DAF-FM Fluorescence Research Articles

Role of superoxide–nitric oxide interactions in the accelerated age‐related loss of muscle mass in mice lacking Cu,Zn superoxide dismutase

SummaryMice lacking Cu,Zn superoxide dismutase (SOD1) show accelerated, age-related loss of muscle mass. Lack of SOD1 may lead to increased superoxide, reduced nitric oxide (NO), and increased peroxynitrite, each of which could initiate muscle fiber loss. Single muscle fibers from flexor digitorum brevis of wild-type (WT) and Sod1−/− mice were loaded with NO-sensitive (4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate, DAF-FM) and superoxide-sensitive (dihydroethidium, DHE) probes. Gastrocnemius muscles were analyzed for SOD enzymes, nitric oxide synthases (NOS), and 3-nitrotyrosine (3-NT) content. A lack of SOD1 did not increase superoxide availability at rest because no increase in ethidium or 2-hydroxyethidium (2-HE) formation from DHE was seen in fibers from Sod1−/− mice compared with those from WT mice. Fibers from Sod1−/− mice had decreased NO availability (decreased DAF-FM fluorescence), increased 3-NT in muscle proteins indicating increased peroxynitrite formation and increased content of peroxiredoxin V (a peroxynitrite reductase), compared with WT mice. Muscle fibers from Sod1−/− mice showed substantially reduced generation of superoxide in response to contractions compared with fibers from WT mice. Inhibition of NOS did not affect DHE oxidation in fibers from WT or Sod1−/− mice at rest or during contractions, but transgenic mice overexpressing nNOS showed increased DAF-FM fluorescence and reduced DHE oxidation in resting muscle fibers. It is concluded that formation of peroxynitrite in muscle fibers is a major effect of lack of SOD1 in Sod1−/− mice and may contribute to fiber loss in this model, and that NO regulates superoxide availability and peroxynitrite formation in muscle.

Urocortin II Causes Phosphorylation of eNOS and Stimulation of NO Production in Cardiac Myocytes

AIM: Urocortin II (UcnII) exerts beneficial effects in heart failure. In cardiac myocytes, UcnII exerts positive inotropic and lusitropic effects through a PKA-dependent pathway. We tested the hypothesis that, in addition, UcnII stimulates endothelial NO synthase (eNOS) and evaluated the underlying signaling pathways and mechanisms.METHODS: UcnII-induced phosphorylation of Akt and eNOS was measured using phospho-specific antibodies. Isolated cardiac myocytes were loaded with 5x10-6M DAF-FM and UcnII-induced changes in NO production were assessed by changes in DAF-FM fluorescence in electrically paced myocytes (0.5 Hz, room temperature) by means of confocal microscopy.RESULTS: In rabbit ventricular myocytes, UcnII caused increases in phosphorylation of Akt at Ser473 (+89.4±21.4%) and Thr308 (+60.4±39.7%) and phosphorylation of eNOS at Ser1177 (+49.6±25.9%; n=6-11, all P<0.05 vs untreated controls). Wortmannin (300nM) and LY294002 (10-5M), inhibitors of PI3K, largely reduced UcnII-induced phosphorylation of Akt and eNOS, respectively (n=11-20, P<0.05). Cellular NO production increased by 41.1±5.5% (n=20, P<0.01), which was inhibited by eNOS inhibitors, L-NIO (10-5M) and L-NAME (1mM) by ∼30% (n=4-8, P<0.05). Inhibition of PKA by H89 (5x10-6M) also reduced eNOS phosphorylation (n=11, P<0.05) but did not affect Akt phosphorylation. Direct stimulation of cAMP/PKA signaling via forskolin (10-5M) increased phosphorylation of eNOS (n=5, P<0.05) but not of Akt. When both PI3K/Akt (LY294002 10-5M) and cAMP/PKA (H89 10-6M) signaling were inhibited, the UcnII-induced increase of [NO]i was attenuated by ∼20% (n=10, P<0.01). UcnII also increased [NO]i in mouse, rat, and human ventricular myocytes as well as in rabbit atrial myocytes (n=4-12).SUMMARY: We conclude that, in cardiac myocytes, UcnII causes eNOS phosphorylation to stimulate cellular NO production via both cAMP/PKA and PI3K/Akt signaling.

