstrated M(3) and M(4) receptors expression in MCF-7 cells. T1N0Mx-IgG promotes cell proliferation, mimicking the action of the muscarinic agonist carbachol. This effect was preferentially due to M(3) receptor activation in tumor cells via phospholipase C-induced nitric oxide liberation by calcium-dependent nitric oxide synthases. IgG from control patients was unable to produce this effect. IgG from patients with breast cancer in early stages could be promoting tumor progression by muscarinic activation, and its presence could be determining the prognosis of this illness.

Pharmacological inhibition of PTEN limits myocardial infarct size and improves left ventricular function postinfarction

Phosphoinositide 3-kinase (PI3K) mediates myocardium protective signaling through phosphorylation of phosphatidylinositol (Ptdins) to produce Ptdins(3,4,5)P(3). Lipid phosphatase and tensin homolog on chromosome 10 (PTEN) antagonizes PI3K activity by dephosphorylating Ptdins(3,4,5)P(3); therefore, the inhibition of PTEN enhances PI3K/Akt signaling and could prevent myocardium from ischemia-reperfusion (I/R) injury. Here we studied 1) whether the pharmacological inhibition of PTEN by bisperoxovanadium molecules [BpV(HOpic)] attenuates simulated I/R (SIR) injury in vitro and 2) whether the administration of BpV(HOpic) either before or after ischemia limits myocardial infarct size (IS) and ameliorates cardiodysfunction caused by infarction. First, adult rat cardiomyocytes were treated with or without BpV(HOpic) and then exposure to SIR. Second, anesthetized rats received BpV(HOpic) either before or after ischemia. IS was assessed at 4 h reperfusion, and left ventricular function was evaluated by echocardiography at 28 days postreperfusion. As a result, BpV(HOpic) decreased cell death, improved 3-[4,5-yl]-2,5-diphenyltetrazolium bromide (MTT) viability, and reduced apoptosis in cells exposed to SIR. These protective effects of BpV(HOpic) are associated with increased phospho-Akt and the repression of caspase-3 activity. Second, the administration of BpV(HOpic) significantly reduced IS and suppressed caspase-3 activity following I/R injury and consequentially improved cardiac function at 28 day postinfarction. These beneficial effects of BpV(HOpic) are attributed to increases in myocardial levels of phosphorylation of Akt/endothelial nitric oxide synthase (eNOS), ERK-1/2, and calcium-dependent nitric oxide synthase activity. In conclusion, the pharmacological inhibition of PTEN protects against I/R injury through the upregulation of the PI3K/Akt/eNOS/ERK prosurvival pathway, suggesting a new therapeutic strategy to combat I/R injury.

Mitochondrial-Driven Ubiquinone Enhances Extracellular Calcium-Dependent Nitric Oxide Production and Reduces Glycochenodeoxycholic Acid-Induced Cell Death in Hepatocytes

Ca(2+) mobilization, nitric oxide (NO), and oxidative stress have been involved in cell death induced by hydrophobic bile acid in hepatocytes. The aim of the study was the elucidation of the effect of the antioxidant mitochondrial-driven ubiquinone (Mito Q) on the intracellular Ca(2+) concentration, NO production, and cell death in glycochenodeoxycholic acid (GCDCA)-treated HepG2 cells. The role of the regulation of the intracellular Ca(2+) concentration by Ca(2+) chelators (EGTA or BAPTA-AM), agonist of Ca(2+) entrance (A23187) or NO (L-NAME or NO donor), was assessed during Mito Q cytoprotection in GCDCA-treated HepG2 cells. Cell death, NO synthase (NOS)-1, -2, and -3 expression, Ca(2+) mobilization, and NO production were evaluated. GCDCA reduced the intracellular Ca(2+) concentration and NOS-3 expression and enhanced cell death in HepG2. NO donor prevented and L-NAME enhanced GCDCA-induced cell death. The reduction of Ca(2+) entry by EGTA, but not its release from intracellular stores by BAPTA-AM, reduced the expression of NOS-3 and enhanced cell death in control and GCDCA-treated cells. Mito Q prevented the reduction of intracellular Ca(2+) concentration, NOS-3 expression, NO production, and cell death in GCDCA-treated HepG2 cells. The conclusion is that the recovery of Ca(2+)-dependent NOS-3 expression by Mito Q may be considered an additional cytoprotective property of an antioxidant.

