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1.  Brown Adipose Tissue Blood Flow and Mass in Obesity: A Contrast Ultrasound Study in Mice 
When activated by the sympathetic nervous system, brown adipose tissue (BAT) increases energy expenditure to produce heat. Augmenting BAT mass or increasing BAT activation could potentially be used to decrease obesity. Noninvasive methods to detect and monitor BAT mass are needed. Contrast ultrasound can estimate BAT blood flow and is able to measure the perfused volume of an organ and thus its mass. The objective of this study was to evaluate whether contrast ultrasound could characterize BAT mass in two mouse models of obesity: wild-type mice fed a high-fat diet and mutant db/db mice.
Contrast ultrasound of BAT (Definity 2 μL/min; 14-MHz linear probe) was performed before and after stimulation of BAT with norepinephrine (NE). BAT replenishment curves were obtained, and blood flow was estimated by the product of the curve’s plateau and slope. Additionally, consecutive two-dimensional images of perfused BAT were acquired at 1-mm intervals after stimulation with NE and used to assess BAT volume and mass.
BAT blood flow increased after NE infusion in all mice studied. Blood flow response to NE was similar in wild-type mice fed either a low-fat diet or a high-fat diet. BAT blood flow was lower in db/db mice than in wild-type mice (P = .02). Contrast ultrasound–derived BAT mass was correlated with BAT mass obtained at necropsy (R2 = 0.83, P < .001). BAT mass was higher in mice fed a high-fat diet than in those fed a low-fat diet.
Contrast ultrasound can be used to estimate BAT mass in mice when BAT vascularization is not significantly impaired. This noninvasive technique may potentially allow the serial evaluation of therapies designed to augment BAT mass.
PMCID: PMC3954535  PMID: 23993691
Brown adipose tissue; Imaging; Obesity; Contrast ultrasound; db/db mice
2.  MAPK-activated protein kinase 2-deficiency causes hyperacute tumor necrosis factor-induced inflammatory shock 
BMC Physiology  2014;14(1):5.
MAPK-activated protein kinase 2 (MK2) plays a pivotal role in the cell response to (inflammatory) stress. Among others, MK2 is known to be involved in the regulation of cytokine mRNA metabolism and regulation of actin cytoskeleton dynamics. Previously, MK2-deficient mice were shown to be highly resistant to LPS/d-Galactosamine-induced hepatitis. Additionally, research in various disease models has indicated the kinase as an interesting inhibitory drug target for various acute or chronic inflammatory diseases.
We show that in striking contrast to the known resistance of MK2-deficient mice to a challenge with LPS/D-Gal, a low dose of tumor necrosis factor (TNF) causes hyperacute mortality via an oxidative stress driven mechanism. We identified in vivo defects in the stress fiber response in endothelial cells, which could have resulted in reduced resistance of the endothelial barrier to deal with exposure to oxidative stress. In addition, MK2-deficient mice were found to be more sensitive to cecal ligation and puncture-induced sepsis.
The capacity of the endothelial barrier to deal with inflammatory and oxidative stress is imperative to allow a regulated immune response and maintain endothelial barrier integrity. Our results indicate that, considering the central role of TNF in pro-inflammatory signaling, therapeutic strategies examining pharmacological inhibition of MK2 should take potentially dangerous side effects at the level of endothelial barrier integrity into account.
PMCID: PMC4160542  PMID: 25185746
MK2; Inflammatory shock; Tumor necrosis factor; Reactive oxygen species; Actin cytoskeleton; Endothelial permeability; Cecal ligation and puncture
3.  In Vivo Noninvasive Characterization of Brown Adipose Tissue Blood Flow by Contrast Ultrasound in Mice 
Interventions to increase brown adipose tissue (BAT) volume and activation are being extensively investigated as therapies to decrease the body weight in obese subjects. Noninvasive methods to monitor these therapies in animal models and humans are rare. We investigated whether contrast ultrasound (CU) performed in mice could detect BAT and measure its activation by monitoring BAT blood flow. After validation, CU was used to study the role of uncoupling protein 1 (UCP1) and nitric oxide synthases in the acute regulation of BAT blood flow.
