Enhanced NO Production Research Articles

Iron induces protection and necrosis in cultured cardiomyocytes: Role of reactive oxygen species and nitric oxide

We investigate here the role of reactive oxygen species and nitric oxide in iron-induced cardiomyocyte hypertrophy or cell death. Cultured rat cardiomyocytes incubated with 20 μM iron (added as FeCl 3–Na nitrilotriacetate, Fe–NTA) displayed hypertrophy features that included increased protein synthesis and cell size, plus realignment of F-actin filaments along with sarcomeres and activation of the atrial natriuretic factor gene promoter. Incubation with higher Fe–NTA concentrations (100 μM) produced cardiomyocyte death by necrosis. Incubation for 24 h with Fe–NTA (20–40 μM) or the nitric oxide donor Δ-nonoate increased iNOS mRNA but decreased iNOS protein levels; under these conditions, iron stimulated the activity and the dimerization of iNOS. Fe–NTA (20 μM) promoted short- and long-term generation of reactive oxygen species, whereas preincubation with l-arginine suppressed this response. Preincubation with 20 μM Fe–NTA also attenuated the necrotic cell death triggered by 100 μM Fe–NTA, suggesting that these preincubation conditions have cardioprotective effects. Inhibition of iNOS activity with 1400 W enhanced iron-induced ROS generation and prevented both iron-dependent cardiomyocyte hypertrophy and cardioprotection. In conclusion, we propose that Fe–NTA (20 μM) stimulates iNOS activity and that the enhanced NO production, by promoting hypertrophy and enhancing survival mechanisms through ROS reduction, is beneficial to cardiomyocytes. At higher concentrations, however, iron triggers cardiomyocyte death by necrosis.

Abstract 5222: Genetic Deficiency of Arginase II Reduces Endothelial Dysfunction and Atherosclerosis in ApoE-Knockout Mice

Introduction: Arginase II is known to be one of the key mediator in vascular pathophysiology of cholesterol-medicated endothelial dysfunction by reciprocal regulation of NOS and NO production through substrate competition. We aimed to investigate whether the transgenic Arginase II inhibition could reduce the endothelial dysfunction and atherosclerosis in apolipoprotein E-knockout (ApoE-KO) mice. Methods and Results: ApoE-KO mice were bred on an arginase II KO (ArgII-KO) background. Mice that were homozygous deleted at both alleles were used in the study. Male 8 – 12 wk old Arg-KO mice, ApoE-KO mice, Arg/ApoE-KO (Dbl-KO) mice fed high cholesterol diet for 16 weeks, and age-matched wild type (WT) mice fed normal diet were enrolled. Arginase activity was significantly higher in the aorta of ApoE-KO mice than compared to WT mice (1612.2±445.9 pmol Urea/mg protein/min vs. 836.3±12.2 pmol Urea/mg protein/min, n=5, p < 0.01), but not detected in Arg-KO and Dbl-KO mice. We measured NO an ROS in vascular strips pinned down endothelial side up using the fluorescent probes DAF-FM diacetate and DHE. Dbl-KO mice showed significantly higher NO production (0.0737±0.0056 intensity/sec vs. 0.0256±0.0029 intensity/sec, n=6, p < 0.05) and lower ROS production than ApoE-KO mice (0.0676±0.0065 intensity/sec vs. 0.1147±0.01619 intensity/sec, n=6, p < 0.05). We tested endothelial dependent vasorelaxation in aortic rings in organ chambers. The vasorelaxation response to acetylcholine (Ach) in Dbl-KO mice was greater than in ApoE-KO mice (log EC 50 , −5.68 vs. −5.13, p <0.05), although it was less than in Arg-KO and WT mice. N G -nitro-L-arginine methyl ester (L-NAME) incubation didn’t only impair Ach response, but also abolished the inter-group difference. The aortic atheromatous plaque burden as measured by Sudan IV staining was significantly reduced in Dbl-KO group compared to ApoE-KO group (38.4±3.8% vs. 52.0±2.8%, n=6, p <0.01), No significant plaque was detectable in Arg-KO and WT mice. Conclusion: The genetic inhibition of Arginase II enhanced NO production, reduced ROS produnction restored endothelial function and lowered atheromatous plaque burden. Therefore, Arginase II represents a crtical novel target for atherosclerosis therapy.