Bradykinin B2 Receptor KO Mice Are Protected From Thrombosis by A Platelet Spreading Defect

AbstractAbstract 3198 Background:Bradykinin B2 receptor KO mice (B2R KO) have delayed rose bengal and ferric chloride carotid artery thrombosis times and long tail bleeding times. In B2R KO mice, elevated serum angiotensin II (AngII) binds to an over-expressed angiotensin receptor 2 (AT2R) to increase plasma NO and PGI2 (Blood 108:192, 2006). The combined presence of elevated AngII and AT2R was essential to have the thrombosis protection phenotype. However, thrombosis remained delayed in B2R KO mice despite reduction in AngII levels from 258+64 to 40+15 pg/ml using losartan, an angiotension 1 receptor antagonist. These data suggested an additional pathway for thrombosis protection in B2R KO mice. Methods and Results:B2R KO mice have elevated bradykinin and bradykinin 1–5 levels with normal thrombin generation times. Losartan treatment lowered ACE mRNA in mice. B2R KO mice also had elevated Mas mRNA, the receptor for angiotensin1-7, with overexpressed renal AT2R and Mas antigens. Treatment of B2R KO with A-779, a Mas antagonist, shortened thrombosis time (58+4 to 38+4 min) and tail bleeding time (170+13 to 88+8 sec) and lowered plasma concentrations of nitrate (21.5+3.6 to 15+5 micromolar) and 6-keto-PGF1alpha (259+103 to 132+58 pg/ml). Immunofluorescent staining and immunoblot demonstrate AT2R and Mas antigen on murine platelets. “Threshold” ADP- and thrombin-induced platelet aggregation was identical for B2R KO and wild type platelets. Platelet activation by arachidonic acid, ionomycin, thrombin, or collagen as measured by flow cytometry (the activated α2bβ3 integrin complex and P-selectin) was similar between B2R KO and wild type platelets. Although static and flow adherence to collagen was normal, B2R KO platelets had defects in spreading on collagen (20+0.6 versus 27+0.4 microns WT, p<0.0001) under both static and flow conditions. B2R KO platelets had increased DAF-FM fluorescence (649+41 versus 405+36 AFU, p=0.0012), suggesting increased platelet NO. Increasing concentrations of GSNO, a NO donor, or carbaprostacyclin, a stable derivative of prostacyclin, inhibited mouse platelet spreading in a concentration-dependent manner. B2R KO and wild type platelets did not show a difference in cGMP [631+143 (Mean+SD) vs 594+80 fmol/108 plts] or cAMP [505+227 vs 568+151 fmol/108 plts] levels. However, B2R KO platelets had increased constitutive and GSNO-induced total protein nitrosylation on biotin-switch assay. N-acetylcysteine, a protein nitrosylation inhibitor and antioxidant, corrected the spreading defect in B2R KO platelets. Conclusion:B2R KO mice are protected from thrombosis by over-expression of two receptors from the renin-angiotensin system that results in increased NO and prostacyclin and an acquired platelet spreading defect. These data suggest that a platelet spreading defect alone protects from thrombosis and indicate that the receptors AT2R and MAS compensate the absence of the B2R. Last, these studies show a novel mechanism(s) by which receptors in the renin-angiotensin system influence platelet function and arterial thrombosis risk in vivo. Disclosures:No relevant conflicts of interest to declare.

Nitric oxide release follows endothelial nanomechanics and not vice versa

In the vascular endothelium, mechanical cell stiffness (К) and nitric oxide (NO) release are tightly coupled. "Soft" cells release more NO compared to "stiff" cells. Currently, however, it is not known whether NO itself is the primary factor that softens the cells or whether NO release is the result of cell softening. To address this question, a hybrid fluorescence/atomic force microscope was used in order to measure changes in К and NO release simultaneously in living vascular endothelial cells. Aldosterone was applied to soften the cells transiently and to trigger NO release. NO synthesis was then either blocked or stimulated and, simultaneously, К was measured. Cell indentation experiments were performed to evaluate К, while NO release was measured either by an intracellular NO-dependent fluorescence indicator (DAF-FM/DA) or by NO-selective electrodes located close to the cell surface. After the application of aldosterone, К decreases, within 10 min, to 80.5 ± 1.7% of control (100%). DAF-FM fluorescence intensity increases simultaneously to 132.9 ± 2.2%, which indicates a significant increase in the activity of endothelial NO synthase (eNOS). Inhibition of eNOS (by N (ω)-nitro-L: -arginine methyl ester) blocks the NO release, but does not affect the aldosterone-induced changes in К. Application of an eNOS-independent NO donor (NONOate/AM) raises intracellular NO concentration, but, again, does not affect К. Data analysis indicates that a decrease of К by about 10% is sufficient to induce a significant increase of eNOS activity. In conclusion, these nanomechanic properties of endothelial cells in vascular endothelium determine NO release, and not vice versa.