Methods and Results
Blood flow of interscapular BAT was assessed in mice (n=64) with CU by measuring the signal intensity of continuously infused contrast microbubbles. Blood flow of BAT estimated by CU was 0.5±0.1 (mean±SEM) dB/s at baseline and increased 15-fold during BAT stimulation by norepinephrine (NE, 1 μg·kg−1·min−1). Assessment of BAT blood flow using CU was correlated to that performed with fluorescent microspheres (R2=0.86, p<0.001). To evaluate whether intact BAT activation is required to increase BAT blood flow, CU was performed in UCP1-deficient (UCP1−/−) mice with impaired BAT activation. Norepinephrine infusion induced a smaller increase in BAT blood flow in UCP1−/− mice than in wild-type mice. Finally, we investigated whether NOS played a role in acute NE-induced changes of BAT blood flow. Genetic and pharmacologic inhibition of NOS3 attenuated the NE-induced increase in BAT blood flow.
These results indicate that CU can detect BAT in mice, and estimate BAT blood flow in mice with functional differences in BAT.
PMCID: PMC3471995  PMID: 22776888
brown adipose tissue; imaging; contrast ultrasound; uncoupling protein; nitric oxide synthase
6.  The Soluble Guanylate Cyclase Activator BAY 58-2667 Protects against Morbidity and Mortality in Endotoxic Shock by Recoupling Organ Systems 
PLoS ONE  2013;8(8):e72155.
Sepsis and septic shock are associated with high mortality rates and the majority of sepsis patients die due to complications of multiple organ failure (MOF). The cyclic GMP (cGMP) producing enzyme soluble guanylate cyclase (sGC) is crucially involved in the regulation of (micro)vascular homeostasis, cardiac function and, consequently, organ function. However, it can become inactivated when exposed to reactive oxygen species (ROS). The resulting heme-free sGC can be reactivated by the heme- and nitric oxide (NO)-independent sGC activator BAY 58-2667 (Cinaciguat). We report that late (+8 h) post-treatment with BAY 58-2667 in a mouse model can protect against lethal endotoxic shock. Protection was associated with reduced hypothermia, circulating IL-6 levels, cardiomyocyte apoptosis, and mortality. In contrast to BAY 58-2667, the sGC stimulator BAY 41-2272 and the phosphodiesterase 5 inhibitor Sildenafil did not have any beneficial effect on survival, emphasizing the importance of the selectivity of BAY 58-2667 for diseased vessels and tissues. Hemodynamic parameters (blood pressure and heart rate) were decreased, and linear and nonlinear indices of blood pressure variability, reflective for (un)coupling of the communication between the autonomic nervous system and the heart, were improved after late protective treatment with BAY 58-2667. In conclusion, our results demonstrate the pivotal role of the NO/sGC axis in endotoxic shock. Stabilization of sGC function with BAY 58-2667 can prevent mortality when given in the correct treatment window, which probably depends on the dynamics of the heme-free sGC pool, in turn influenced by oxidative stress. We speculate that, considering the central role of sGC signaling in many pathways required for maintenance of (micro)circulatory homeostasis, BAY 58-2667 supports organ function by recoupling inter-organ communication pathways.
PMCID: PMC3756074  PMID: 24015214
7.  Soluble Guanylate Cyclase α1–Deficient Mice: A Novel Murine Model for Primary Open Angle Glaucoma 
PLoS ONE  2013;8(3):e60156.
Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide. The molecular signaling involved in the pathogenesis of POAG remains unknown. Here, we report that mice lacking the α1 subunit of the nitric oxide receptor soluble guanylate cyclase represent a novel and translatable animal model of POAG, characterized by thinning of the retinal nerve fiber layer and loss of optic nerve axons in the context of an open iridocorneal angle. The optic neuropathy associated with soluble guanylate cyclase α1–deficiency was accompanied by modestly increased intraocular pressure and retinal vascular dysfunction. Moreover, data from a candidate gene association study suggests that a variant in the locus containing the genes encoding for the α1 and β1 subunits of soluble guanylate cyclase is associated with POAG in patients presenting with initial paracentral vision loss, a disease subtype thought to be associated with vascular dysregulation. These findings provide new insights into the pathogenesis and genetics of POAG and suggest new therapeutic strategies for POAG.