Sodium ferulate treatment and interventional mechanism reverse erectile dysfunction in streptozotocin-induced diabetic rats

To investigate the effect and mechanism of sodium ferulate (SF) on reversing erectile dysfunction in diabetes mellitus (DM) rats. Forty-four male adult Sprague-Dawley rats were induced for diabetes by an intraperitoneal injection of streptozotocin. Then the successful models were randomly divided into DM + SF group and DM group (22 rats each respectively). The rats in DM +SF group were treated with sodium ferulate (100 mg x kg(-1) x d(-1)) through a daily gastric lavage. At Week 12, the erectile function of all rats was evaluated and serum samples were harvested. The SOD, CAT, NOS, MDA and NO levels in corpus cavernosum and serum were all measured. The pathologic change in penile cavernous body was observed microscopically. The diabetic rat models were successfully established. The erectile function was much better in normal control group and DM + SF group than that in DM group. And the penile erection rates in three groups were 100%, 66% and 22% respectively. The activity levels of SOD, CAT and NOS were markedly decreased in DM group as compared to those in normal control group and DM + SF group (P < 0.01). The NO content was approximately equal in normal control group and DM + SF group (112 +/- 28) nmol/ml vs (137 +/- 25) nmol/ml. But both were much higher than that in DM group (56 +/- 10) nmol/ml, both P < 0.01. The MDA content was markedly increased in DM group as compared to those in normal control group and DM + SF group (both P < 0.01). Microscopically, muscle fibers in penile cavernous body arranged disorderly, muscular mantle damaged and desmoplasia scattered among muscle fibers in DM group. Sodium ferulate may play interventional roles in reversing diabetic erectile dysfunction through metabolic regulation of free radicals, antagonism of oxidative insults and enhancement of NO production.

Integrin alpha9beta1 mediates enhanced cell migration through nitric oxide synthase activity regulated by Src tyrosine kinase.

Integrins are important mediators of cell adhesion and migration, which in turn are essential for diverse biological functions, including wound healing and cancer metastasis. The integrin alpha9beta1 is expressed on numerous mammalian tissues and can mediate accelerated cell migration. As the molecular signaling mechanisms that transduce this effect are poorly defined, we investigated the pathways by which activated integrin alpha9beta1 signals migration. We found for the first time that specific ligation of integrin alpha9beta1 rapidly activates Src tyrosine kinase, with concomitant tyrosine phosphorylation of p130Cas and activation of Rac-1. Furthermore, activation of integrin alpha9beta1 also enhanced NO production through activation of inducible nitric oxide synthase (iNOS). Inhibition of Src tyrosine kinase or NOS decreased integrin-alpha9beta1-dependent cell migration. Src appeared to function most proximal in the signaling cascade, in a FAK-independent manner to facilitate iNOS activation and NO-dependent cell migration. The cytoplasmic domain of integrin alpha9 was crucial for integrin-alpha9beta1-induced Src activation, subsequent signaling events and cell migration. When taken together, our results describe a novel and unique mechanism of coordinated interactions of the integrin alpha9 cytoplasmic domain, Src tyrosine kinase and iNOS to transduce integrin-alpha9beta1-mediated cell migration.

β 2 -Adrenoceptor Antagonist ICI 118,551 Decreases Pulmonary Vascular Tone in Mice via a G i/o Protein/Nitric Oxide-Coupled Pathway

beta(2)-adrenoceptors are important modulators of vascular tone, particularly in the pulmonary circulation. Because neurohormonal activation occurs in pulmonary arterial hypertension, we have investigated the effect of different adrenergic vasoactive substances on tone regulation in large and small pulmonary arteries, as well as in systemic vessels of mice. We found that the beta(2)-adrenoceptor antagonist ICI 118,551 (ICI) evoked a decrease of vascular tone in large pulmonary arteries and reduced the sensitivity of pulmonary arteries toward different contracting agents, eg, norepinephrine, serotonin, or endothelin. ICI proved to act specifically on pulmonary vessels, because it shifted the dose-response curve of norepinephrine to the right in pulmonary arteries, whereas there was no effect in the aorta. Pharmacological experiments proved that the right shift of the norepinephrine dose-response curve by ICI was mediated via a beta(2)-adrenoceptor/G(i/o) protein-dependent pathway enhancing NO production in the endothelium; these results were corroborated in beta-adrenoceptor and endothelial NO synthase knockout mice where ICI had no effect. ICI increased vascular lumen diameter in lung sections and reduced pulmonary arterial pressure under normoxia and under hypoxia in the isolated perfused lung model. These effects were found to be physiologically relevant, because ICI specifically decreased pulmonary but not systemic blood pressure in vivo. Thus, the beta(2)-adrenoceptor antagonist ICI is a pulmonary arterial-specific vasorelaxant and, therefore, a potentially interesting novel therapeutic agent for the treatment of pulmonary arterial hypertension.