Abstract 4105: Nitric-oxide-mediated inhibition of Na+/H+ exchange (NHE) is a novel mechanism of bile acid induced damage: Relevance to esophageal carcinogenesis

Abstract Background: Barrett's esophagus (BE) is a premalignant condition that is associated with gastroesophageal reflux disease (GERD). Gastric acid and bile acids are two components of refluxate that induce proliferation, DNA damage and oxidative stress. In combination, the effects of gastric acid and bile acids are amplified and synergistic. The major goal of this study was to evaluate the effect of bile acids alone or in combination with acid on intracellular pH (pHi). A mechanism responsible for bile acid-induced pHi alterations was examined. Methods: CP-A cells (non-dysplastic Barrett's esophagus cells) were treated with medium acidified to pH 5.5 and/or 0.5mM bile acid cocktail in the presence/absence of inhibitors of nitric oxide synthase (NOS) or NHE (sodium-hydrogen exchanger). pHi was measured by BCECF microfluorimetry in perfused cells, nitric oxide by DAF-FM fluorescence and DNA damage by the comet assay. Western blot analysis was used to detect the expression of phosphorylated (activated) eNOS, iNOS and NHE1. Results: A dose-dependent decrease in pHi was observed in CP-A cells exposed to bile acids. Bile acid-induced intracellular acidification was abolished when NO production was inhibited by a nonspecific NOS inhibitor L-NAME or by thea specific iNOS inhibitor 1400W. In contrast the NHE inhibitor DMA, significantly decreased the bile acid-mediated pHi response. Exposure of the cells to acidified medium (pH5.5) containing a 0.5mM bile acid cocktail, markedly decreased pHi pHi to a value below extracellular pH. Importantly, medium acidified to pH5.5 containing 0.5mM bile acids induced a similar decrease in pHi as medium acidified to pH 4.5 without bile acids. This The same treatment resulteds in a 250% increase in acid-mediated DNA damage compared to medium acidified to pH5.5 in CP-A cells. Conclusion: In summary, a new mechanism of bile acid induced damage was identified. Bile acids alone or in combination with acidic pH significantly decrease intracellular pH in esophageal cells. The results suggest the effect ofresponse tpo bile acids is the consequence of NOS activation and that leads to NHE inhibition. The mMarked increase in DNA damage after suggests exposure to bile acids in combination with acid may play a crucial role in esophageal carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4105.

Angiotensin‐(1–7) increases neuronal potassium current via a nitric oxide‐dependent mechanism

Angiotensin 1–7 [Ang‐(1–7)] signaling in the brain may play an important protective role in cardiovascular diseases by decreasing sympathetic output. It is generally well‐accepted that nitric oxide (NO•) in the brain functions as a sympatho‐inhibitory molecule. We hypothesized that Ang‐(1–7) increases NO• in neurons and modulates neuronal activity. We tested this hypothesis in catecholaminergic CATH.a neurons. Protein analysis revealed these neurons express the Ang‐(1–7) receptor (MasR) and three isoforms of NO• synthase (nNOS, eNOS, iNOS). Expression of MasR, nNOS, and eNOS protein markedly increased in CATH.a neurons following six days of differentiation with N6,2′‐O‐Dibutyryladenosine 3′,5′‐cyclic monophosphate sodium salt (dbcAMP, 1 mM). As measured by DAF‐FM fluorescence and confocal microscopy, Ang‐(1–7) (100 nM) increased NO• levels in differentiated CATH.a neurons (31 ± 3%; P&lt;0.05 vs. baseline) and this response was attenuated (P&lt;0.05) by the MasR antagonist A779 (5 ± 3%), non‐specific NOS inhibitor (L‐NAME; −5 ± 1%), and nNOS inhibitor (SMTC; −5 ± 7%). Using the whole‐cell patch‐clamp technique, we observed that Ang‐(1–7) increased outward K+ current (IKv) in differentiated CATH.a neurons (53 ± 6% increase; P&lt;0.05 vs. vehicle). These findings indicate that Ang‐(1–7) increases nNOS‐dependent NO• levels via MasR activation and elevates IKv in differentiated CATH.a neurons. NIH P01HL062222

The effect of nNOS overexpression on intracellular ROS activities in skeletal muscle fibres at rest and following a period of contractile activity

ROS serve as important signaling molecules and interact with multiple cell signaling and regulatory pathways to modulate changes in gene expression. The aim was to examine the effect of nNOS overexpression on ROS activities in skeletal muscle fibres at rest and following a period of contractile activity. To elucidate the roles of ROS, we examined their intracellular activities by using fluorescence microscopy. Mice overexpressing nNOS and age‐matched wild type (WT) mice were used in this study. Flexor digitorum brevis muscles were dissected and incubated in collagenase to isolate single muscle fibres. Fibres were plated on culture dishes and were loaded with DAF‐FM DA, DHE or DCFH DA, fluorescent probes for the assessment of nitric oxide (NO·) superoxide (O2·) and a general probe for ROS respectively. Contractile activity was induced by electrical stimulation. Control non‐stimulated fibres from the nNOS mice showed a higher rate of increase in DAF‐FM fluorescence compared with the WT group implying a higher NO· formation. Fibres from nNOS overexpressors showed a reduced ethidium fluorescence compared with fibres from WT mice at rest indicating a lower O2· generation. No differences were observed between groups following contractions. These results suggest that overexpression of nNOS can increase NO· ‘bioavailability’ in skeletal muscle cells and reduce O2· toxicity. Supported by the A Onassis Foundation (GR).