PMCID: PMC3603933  PMID: 23527308
8.  Nitric Oxide Regulates Pulmonary Vascular Smooth Muscle Cell Expression of the Inducible cAMP Early Repressor Gene 
Nitric oxide (NO) regulates vascular smooth muscle cell (VSMC) structure and function, in part by activating soluble guanylate cyclase (sGC) to synthesize cGMP. The objective of this study was to further characterize the signaling mechanisms by which NO regulates VSMC gene expression using transcription profiling. DNA microarrays were hybridized with RNA extracted from rat pulmonary artery smooth muscle cells (RPaSMC) exposed to the NO donor compound, S-nitroso-glutathione (GSNO). Many of the genes, whose expression was induced by GSNO, contain a cAMP-response element (CRE), of which one encoded the inducible cAMP early repressor (ICER). sGC and cAMP-dependent protein kinase, but not cGMP-dependent protein kinase, were required for NO-mediated phosphorylation of CRE-binding protein (CREB) and induction of ICER gene expression. Expression of a dominant-negative CREB in RPaSMC prevented the NO-mediated induction of CRE-dependent gene transcription and ICER gene expression. Pre-treatment of RPaSMC with the intracellular calcium (Ca2+) chelator, BAPTA-AM, blocked the induction of ICER gene expression by GSNO. The store-operated Ca2+ channel inhibitors, 2-ABP and SKF-96365, reduced the GSNO-mediated increase in ICER mRNA levels, while 2-ABP did not inhibit GSNO-induced CREB phosphorylation. Our results suggest that induction of ICER gene expression by NO requires both CREB phosphorylation and Ca2+ signaling. Transcription profiling of RPaSMC exposed to GSNO revealed important roles for sGC, PKA, CREB, and Ca2+ in the regulation of gene expression by NO. The induction of ICER in GSNO-treated RPaSMC highlights a novel cross-talk mechanism between cGMP and cAMP signaling pathways.
PMCID: PMC3466086  PMID: 21642009
nitric oxide; vascular smooth muscle; cAMP-response element; cyclic GMP; protein kinase A
9.  Inhaled Nitric Oxide Improves Outcomes After Successful Cardiopulmonary Resuscitation in Mice 
Circulation  2011;124(15):1645-1653.
Sudden cardiac arrest (CA) is a leading cause of death worldwide. Breathing nitric oxide (NO) reduces ischemia-reperfusion (IR) injury in animal models and in patients. The objective of this study was to learn whether inhaled NO improves outcomes after CA and cardiopulmonary resuscitation (CPR).
Methods and Results
Adult male mice were subjected to potassium-induced CA for 7.5 min whereupon CPR was performed with chest compression and mechanical ventilation. One hour after CPR, mice were extubated and breathed air alone or air supplemented with 40 parts per million (ppm) NO for 23h. Mice that were subjected to CA/CPR and breathed air exhibited a poor 10-day survival rate (4/13), depressed neurological and left ventricular (LV) function, and increased caspase-3 activation and inflammatory cytokine induction in the brain. Magnetic resonance imaging revealed brain regions with marked water diffusion abnormality 24h after CA/CPR in mice that breathed air. Breathing air supplemented with NO for 23h starting 1h after CPR attenuated neurological and LV dysfunction 4 days after CA/CPR and markedly improved 10-day survival rate (11/13, P=0.003 vs Air). The protective effects of inhaled NO on the outcome after CA/CPR were associated with reduced water diffusion abnormality, caspase-3 activation, and cytokine induction in the brain and increased serum NOx levels. Deficiency of the α1 subunit of soluble guanylate cyclase (sGC), a primary target of NO, abrogated the ability of inhaled NO to improve outcomes after CA/CPR.
These results suggest that NO inhalation after CA and successful CPR improves outcome via sGC-dependent mechanisms.
PMCID: PMC3199136  PMID: 21931083
cardiopulmonary resuscitation; heart arrest; neurological function; magnetic resonance imaging; nitric oxide synthase; physiology
11.  The alpha1 isoform of soluble guanylate cyclase regulates cardiac contractility but is not required for ischemic preconditioning 
Basic Research in Cardiology  2011;106(4):635-643.