ENOS Activation by Physical Forces: From Short-Term Regulation of Contraction to Chronic Remodeling of Cardiovascular Tissues

Nitric oxide production in response to flow-dependent shear forces applied on the surface of endothelial cells is a fundamental mechanism of regulation of vascular tone, peripheral resistance, and tissue perfusion. This implicates the concerted action of multiple upstream "mechanosensing" molecules reversibly assembled in signalosomes recruiting endothelial nitric oxide synthase (eNOS) in specific subcellular locales, e.g., plasmalemmal caveolae. Subsequent short- and long-term increases in activity and expression of eNOS translate this mechanical stimulus into enhanced NO production and bioactivity through a complex transcriptional and posttranslational regulation of the enzyme, including by shear-stress responsive transcription factors, oxidant stress-dependent regulation of transcript stability, eNOS regulatory phosphorylations, and protein-protein interactions. Notably, eNOS expressed in cardiac myocytes is amenable to a similar regulation in response to stretching of cardiac muscle cells and in part mediates the length-dependent increase in cardiac contraction force. In addition to short-term regulation of contractile tone, eNOS mediates key aspects of cardiac and vascular remodeling, e.g., by orchestrating the mobilization, recruitment, migration, and differentiation of cardiac and vascular progenitor cells, in part by regulating the stabilization and transcriptional activity of hypoxia inducible factor in normoxia and hypoxia. The continuum of the influence of eNOS in cardiovascular biology explains its growing implication in mechanosensitive aspects of integrated physiology, such as the control of blood pressure variability or the modulation of cardiac remodeling in situations of hemodynamic overload.

Mitochondrial dysfunction in endothelium impairs vasodilation in Zucker obese rats with insulin resistance

Mitochondria affect cerebrovascular tone by activation of mitochondrial ATP‐sensitive K+ channels (mitoKATP). Insulin resistance (IR) causes mitochondrial dysfunction, therefore, we evaluated the effects of diazoxide, a mitoKATP activator, on cerebral arteries of Zucker obese (ZO) rats and lean (ZL) controls. Diameter studies showed reduced vasodilation to diazoxide in ZO compared to ZL. Endothelial denudation reduced vasodilation to diazoxide in all arteries implicating endothelial mitoKATP. Inhibition of nitric oxide synthase (NOS) reduced vasodilation in intact arteries from ZL but not ZO, indicating diminished NO effects. Paradoxically, NOS inhibition in endothelium denuded arteries enhanced vasodilation in ZO implying the expression and uncoupling of NOS in vascular smooth muscle (VSM). Inhibition of cyclooxygenase (COX) enhanced vasodilation in intact arteries from ZL but not ZO. In contrast, endothelial denudation enhanced vasodilation in ZO but not ZL. Thus, ZO arteries exhibit enhanced production of both vasoconstrictor and vasodilator prostanoids. Fluorescence studies of rat cerebral microvascular endothelial cells showed enhanced NO production in response to diazoxide and also confirmed reduced NO generation in ZO arteries. Thus, endothelial mitochondrial dysfunction in ZO arteries results in the impairment of NOS and COX activity. Support: NIH HL077731, HL030260, HL 065380.