Abstract 4883: Insulin-like Growth Factor Binding Protein-3 (IGFBP3) Modulates Vascular Tone via Scavenging Receptor-B1 (SRB1)-Dependent Nitric Oxide Release

Purpose: We recently reported the vascular protective effect of IGFBP3 in experimental models of ischemic vascular injury involving increased homing of circulating endothelial progenitor cells and activation of nitric oxide synthase (eNOS). The vascular protective potential of a molecule is determined in part by its ability to facilitate perfusion to the ischemic areas by dilation of the resident vasculature. Therefore, we hypothesized that IGFBP3 regulates vascular tone by causing vasodilation and thereby, enhances the perfusion of vascular bed. Methods: We evaluated the effect of intraluminal IGFBP3 on the pressure- and agonist-induced constrictions in rat posterior cerebral arteries (PCAs) and compared with that of HDL, a known activator of SRB1. NO release and endothelial intracellular Ca2+ ([Ca2+]i) were evaluated by DAF-FM and Fura-2 fluorescence microscopy, respectively, in intact arteries and human microvascular endothelial cells (HMVECs). Signaling pathways mediating the effects of IGFBP3 were delineated in intact arteries and HMVECs. Activation of eNOS by IGFBP3 was evaluated by Western blotting of ser1177 phosphorylated eNOS in HMVECs. IGF-independent effect was confirmed by using a non-IGF-binding mutant of IGFBP3. Results: Intraluminal IGFBP3 (100ng/ml) or HDL (1mg/ml) caused significant decrease in pressure- (40, 70 and 100 mmHg) and serotonin-induced constrictions of PCAs compared to the vehicle-control. These effects were blocked by L-NAME or SRB1-function blocking antibody (SRB1-ab). NO release was stimulated in PCAs by intraluminal IGFBP3 and HDL with no change in the [Ca2+]i. NO release by IGFBP3, with no change in the [Ca2+]i, in HMVECs was blocked by SRB1-ab, PP2 (cSrc inhibitor), LY294002 (PI3-kinase inhibitor) and triciribin (akt inhibitor) not by KN-92 (CamKII inhibitor). Non-IGF-binding mutant and wild type IGFBP3 showed similar effect in stimulating NO release in HMVECs. Phosphorylation of eNOS at ser1177 was increased in a time-dependent manner by IGFBP3 that was blocked by SRB1-ab. Conclusions: These results suggest that IGFBP3 modulates vascular tone by NO release via SRB1-dependent cSrc/PI3-kinase/akt pathway and this response may significantly contribute to the vascular protective effect of IGFBP3.

Effects of epigallocatechin gallate on rotenone-injured murine brain cultures

Green tea polyphenol epigallocatechin-3-gallate (EGCG) is reported to have antioxidant abilities and to counteract beneficially mitochondrial impairment and oxidative stress. The present study was designed to investigate neuroprotective effects of EGCG on rotenone-treated dissociated mesencephalic cultures and organotypic striatal cultures. Rotenone is a potent inhibitor of complex I of the respiratory chain, which in vitro causes pathological and neurochemical characteristics of diseases in which mitochondrial impairment is involved, e.g., Parkinson's disease. Treatment with EGCG (0.1, 1, 10 muM) alone had no significant effects on mesencephalic cultures. In striatal slice cultures, EGCG led to a significant increase of propidium iodide (PI) uptake and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM), but not dihydroethidium (DHE) fluorescence intensity. Rotenone (20 nM on the eighth DIV for 48 h) significantly decreased the numbers and the neurite lengths of TH ir neurons by 23 and 34% in dissociated mesencephalic cell cultures compared to untreated controls. Exposure of striatal slices to rotenone (0.5 mM for 48 h) significantly increased PI uptake, and DAF-FM and DHE fluorescence intensities by 41 and 136 and 19%, respectively, compared to controls. Against rotenone, in dissociated mesencephalic cultures, EGCG produced no significant effect on either the number or neurite lengths of THir neurons compared to rotenone-treated cultures, but EGCG significantly decreased PI uptake by 19% and DAF-FM fluorescence intensity by 19 and 58%, respectively, compared to increase in rotenone-exposed striatal slices. On the other hand, EGCG did not affect superoxide (O(2) (-)) formation as detected with DHE. These data indicate that EGCG slightly protects striatal slices by counteracting nitric oxide (NO(.)) production by rotenone. In conclusion, EGCG partially protects striatal slices but not dissociated cells against rotenone toxicity.