Nitric oxide (NO)-dependent soluble guanylate cyclase (sGC) activation is an important component of cardiac signal transduction pathways, including the cardioprotective signaling cascade induced by ischemic preconditioning (IPC). The sGCα subunit, which binds to the common sGCβ1 subunit, exists in two different isoforms, sGCα1 and sGCα2, but their relative physiological roles remain unknown. In the present study, we studied Langendorff-perfused isolated hearts of genetically engineered mice lacking functional sGCα1 (sGCα1KO mice), which is the predominant isoform in the heart. Our results show that the loss of sGCα1 has a positive inotropic and lusitropic effect on basal cardiac function, indicating an important role for sGCα1 in regulating basal myocardial contractility. Surprisingly, IPC led to a similar 35–40% reduction in infarct size and concomitant protein kinase Cε (PKCε) phosphorylation in both wild-type (WT) and sGCα1KO hearts subjected to 40 min of global ischemia and reperfusion. Inhibition of the activation of all sGC isoforms by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ, 10 μmol/L) completely abolished the protection by IPC in WT and sGCα1KO hearts. NO-stimulated cGMP production was severely attenuated in sGCα1KO hearts compared to WT hearts, indicating that the sGCα2 isoform only produces minute amounts of cGMP after NO stimulation. Taken together, our results indicate that although sGCα1 importantly regulates cardiac contractility, it is not required for cardioprotection by IPC. Instead, our results suggest that possibly only minimal sGC activity, which in sGCα1KO hearts is provided by the sGCα2 isoform, is sufficient to transduce the cardioprotective signal induced by IPC via phosphorylation of PKCε.
PMCID: PMC3375061  PMID: 21394564
Soluble guanylate cyclase; Ischemic preconditioning; Isolated heart; Contractility; Nitric oxide
12.  Genetic modifiers of hypertension in soluble guanylate cyclase α1–deficient mice 
The Journal of Clinical Investigation  2012;122(6):2316-2325.
Nitric oxide (NO) plays an essential role in regulating hypertension and blood flow by inducing relaxation of vascular smooth muscle. Male mice deficient in a NO receptor component, the α1 subunit of soluble guanylate cyclase (sGCα1), are prone to hypertension in some, but not all, mouse strains, suggesting that additional genetic factors contribute to the onset of hypertension. Using linkage analyses, we discovered a quantitative trait locus (QTL) on chromosome 1 that was linked to mean arterial pressure (MAP) in the context of sGCα1 deficiency. This region is syntenic with previously identified blood pressure–related QTLs in the human and rat genome and contains the genes coding for renin. Hypertension was associated with increased activity of the renin-angiotensin-aldosterone system (RAAS). Further, we found that RAAS inhibition normalized MAP and improved endothelium-dependent vasorelaxation in sGCα1-deficient mice. These data identify the RAAS as a blood pressure–modifying mechanism in a setting of impaired NO/cGMP signaling.
PMCID: PMC3366402  PMID: 22565307
13.  Toll-like Receptor 4 Signaling Confers Cardiac Protection Against Ischemic Injury via Inducible Nitric Oxide Synthase- and Soluble Guanylate Cyclase-dependent Mechanisms 
Anesthesiology  2011;114(3):603-613.
Prior administration of a small dose of lipopolysaccharide confers a cardiac protection against ischemia-reperfusion injury. However, the signaling mechanisms that control the protection are incompletely understood. We tested the hypothesis that TLR4 mediates the ability of lipopolysaccharide to protect against cardiac ischemia-reperfusion injury through distinct intracellular pathways involving myeloid differentiation factor 88 (MyD88), TIR-domain-containing adaptor protein inducing interferon-β–mediated transcription-factor (Trif), inducible nitric-oxide synthase (iNOS), and soluble guanylate cyclase (sGC).
Wild-type mice and the genetically modified mice, i.e., TLR4-deficient (TLR4-def), TLR2 knockout (TLR2−/−), MyD88−/−, Trif−/−, iNOS−/−, and sGCα1−/−, were treated with normal saline or 0.1 mg/kg of lipopolysaccharide, intraperitoneally. Twenty-four hours later, isolated hearts were perfused in a Langendorff apparatus and subsequently subjected to 30 min of global ischemia and reperfusion for up to 60 min. Left ventricular function and myocardial infarction sizes were examined.