Estrogen‐Dependent Enhancement of NO Production in the Nucleus Tractus Solitarius Contributes to Ethanol‐Induced Hypotension in Conscious Female Rats

Our previous pharmacological and cellular studies showed that peripheral (cardiac and vascular) nitric oxide synthase (NOS)-derived NO is implicated in the estrogen (E(2))-dependent hypotensive action of ethanol in female rats. The objective of this study was to test the hypothesis that enhanced NO production in the nucleus tractus solitarius (NTS) is implicated in the E(2)-dependent hypotensive action of ethanol. To achieve this goal, we utilized in vivo electrochemistry to measure real time changes in neuronal NO to investigate the acute effects of intragastric ethanol (0, 0.5, or 1 g/kg) on NO in NTS neurons, blood pressure (BP), and heart rate (HR) in conscious female rats in the absence (ovariectomized, OVX, rats) or presence of E(2). In sham operated (SO) rats, ethanol elicited dose-related increase in NTS NO and reduction in BP. These neurochemical and BP effects of ethanol were absent in OVX rats. Whether the neurochemical effect of ethanol and the associated hypotension are dependent on rapid E(2) signaling was investigated. In OVX rats pretreated, 30 minutes earlier, with E(2) (1 microg/kg), intragastric ethanol (1 g/kg) increased NTS NO and reduced BP and these responses were comparable to those obtained in SO rats. The present findings suggest that increased production of NO in NTS neurons contributes to ethanol-evoked hypotension in female rats. Further, ethanol enhancement of neuronal NO production in the brainstem is dependent on rapid E(2) signaling.

Apoptosis-inducing effect of Akebia saponin D from the roots of Dipsacus asper Wall in U937 cells

Methanol extracts of the root of Dipsacus asper Wall (Dipsacaceae) were found to exhibit apoptosis-inducing activities in U937 (human monocyte-like histiocytic) cells. Investigation of the active n-BuOH fraction led to the isolation of akebia saponin D (ASD). Structure was established by spectroscopic methods. Treatment of U937 cells with ASD induced apoptosis in a dose dependent manner. ASD exerted strong cytotoxicity against human and murine leukemia cells. It is significantly increased the subG1 cell population and expression of p53 and Bax gene. And also ASD enhanced NO production from RAW264.7 macrophage cells. Taken together, these results strongly indicate that ASD may exert apoptosis-inducing activity via induction of apoptosis through activation chiefly via the nitric oxide and apoptosis-related p53 and Bax gene expression. These data provide scientific evidence that Dipsacus asper Wall can be useful as a chemopreventive agent.

Phosphorylation within an Autoinhibitory Domain in Endothelial Nitric Oxide Synthase Reduces the Ca2+ Concentrations Required for Calmodulin To Bind and Activate the Enzyme

We have investigated the effects of phosphorylation at Ser-617 and Ser-635 within an autoinhibitory domain (residues 595-639) in bovine endothelial nitric oxide synthase on enzyme activity and the Ca (2+) dependencies for calmodulin binding and enzyme activation. A phosphomimetic S617D substitution doubles the maximum calmodulin-dependent enzyme activity and decreases the EC 50(Ca (2+)) values for calmodulin binding and enzyme activation from the wild-type values of 180 +/- 2 and 397 +/- 23 nM to values of 109 +/- 2 and 258 +/- 11 nM, respectively. Deletion of the autoinhibitory domain also doubles the maximum calmodulin-dependent enzyme activity and decreases the EC 50(Ca (2+)) values for calmodulin binding and calmodulin-dependent enzyme activation to 65 +/- 4 and 118 +/- 4 nM, respectively. An S635D substitution has little or no effect on enzyme activity or EC 50(Ca (2+)) values, either alone or when combined with the S617D substitution. These results suggest that phosphorylation at Ser-617 partially reverses suppression by the autoinhibitory domain. Associated effects on the EC 50(Ca (2+)) values and maximum calmodulin-dependent enzyme activity are predicted to contribute equally to phosphorylation-dependent enhancement of NO production during a typical agonist-evoked Ca (2+) transient, while the reduction in EC 50(Ca (2+)) values is predicted to be the major contributor to enhancement at resting free Ca (2+) concentrations.

Post‐transcriptional regulation of inducible nitric oxide synthase (iNOS) by Protein Interacting with Never in Mitosis Gene A‐1 (PIN1) in murine aortic endothelial cells