Involvement of scavenging receptor‐B1 in NO release by insulin‐like growth factor binding protein‐3 (IGFBP3) in human endothelial and CD34 cells

We recently reported the vascular protective potential of IGFBP3 involving recruitment of circulating endothelial progenitor cells (CD34) to the sites of vascular injury. NO plays an important role in the migration of CD34 cells and therefore, we hypothesized that NO is involved in the effects of IGFBP3. NO release was quantified by DAF‐FM fluorescence in human cord blood‐derived CD34 cells, umbilical vein endothelial cells (HUVECs), pulmonary microvascular endothelial cells (HMVECs) and human retinal ganglionic cells (HRGCs). IGFBP3 (100 ng/ml) increased NO release in all four cell types. Pretreatment with scavenging receptor‐B1 (SRB1) antibody inhibited NO release in endothelial cells but not in HRGCs. HDL induced NO release in endothelial and CD34 cells but not in HRGCs in which mRNA for SRB1 was not detected. Co‐treatment with IGFBP3 and HDL caused a greater release of NO than with either individual treatment. DMS, a sphingosine kinase inhibitor blocked NO release in CD34 cells. IGFBP3 caused eNOS and vasodilator‐stimulated peptide (VASP) phosphorylation and redistribution of VASP in endothelial cells that was inhibited by L‐NAME. IGFBP3 and HDL caused relaxation of phenylephrine‐preconstricted rat mesenteric arterial segments that was inhibited by L‐NAME and combination of L‐NAME and indomethacin, respectively. These results suggest involvement of NO in the vascular protective effect of IGFBP3.

Endothelial cell-derived nitric oxide mobilization is attenuated in copper-deficient rats

The attenuation of endothelium-dependent nitric oxide (NO) mediated vasodilation is a consistent finding in both conduit and resistance vessels during dietary copper (Cu) deficiency. Although the effect is well established, evidence for the mechanism remains circumstantial. This study was designed to determine the relative amount of NO produced in and released from the vascular endothelium. Using the fluorescent NO indicator, 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM), we now demonstrate the effect of a Cu-deficient diet on the production of NO from the endothelium of resistance arterioles. In one group of experiments, control and Cu-chelated lung microvascular endothelial cells (ECs) were used to assay NO production and fluorescence was observed by confocal microscopy. Weanling Sprague-Dawley rats were fed purified diets that were either Cu adequate (6.3 micrograms Cu per gram of food) or Cu deficient (0.3 micrograms Cu per gram of food) for 4 weeks. In the second series of experiments, first-order arterioles were microsurgically isolated from the rat cremaster muscle, cannulated, and pressurized with (3[N-morpholino]propanesulfonic acid) physiologic salt solution (MOPS-PSS). DAF-FM (5 micromol.L-1) was added in the lumen of the vessel to measure NO release. Baseline DAF-FM fluorescence was significantly lower in Cu-chelated ECs than in controls. In response to 10-6 mol.L-1 acetylcholine, fluorescent intensity was significantly less in chelated ECs and in the lumen of Cu-deficient arterioles. The results suggest that production and release of NO by the vascular endothelium is inhibited by a restriction of Cu. This inhibition may account for the attenuated vasodilation previously reported in Cu-deficient rats.

Abstract 1746: Insulin Like Growth Factor Binding Protein-3 (IGFBP-3) Mediates Vascular Repair By HDL Receptor Activation And Sphingosine Kinase (SK-1)/Sphingosine 1-Phosphate (S1P) Dependent Nitric Oxide (NO) Generation

Purpose: We demonstrated that IGFBP-3 stimulates hematopoietic stem cells (HSC) to differentiate into endothelial cells, form capillaries, and stabilize the vasculature (Chang, et al, PNAS 2007). Local IGFBP- 3 production is increased by hypoxia and facilitates the homing of HSC to areas of injury. In the circulation, IGFBP-3 is bound to HDL. In this study, we investigated the signaling pathways responsible for the robust migratory effects of IGFBP-3. Methods: The effects of IGFBP-3 on NO generation in human vascular precursors (CD 34 + , CD14 − ), human lung microvascular endothelial cells, and human umbilical vein endothelial cells were examined using DAF-FM fluorescence. Western analysis was use for detection of eNOS and vasodilator-stimulated phosphoprotein (VASP), which redistributes to lamellipodia forming an active motor complex that supports motility and is phosphorylated in response to NO. Localization of VASP was performed by immunohistochemistry. SK-1 was assessed following IGFBP-3 stimulation. Results: In CD34 + cells and endothelial cells, IGFBP-3 stimulated eNOS phosphorylation at Ser1177 (102 ± 1.8%, P = 0.0002) and increased NO generation (275 ± 50%, P = 0.05) by increasing SK-1 and S1P generation. IGFBP-3 was bound and internalized by the HDL receptor, scavenger receptor 1B (SR1B). NO generation following IGFBP-3 exposure was reduced by SK inhibitors or SR-1B blocking antibody pretreatment (35 ± 5%, P &lt; 0.02). IGFBP-3 generated NO increased phosphorylation of VASP at Ser239 and promoted the redistribution of VASP to lamellipodia. Conclusions: IGFBP-3 effects on cell migration are NO dependent and mediated in part by activation of the HDL receptor SR1B suggesting that some of the beneficial effects of HDL are mediated by the association of IGFBP-3.