Compared to saline-treated mice, lipopolysaccharide-treated mice had markedly improved left ventricular developed pressure and dP/dtmax (P < 0.01) and reduced MI sizes (37.2 ± 3.4% vs. 19.8 ± 4.9%, P < 0.01) after ischemia-reperfusion. The cardiac protective effect of lipopolysaccharide was abolished in the TLR4-def and MyD88−/− mice, but remained intact in TLR2−/− or Trif−/− mice. iNOS−/− mice or wild-type mice treated with the iNOS inhibitor 1400W failed to respond to the TLR4-induced nitric oxide production and were not protected by the lipopolysaccharide preconditioning. While sGC 1−/− mice had robust nitric oxide production in response to lipopolysaccharide, they were not protected by the TLR4-elicited cardiac protection.
TLR4 activation confers a potent cardiac protection against ischemia-reperfusion injury via a MyD88-dependent, but Trif-independent mechanism. iNOS/sGC are essential for the TLR4-induced cardiac protection.
PMCID: PMC3044772  PMID: 21270629
14.  Do-it-yourself: construction of a custom cDNA macroarray platform with high sensitivity and linear range 
BMC Biotechnology  2011;11:97.
Research involving gene expression profiling and clinical applications, such as diagnostics and prognostics, often require a DNA array platform that is flexibly customisable and cost-effective, but at the same time is highly sensitive and capable of accurately and reproducibly quantifying the transcriptional expression of a vast number of genes over the whole transcriptome dynamic range using low amounts of RNA sample. Hereto, a set of easy-to-implement practical optimisations to the design of cDNA-based nylon macroarrays as well as sample 33P-labeling, hybridisation protocols and phosphor screen image processing were analysed for macroarray performance.
The here proposed custom macroarray platform had an absolute sensitivity as low as 50,000 transcripts and a linear range of over 5 log-orders. Its quality of identifying differentially expressed genes was at least comparable to commercially available microchips. Interestingly, the quantitative accuracy was found to correlate significantly with corresponding reversed transcriptase - quantitative PCR values, the gold standard gene expression measure (Pearson's correlation test p < 0.0001). Furthermore, the assay has low cost and input RNA requirements (0.5 μg and less) and has a sound reproducibility.
Results presented here, demonstrate for the first time that self-made cDNA-based nylon macroarrays can produce highly reliable gene expression data with high sensitivity and covering the entire mammalian dynamic range of mRNA abundances. Starting off from minimal amounts of unamplified total RNA per sample, a reasonable amount of samples can be assayed simultaneously for the quantitative expression of hundreds of genes in an easily customisable and cost-effective manner.
PMCID: PMC3217856  PMID: 22026914
15.  Soluble Guanylate Cyclase α1β1 Limits Stroke Size and Attenuates Neurological Injury 
Background and Purpose
Nitric oxide (NO) mediates endothelium-dependent vasodilation, modulates cerebral blood flow (CBF), and determines stroke outcome. NO signals in part by stimulating soluble guanylate cyclase (sGC) to synthesize cGMP. To study the role of sGC in stroke injury, we compared the outcome of cerebral ischemia and reperfusion in mice deficient in the α1 subunit of sGC (sGCα1−/−) to that on wild-type (WT) mice.
Blood pressure, cerebrovascular anatomy, and vasoreactivity of pressurized carotid arteries were compared in both mouse genotypes. CBF was measured before and during middle cerebral artery (MCA) occlusion and reperfusion. We then assessed neurological deficit and infarct volume after 1 hour of occlusion and 23 hours of reperfusion, and after 24 hours of occlusion.
Blood pressure and cerebrovascular anatomy were similar between genotypes. We found that vasodilation of carotid arteries in response to acetylcholine or sodium nitroprusside was diminished in sGCα1−/− compared to WT mice. CBF deficits did not differ between the genotypes during occlusion, but during reperfusion, CBF was 45% less in sGCα1−/− mice. Infarct volumes and neurological deficits were similar after 24 hours of occlusion in both genotypes. After 1 hour of ischemia and 23 hours of reperfusion, infarct volumes were two-fold larger and neurological deficits were worse in sGCα1−/− than in the WT mice.
sGCα1 deficiency impairs vascular reactivity to NO, and is associated with incomplete reperfusion, larger infarct size and worse neurological damage, suggesting that cGMP generated by sGCα1β1 is protective in ischemic stroke.