PIN1, a peptidyl‐proline isomerase, increases the level or activity of several transcription factors that can induce iNOS. Thus it was hypothesized that depletion of PIN1 would reduce induction of iNOS by lipopolysaccharide (LPS) and interferon‐gamma (IFN). Murine aortic endothelial cells (EC) were stably transfected with a vector producing a PIN1 short hairpin RNA (shRNA) or an inactive control sequence. PIN1 shRNA reduced PIN1 levels &gt;85% compared to control. Unexpectedly, suppression of PIN 1 enhanced NO production in response to LPS/IFN, and iNOS protein was induced 4.7‐fold more. The increased iNOS was cytotoxic to EC lacking PIN1, but not to control EC, in a crystal violet staining assay. Furthermore, cytotoxicity was prevented by a nitric oxide synthase inhibitor, L‐NAME. LPS/IFN increased iNOS/β‐actin mRNA ratio as expected, but PIN1 knockdown did not affect iNOS mRNA levels measured by RT‐PCR. Instead, PIN1 depletion stabilized iNOS protein in cycloheximide‐treated EC such that the time to a 50% decrease was extended from 8 h to more than 24 h. The loss of iNOS was antagonized by the calpain inhibitor, MDL 28170, but not by the selective proteasome inhibitor, epoxomicin. These results suggest that PIN1 normally restrains the induction of iNOS by facilitating its calpain‐mediated degradation in EC. Modulation of PIN1 may significantly affect iNOS‐dependent vascular activity and inflammatory diseases.

Increased Nitric Oxide Production and GFAP Expression in the Brains of Influenza A/NWS Virus Infected Mice

The cause of influenza to the brain was investigated using the A/NWS/33 influenza virus infected BALB/c mouse model. NOS-2 mRNA levels in the infected mouse brain was greater than in control mice in all brain regions examined, particularly in the olfactory bulb and hippocampus by 1 day p.i. On the contrary, no differences in NOS-1 or NOS-3 mRNA levels were found between infected and control mice. There was also a marked increase in the levels of metabolites of nitric oxide in the olfactory bulb and hippocampus. Immunohistochemistry showed positive staining for anti-NOS-2 primarily in the hippocampus of infected mice. Further, anti-NOS-2 and GFAP staining was mostly found around capillary blood vessels of the hippocampus starting early in the course of the disease. These results indicate that the NWS enhances the activation of astrocytes and NOS-2 expression which in turn enhances NO production and the expansion of capillary blood vessels.

IL-13 pre-treatment of murine peritoneal macrophages increases their anti-Toxoplasma gondii activity induced by lipopolysaccharides

Th1 cytokines and microbial lipopolysaccharides (LPS) activate macrophages to produce inflammatory mediators and effector molecules. Althrough Th2 cytokines often have an opposite action to Th1 cytokines and down-modulate the inflammatory response of macrophages, they can induce a distinct alternative activation that is beneficial in host defence. In this study, we report that IL-13 enhances the anti-Toxoplasma activity of LPS-activated murine macrophages. The inhibition of parasite proliferation was not related to reduced Toxoplasma gondii penetration into the cells, nor to the conversion of tachyzoites into bradyzoites. Used alone, IL-13 triggers the polarisation of macrophages towards type 2. However, in LPS-activated macrophages, we show the priming capacity of this cytokine to enhance the expression of inducible nitric oxide synthase (iNOS), a major marker of type 1 macrophages. This effect of IL-13 was not dependent on the activation state of macrophages (resident versus thioglycolate-elicited) or the timing of pre-treatment. We demonstrate a correlation between the enhancement of NO production and upgrading of the microbicidal effectiveness of the macrophages. Thus, both Th2 and Th1 cytokines could activate macrophages to control infections.

Inhibitory effect of oral intake of natural Phellinus linteus fruit body on growth and pulmonary metastasis of B16/BL6 melanoma

In the present study, the immunotherapeutic activity of oral intake of natural Phellinus linteus fruit body (nPF) against growth and pulmonary metastasis of injected B16/BL6 melanoma cells has been examined. The nPF powder was orally administered to B16/BL6-bearing mice, a model for the pulmonary metastasis of melanoma, at doses of 83, 415 and 2,075 mg/kg day in their diet. The dietary intake of nPF powder significantly prolonged the survival time of the B16/BL6-bearing mice. Furthermore, ingestion of nPF dose-dependently enhanced NO production by macrophages prepared from the tumor-bearing, nPF-treated animals. Simultaneously, nPF did not induce direct cytotoxicity against B16/BL6. These results suggest that the oral intake of nPF powder may be useful in immunochemotherapy for cancer because of its life prolongation through the inhibitory effect on growth and metastasis of cancer.