Nitric oxide dependence of glutamate‐mediated modulation at a vertebrate neuromuscular junction

Recent evidence has revealed a contribution of glutamate in the stereotyped cholinergic neuromuscular transmission. Indeed, receptors, transporters and glutamate itself are present at the neuromuscular junction (NMJ) while glutamate activation of metabotropic receptors (mGluRs) decreases synaptic transmission and mediates depression through presynaptic mechanisms. However, we have shown that the mGluRs are located postsynaptically, inconsistent with the presynaptic action of glutamate. In the present study, we tested whether nitric oxide (NO) serves as a retrograde messenger mediating the distant effect of glutamate. Glutamate or an mGluR agonist [trans-(1S,3R)-aminocyclopentanedicarboxylic acid (ACPD)] failed to reduce synaptic transmission in the presence of an NOS inhibitor (3Br7NINa, 3-bromo-7-nitroindazole sodium salt). Moreover, application of 3Br7NINa precluded the effect of the mGluR antagonist MCPG [(S)-alpha-methyl-4-carboxyphenylglycine] on high-frequency-induced synaptic depression. Iontophoretic injections of BAPTA [1,2-bis(2-aminophenoxy)ethane-N,N,N'-tetraacetic acid] in muscle fibres abolished the effect of trans-ACPD on synaptic transmission and blocked the mGluR component of depression, indicating the involvement of muscular calcium in mGluR-induced depression. Also, the use of this protocol unveiled a muscular calcium-dependent potentiating pathway dependent on cyclo-oxygenase activity. In addition, local application of trans-ACPD induced an increase in NO production by muscle fibres visualized with the indicator DAF-FM (4-amino-5-methylamino-2',7'-difluorofluorescein). This was prevented by 3Br7NINa or the iontophoretic injection of BAPTA. Moreover, motor nerve stimulation (50 Hz, 30 s) induced an increase in DAF-FM fluorescence that was abolished by 3Br7NINa and MCPG. Hence, the data suggest that the production of the retrograde molecule NO depends on the postsynaptic calcium-dependent activation of nitric oxide synthase following mGluRs stimulation and is essential for the glutamatergic modulation of synaptic efficacy and plasticity at the NMJ.

Both cell volume and calmodulin contribute to the regulation of macula densa nitric oxide

Elevations in luminal sodium chloride concentration ([NaCl]L) alter macula densa (MD) intracellular calcium concentration and result in the release of nitric oxide (NO). Also, concomitant elevations in [NaCl]L and luminal osmolality (osmL) lead to shrinkage of macula densa cells, while increases in [NaCl]L at constant osmL produce cell swelling. Currently, there is little information concerning the intracellular signaling event that leads to the activation of MD NO synthase. Current studies were performed to determine whether changes in MD cell volume and/or calmodulin‐dependent signaling activate MD NO synthase. Thick ascending limb‐glomerulus preparations were isolated and perfused from rabbits, and MD NO release was detected with fluorescence microscopy using DAF‐FM. Elevations in [NaCl]L (from 0 to 150 mmol/L) at constant osmL produced an increase in DAF‐FM fluorescence of 5±0.5%, while concomitant elevations in [NaCl]L and osmL led to an increase of 12±0.7%. Luminal application of furosemide, a blocker of Na+:2Cl‐:K+ cotransport, reversed [NaCl]L‐dependent NO release (‐0.04±0.2%). Also, application of the calmodulin‐inhibitor calmidazolium blocked [NaCl]L‐dependent NO release (1.8±1.8%). Thus, [NaCl]L‐dependent NO production by MD cells may be modulated by changes in cell volume and requires activation of calmodulin signaling.

Abstract 1333: Modulation Of Cardiac Beta-adrenergic Responsiveness By The Neuronal Nitric Oxide Synthase/Sarcolemmal Calcium Pump Complex