Calcium-dependent nitric oxide production is involved in the cytoprotective properties of n-acetylcysteine in glycochenodeoxycholic acid-induced cell death in hepatocytes

The intracellular oxidative stress has been involved in bile acid-induced cell death in hepatocytes. Nitric oxide (NO) exerts cytoprotective properties in glycochenodeoxycholic acid (GCDCA)-treated hepatocytes. The study evaluated the involvement of Ca 2+ on the regulation of NO synthase (NOS)-3 expression during N-acetylcysteine (NAC) cytoprotection against GCDCA-induced cell death in hepatocytes. The regulation of Ca 2+ pools (EGTA or BAPTA-AM) and NO ( l-NAME or NO donor) production was assessed during NAC cytoprotection in GCDCA-treated HepG2 cells. The stimulation of Ca 2+ entrance was induced by A23187 in HepG2. Cell death, Ca 2+ mobilization, NOS-1, -2 and -3 expression, AP-1 activation, and NO production were evaluated. GCDCA reduced intracellular Ca 2+ concentration and NOS-3 expression, and enhanced cell death in HepG2. NO donor prevented, and l-NAME enhanced, GCDCA-induced cell death. The reduction of Ca 2+ entry by EGTA, but not its release from intracellular stores by BAPTA-AM, enhanced cell death in GCDCA-treated cells. The stimulation of Ca 2+ entrance by A23187 reduced cell death and enhanced NOS-3 expression in GCDCA-treated HepG2 cells. The cytoprotective properties of NAC were related to the recovery of intracellular Ca 2+ concentration, NOS-3 expression and NO production induced by GCDCA-treated HepG2 cells. The increase of NO production by Ca 2+-dependent NOS-3 expression during NAC administration reduces cell death in GCDCA-treated hepatocytes.

Effects of Calcitonin Gene-Related Peptide on the Expression and Activity of Nitric Oxide Synthase During Mandibular Bone Healing in Rabbits: An Experimental Study

Previous studies have found that calcitonin gene-related peptide (CGRP) takes part in the local regulation of bone growth and metabolism. However, the detailed regulatory mechanism of CGRP in the bone healing has not been explored. The objective of this study was to investigate the effects and the regulatory mechanism of CGRP on the expression and activity of nitric oxide synthase (NOS) in bony callus during mandibular defect healing. To determine the effect of CGRP on bony callus, a bone defect in the left mandible was created in 48 adult rabbits (divided randomly into experimental and control group) and half of them underwent inferior alveolar nerve amputation. The bony callus were collected 4, 7, 14, and 28 days after operation, and the expressions of CGRP and NOS in the paraffin slices were analyzed with immunohistochemical staining. The activity of calcium-dependent nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) in the fresh specimens were measured with the NOS detecting kit. In the immunohistochemical analysis of bony callus, the immunohistochemical stain of CGRP was lower in experimental groups than in control groups from 4 to 14 days (P< .05 or P< .01), and the stain of eNOS showed the same phenomena from 4 to 7 days (P< .01), but the stain of iNOS did not show any statistical difference. In the NOS activity analysis, the activity of cNOS was lower in experimental groups than in control groups from 4 to 7 days (P< .05 or P< .01), and the activity of iNOS was lower in experimental groups than in control groups from 7 to 14 days (P< .01). The expression and activity of NOS has a positive correlation with CGRP expression during bone healing. CGRP may promote fracture healing via regulating the expression and the activity of NOS.

Calcium-dependent release of NO from intracellular S-nitrosothiols

The paper describes a novel cellular mechanism for rapid calcium-dependent nitric oxide (NO) release. This release occurs due to NO liberation from S-nitrosothiols. We have analysed the changes of NO concentration in acutely isolated pancreatic acinar cells. Supramaximal acetylcholine (ACh) stimulation induced a Ca(2+)-dependent increase in the fluorescence in the majority of cells loaded with the NO probe DAF-FM via a patch pipette. The ACh-induced NO signals were insensitive to inhibitors of calmodulin and protein kinase C but were inhibited by calpain antagonists. The initial part of the NO signals induced by 10 muM ACh showed little sensitivity to inhibition of NO synthase (NOS); however, cell pretreatment with NO donors (increasing cellular S-nitrosothiol contents) substantially enhanced the initial component of NO responses. Pancreatic acinar cells were able to generate fast calcium-dependent NO responses when stimulated with physiological or supramaximal doses of secretagogues. Importantly, the source of this NO is the already available S-nitrosothiol store rather than de novo synthesis by NOS. A similar mechanism of NO release was found in dorsal root ganglia neurons.