Interleukin-13 primes iNO synthase expression induced by LPS in mouse peritoneal macrophages

Th2 cytokines such as interleukin-13 (IL-13) have both, stimulatory and inhibitory effects on effector functions of macrophages. Reactive nitrogen species are classicaly induced in Th1 cytokines and/or lipopolysaccharides (LPS) activated macrophages and this response is inhibited by IL-13. In contrast, IL-13 primes macrophages to produce NO in response to LPS when IL-13 treatment happens prior to LPS exposure. This mechanism occurs through a complex signalling pathway, which involves the scavenger receptor CD36, the LPS receptor CD14 and the nuclear receptor PPARγ. The enhancement of NO production is the consequence of iNOS induction at mRNA and protein levels. The increase of the NO production induced by LPS in IL-13 pre-treated macrophages is found to potentiate the inhibition of Toxoplasma gondii intracellular replication. These results reveal a novel IL-13 signalling pathway that primes the antimicrobial activity of macrophages induced by LPS caused by overexpression of the iNOS-NO axis.

Glucocorticoids exert opposing effects on macrophage function dependent on their concentration

Glucocorticoids (GCs) are involved in the modulation of macrophage function and thereby control the host's immune responses to pathogens. However, neither the role of hormone concentration nor the differential contribution of the glucocorticoid (GR) and the mineralocorticoid receptors (MR) to these activities are known. Here we show that low levels of corticosterone enhance NO production as well as mRNA expression of pro-inflammatory cytokines, chemokines and enzymes required for mediator synthesis. In contrast, at high corticosterone concentrations macrophage function was strongly repressed. Importantly, inactivation of the GR by lentiviral delivery of siRNAs abrogated both the immunostimulatory and the immunosuppressive GC actions whereas inactivation of the MR had no effect. Furthermore, removal of endogenous GCs by adrenalectomy in vivo induced a preactivated state in macrophages that could be modulated by corticosterone. We conclude that GCs exert distinct effects on macrophage function dependent on their concentration, and that they primarily act through the GR despite concomitant expression of the MR.

The Fas death pathway controls coordinated expansions of type 1 CD8 and type 2 CD4 T cells inTrypanosoma cruziinfection

We investigated the role of the Fas ligand (FasL)/Fas death pathway on apoptosis and cytokine production by T cells in Trypanosoma cruzi infection. Anti-FasL, but not anti-TNF-alpha or anti-TRAIL, blocked activation-induced cell death of CD8 T cells and increased secretion of IL-10 and IL-4 by CD4 T cells from T. cruzi-infected mice. CD4 and CD8 T cells up-regulated Fas/FasL expression during T. cruzi infection. However, Fas expression increased earlier in CD8 T cells, and a higher proportion of CD8 T cells was activated and expressed IFN-gamma compared with CD4 T cells. Injection of anti-FasL in infected mice reduced parasitemia and CD8 T cell apoptosis and increased the ratio of CD8:CD4 T cells recovered from spleen and peritoneum. FasL blockade increased the number of activated T cells, enhanced NO production, and reduced parasite loads in peritoneal macrophages. Injection of anti-FasL increased IFN-gamma secretion by splenocytes responding to T. cruzi antigens but also exacerbated production of type 2 cytokines IL-10 and IL-4 at a late stage of acute infection. These results indicate that the FasL/Fas death pathway regulates apoptosis and coordinated cytokine responses by type 1 CD8 and type 2 CD4 T cells in T. cruzi infection.

Uptake of inhalable microparticles affects defence responses of macrophages infected with Mycobacterium tuberculosis H37Ra

To investigate whether inhalable microparticles containing two anti-tuberculosis agents, isoniazid and rifampicin, evoke host-defence strategies in macrophages in addition to targeting the incorporated drugs. Microparticles were prepared by spray-drying a homogeneous solution of drugs and poly(lactic acid) (PLA; apparent viscosity 1.1 cP). Four parts PLA and three parts rifampicin were dissolved in dichloromethane. One part isoniazid was dissolved in methanol. The two solutions were mixed in the ratio 22 : 3 at which none of the solutes precipitated. These were administered as 'nose-only' inhalations to mice or exposed to cultured J774 mouse macrophages. Targeting to lung macrophages was investigated by transmission electron microscopy. Reactive oxygen species (ROS) were estimated by a cytochrome c assay and flow cytometry. Reactive nitrogen intermediates (RNI) were assayed using Griess reagent. Cytokines in culture supernatants were estimated by ELISA. Treatment with inhalable microparticles targeted lung macrophages in vivo and induced intense Golgi activity in the vicinity of microparticle-containing phagosomes. Microparticles induced a respiratory burst involving NADPH oxidase and enhanced NO production by infected macrophages. Microparticle-induced NADPH oxidase activation required optimal calcium ions. Microparticles efficiently induced tumour necrosis factor-alpha (TNF-alpha) secretion by macrophages recovered from infected mice. Microparticle phagocytosis induces responses in infected murine macrophages that are indicative of activation of innate bactericidal mechanisms, and are inimical to bacterial survival. It is likely that such responses augment straightforward drug action on the bacterium and contribute to the unexpectedly high efficacy of microparticles in experimental tuberculosis.