The pivotal role of neuronal nitric oxide synthase (nNOS) in regulating cardiac function has only recently been unveiled. Notably, others have shown that responsiveness to β-adrenergic stimulation is dependent on nNOS activity. In a cellular model, we showed that the Ca 2+ /calmodulin-dependent nNOS activity is reduced by overexpression of isoform 4b of the plasma membrane Ca 2+ /Calmodulin-dependent Ca 2+ -pump (PMCA4b), which binds to nNOS. We demonstrated that PMCA4b overexpression in the heart reduced β-adrenergic responsiveness in vivo via an nNOS dependent mechanism (Oceandy et al, Circulation 2007). Here we investigated the cellular mechanisms of the regulation of the β-adrenergic response by PMCA4b. We used an adenoviral system to overexpress PMCA4b (PMCA4b cells) or LacZ (control, C) in neonatal rat cardiomyocytes. PMCA4b cells showed an 18±5% and 24±5% reduction in nitric oxide (DAF-FM fluorescence) and cGMP levels, respectively (n=6, p&lt;0.05 each) compared to C demonstrating the regulation of NO production by the PMCA4b in this system. Since nNOS has been shown to regulate phospholamban (PLB) phosphorylation, we examined phosphorylation of PLB at Ser16. PMCA4b cells showed a significant increase in Ser16-PLB at baseline (66±17%, p&lt;0.05) compared to C. As a result of increased baseline Ser16-PLB in PMCA4b cells, β-adrenergic stimulation of PMCA4b cells using 2μM isoproter-enol (IP) showed reduced relative induction in Ser16-PLB (23±10% vs. 78±19% in C; n=5, p&lt;0.05). Further analysis in adult cardiomyocytes isolated from our PMCA4b transgenic mice (PMCA4b TG) demonstrated that PMCA4b TG showed 3-fold higher Ser16-PLB phosphorylation at baseline compared to wild type (WT) myocytes and the relative response following β-adrenergic stimulation was significantly reduced (1.2±0.2 fold induction after IP treatment in PMCA4b TG, vs. 3.1±0.7 in WT, n=5, p&lt;0.05). Thus, PMCA4b regulates NO production from nNOS, which in turn modulates cGMP levels and PLB phosphorylation. These findings provide mechanistic insight into the regulation of the β-adrenergic response in the heart by PMCA4b and place this Ca 2+ -pump upstream of the recently described pathway linking nNOS and Ser16-PLB phosphorylation and downstream of the β-adrenergic receptor(s).

Peroxynitrite formation mediates LPC-induced augmentation of cardiac late sodium currents

Lysophosphatidylcholine (LPC) accumulates in the ischaemic myocardium and is arrhythmogenic. We have examined the mechanisms underlying the effects of LPC on the late cardiac Na+ current (ILNa). Na+ currents were recorded in HEK293 cells expressing NaV1.5 and isolated rat ventricular myocytes. LPC enhanced recombinant ILNa, while it reduced peak Na+ current. Computer modeling of human ventricular myocyte action potentials predicted a marked duration prolonging effect and arrhythmogenic potential due to these effects of LPC on peak and late currents. Enhancement of recombinant ILNa was suppressed by the antioxidant ascorbic acid and by the NADPH oxidase inhibitor DPI. Inhibitors of the mitochondrial electron transport chain (rotenone, TTFA and myxothiazol) were without effect on LPC responses. The superoxide donor pyrogallol was without effect on ILNa. Enhancement of ILNa was abrogated by the NOS inhibitors l-NAME and 7-nitroindazole, while LPC induced an l-NAME-sensitive production of NO, measured as enhanced DAF-FM fluorescence, in both HEK293 cells and ventricular myocytes. Despite this, the NO donors SNAP and SNP caused no change in ILNa. However, SNAP enhanced TTX-sensitive recombinant and native ILNa in the presence of pyrogallol, suggesting peroxynitrite formation as a mediator of the response to LPC. In support of this, the peroxynitrite scavenger FeTPPS prevented the response of ILNa to LPC. Peroxynitrite formation provides a novel mechanism by which LPC regulates the late cardiac Na+ current.

Real‐time measurement of nitric oxide in single mature mouse skeletal muscle fibres during contractions

Nitric oxide (NO) is thought to play multiple roles in skeletal muscle including regulation of some adaptations to contractile activity, but appropriate methods for the analysis of intracellular NO activity are lacking. In this study we have examined the intracellular generation of NO in isolated single mature mouse skeletal muscle fibres at rest and following a period of contractile activity. Muscle fibres were isolated from the flexor digitorum brevis muscle of mice and intracellular NO production was visualized in real-time using the fluorescent NO probe 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA). Some leakage of DAF-FM was apparent from fibres loaded with the probe, but they retained sufficient probe to respond to changes in intracellular NO following addition of the NO donor 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) up to 30 min after loading. Electrically stimulated contractions in isolated fibres increased the rate of change in DAF-FM fluorescence by approximately 48% compared to non-stimulated fibres (P < 0.05) and the rate of change in DAF-FM fluorescence in the stimulated fibres returned to control values by 5 min after contractions. Treatment of isolated fibres with the NO synthase inhibitors NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) or NG-monomethyl-L-arginine (L-NMMA) reduced the increase in DAF-FM fluorescence observed in response to contractions of untreated fibres. Treatment of fibres with the cell-permeable superoxide scavenger 4,5-dihydroxy-1,3-benzenedisulphonic acid (Tiron) also reduced the increase in fluorescence observed during contractions suggesting that superoxide, or more probably peroxynitrite, contributes to the fluorescence observed. Thus this technique can be used to examine NO generation in quiescent and contracting skeletal muscle fibres in real time, although peroxynitrite and other reactive nitrogen species may potentially contribute to the fluorescence values observed.

Protein disulfide-isomerase mediates delivery of nitric oxide redox derivatives into platelets

S-nitrosothiol compounds are important mediators of NO signalling and can give rise to various redox derivatives of NO: nitrosonium cation (NO+), nitroxyl anion (NO-) and NO* radical. Several enzymes and transporters have been implicated in the intracellular delivery of NO from S-nitrosothiols. In the present study we have investigated the role of GPx (glutathione peroxidase), the L-AT (L-amino acid transporter) system and PDI (protein disulfide-isomerase) in the delivery of NO redox derivatives into human platelets. Washed human platelets were treated with inhibitors of GPx, L-AT and PDI prior to exposure to donors of NO redox derivatives (S-nitrosoglutathione, Angeli's salt and diethylamine NONOate). Rapid delivery of NO-related signalling into platelets was monitored by cGMP accumulation and DAF-FM (4-amino-5-methylamino-2'7'-difluorofluorescein) fluorescence. All NO redox donors produced both a cGMP response and DAF-FM fluorescence in target platelets. NO delivery was blocked by inhibition of PDI in a dose-dependent manner. In contrast, inhibition of GPx and L-AT had only a minimal effect on NO-related signalling.PDI activity is therefore required for the rapid delivery into platelets of NO-related signals from donors of all NO redox derivatives. GPx and the L-AT system appeared to be unimportant in rapid NO signalling by the compounds used in the present study. This does not, however, exclude a possible role during exposure of cells to other S-nitrosothiol compounds, such as S-nitrosocysteine. These results further highlight the importance of PDI in mediating the action of a wide range of NO-related signals.

OXIDIZED LOW-DENSITY LIPOPROTEIN DOWN-REGULATES INSULIN-LIKE GROWTH FACTOR 1 RECEPTOR VIA ENHANCED GENERATION OF NITRIC OXIDE AND REACTIVE OXYGEN SPECIES IN HUMAN AORTIC SMOOTH MUSCLE CELLS.

Increased apoptosis and depletion of smooth muscle cells (SMCs) plays an important role in atherosclerotic plaque vulnerability to rupture, a major cause of acute coronary events. We previously reported that the ability of oxidized low-density lipoprotein (oxLDL) to down-regulate insulin-like growth factor 1 receptor (IGF-1R) expression is a critical mediator of oxLDL-induced SMC apoptosis. OxLDL-induced IGF-1R down-regulation was dependent on the generation of reactive oxygen species (ROS), namely peroxide and superoxide as measured by H2DCFDA and dihydroethidium fluorescence. However, an oxidative stress-inducing agent, paraquat (0-800 μM, up to 24 hours), did not stimulate IGF-1R down-regulation, suggesting that another downstream event was critical for receptor down-regulation. To explore mechanisms, we assessed the ability of oxLDL to induce nitric oxide (NO) generation in human aortic SMC culture using DAF-FM fluorescence. OxLDL (60 μg/mL, 4 hours) enhanced NO generation by 2.4-fold vs native LDL (nLDL) control (n = 8, p

ICAM‐1 Governs Angiogenesis via Redox Regulation of PTEN Expression and eNOS activity

eNOS derived NO is critical in the angiogenic process, thus understanding the mechanisms regulating eNOS activity during angiogenesis are crucial. Here we present data showing that ICAM-1 is a novel effector of VEGF164 induced eNOS function, endothelial cell chemotaxis, and angiogenesis. VEGF164 mediated angiogenesis in WT and ICAM-1−/− mice was assessed in vivo using a Millipore disk assay. VEGF164 chemotaxis of mouse aortic endothelial cells (MAEC) was measured by transwell assay. eNOS activity was assessed using western blot analysis of phosphorylation at ser1177 and DAF-FM fluorescence. PTEN expression was assessed by western blot. VEGF164 mediated angiogenesis in vivo was significantly decreased in ICAM-1−/− mice compared to WT mice. ICAM-1−/− MAEC showed impaired VEGF164 mediated chemotaxis, eNOS phosphorylation, and NO production vs. WT MAEC. PTEN expression was significantly elevated in ICAM-1−/− MAEC which was controlled by intracellular glutathione levels, as 150 μM BSO treatment returned PTEN expression to WT levels and restored VEGF164 chemotaxis and eNOS phosphorylation. Likewise, 5 mM glutathione ethyl ester (GEE) supplementation of WT cells recapitulated ICAM-1−/− chemotactic defects, increased PTEN expression, and blocked eNOS phosphorylation. These data suggest a novel role for ICAM-1 in modulating VEGF164 induced angiogenesis and eNOS activity via regulation of intracellular glutathione levels which regulate PTEN expression.