Effect of Magnesium Deficiency on Intracellular ATP Levels in Human Lens Epithelial Cells

Cataractous lenses have an altered distribution of the intracellular ionic environment, and the lens ionic imbalance with increased levels of calcium (Ca2+) and sodium (Na+), coupled with decreased levels of magnesium (Mg2+) and potassium (K+), is related to cataract development in human senile cataracts. We previously found that the decrease of ATP in lenses caused lens ionic imbalance, and probably decrease in ATPase function. In this study, we investigated the effect of Mg2+ deficiency on cataract progression using human lens epithelial (HLE) cells. Expression levels of inducible nitric oxide synthase (iNOS) mRNA in HLE cells were significantly greater in Mg2+-deficient medium (Mg2+ 0.021 mM) than in normal Mg2+ medium (Mg2+ 0.77 mM). The NO release from the HLE cells cultured with Mg2+-deficient medium also increased. On the other hand, the ATP levels in HLE cells 24 h after incubation with Mg2+-deficient medium were lower than that with normal Mg2+ medium. The Ca2+- and Na+/K+-ATPase activities in HLE cells until 24 h incubation with normal Mg2+ or Mg2+-deficient medium did not change. Both diethyldithiocarbamate 10 microM and aminoguanidine 250 microM attenuated the increase of NO release, and caused an increase in ATP levels in HLE cells 24 h after incubation with Mg2+-deficient medium. These results suggest that Mg2+ deficiency enhances NO production via iNOS in the lens. It is possible that the excessive production of NO cause the decrease of ATP levels. These results show that Mg2+ deficiency in the lens may cause an acceleration of the progression of lens opacification.

HSP90/ENOS interactions contribute to maturational and chronic hypoxia‐induced changes in pulmonary vascular reactivity in newborn pigs

Hsp90 is a molecular chaperone involved in regulating eNOS signaling. Concomitant with enhanced NO production, the association of Hsp90 with eNOS increases upon stimulation with agonists such as Acetylcholine (ACh). The role of Hsp90/eNOS interactions in maturational changes in pulmonary vascular reactivity is unclear. Moreover, there are no data on Hsp90/eNOS interactions in newborns with chronic hypoxia-induced pulmonary hypertension. Methods: Piglets were raised in normoxia or hypoxia from day of life 2 to 6. A cannulated artery technique was used to measure ACh responses in resistance pulmonary arteries (RPAs). A coprecipitation technique was used to assess Hsp90/eNOS interactions in RPAs. Results: ACh induced a greater dilation in RPAs from 6-day old pigs raised in normoxia(102+/−5%) than in RPAs from 2 day old pigs (80+/−8%). ACh stimulated a greater increase in Hsp90/eNOS coprecipitation in RPAs from 6-day old pigs raised in normoxia (190+/−64%) than those from 2 day old pigs (18+/−7%). ACh induced dilation was reduced in RPAs from 6-day old pigs raised in hypoxia (30+/−22%) compared with 6-day old pigs raised in normoxia. ACh stimulation failed to increase Hsp90/eNOS coprecipitation in RPAs from pigs raised in hypoxia. Conclusions: Hsp90/eNOS interactions mature during the first week of life and contribute to the enhanced pulmonary vascular responses to NO-dependent agonists observed with advancing postnatal age. Chronic hypoxia alters pulmonary vascular reactivity in part by disrupting the normal maturational enhancement of Hsp90/eNOS interactions. Manipulating Hsp90/eNOS interactions could augment pulmonary vascular NO activity in newborns with chronic hypoxia-induced pulmonary hypertension. Funding: NIH RO1HL075511 and RO1HL068572