PMCID: PMC3047459  PMID: 20595671
cerebral ischemia; gene knockout mice; mouse models
20.  Disruption of the SapM locus in Mycobacterium bovis BCG improves its protective efficacy as a vaccine against M. tuberculosis 
EMBO Molecular Medicine  2011;3(4):222-234.
Mycobacterium bovis bacille Calmette-Guerin (BCG) provides only limited protection against pulmonary tuberculosis. We tested the hypothesis that BCG might have retained immunomodulatory properties from its pathogenic parent that limit its protective immunogenicity. Mutation of the molecules involved in immunomodulation might then improve its vaccine potential. We studied the vaccine potential of BCG mutants deficient in the secreted acid phosphatase, SapM, or in the capping of the immunomodulatory ManLAM cell wall component with α-1,2-oligomannoside. Both systemic and intratracheal challenge of mice with Mycobacterium tuberculosis following vaccination showed that the SapM mutant, compared to the parental BCG vaccine, provided better protection: it led to longer-term survival. Persistence of the SapM-mutated BCG in vivo resembled that of the parental BCG indicating that this mutation will likely not compromise the safety of the BCG vaccine. The SapM mutant BCG vaccine was more effective than the parental vaccine in inducing recruitment and activation of CD11c+MHC-IIintCD40int dendritic cells (DCs) to the draining lymph nodes. Thus, SapM acts by inhibiting recruitment of DCs and their activation at the site of vaccination.
PMCID: PMC3377067  PMID: 21328541
Mycobacterium; SapM; tuberculosis; vaccine; BCG
21.  Nitrite protects against morbidity and mortality associated with TNF- or LPS-induced shock in a soluble guanylate cyclase–dependent manner 
The Journal of Experimental Medicine  2009;206(13):2915-2924.
Nitrite (NO2−), previously viewed as a physiologically inert metabolite and biomarker of the endogenous vasodilator NO, was recently identified as an important biological NO reservoir in vasculature and tissues, where it contributes to hypoxic signaling, vasodilation, and cytoprotection after ischemia–reperfusion injury. Reduction of nitrite to NO may occur enzymatically at low pH and oxygen tension by deoxyhemoglobin, deoxymyoglobin, xanthine oxidase, mitochondrial complexes, or NO synthase (NOS). We show that nitrite treatment, in sharp contrast with the worsening effect of NOS inhibition, significantly attenuates hypothermia, mitochondrial damage, oxidative stress and dysfunction, tissue infarction, and mortality in a mouse shock model induced by a lethal tumor necrosis factor challenge. Mechanistically, nitrite-dependent protection was not associated with inhibition of mitochondrial complex I activity, as previously demonstrated for ischemia–reperfusion, but was largely abolished in mice deficient for the soluble guanylate cyclase (sGC) α1 subunit, one of the principal intracellular NO receptors and signal transducers in the cardiovasculature. Nitrite could also provide protection against toxicity induced by Gram-negative lipopolysaccharide, although higher doses were required. In conclusion, we show that nitrite can protect against toxicity in shock via sGC-dependent signaling, which may include hypoxic vasodilation necessary to maintain microcirculation and organ function, and cardioprotection.
PMCID: PMC2806477  PMID: 19934018
22.  Reactive oxygen species and small-conductance calcium-dependent potassium channels are key mediators of inflammation-induced hypotension and shock 
Septic shock is associated with life-threatening vasodilation and hypotension. To cause vasodilation, vascular endothelium may release nitric oxide (NO), prostacyclin (PGI2), and the elusive endothelium-derived hyperpolarizing factor (EDHF). Although NO is critical in controlling vascular tone, inhibiting NO in septic shock does not improve outcome, on the contrary, precipitating the search for alternative therapeutic targets. Using a hyperacute tumor necrosis factor (TNF)-induced shock model in mice, we found that shock can develop independently of the known vasodilators NO, cGMP, PGI2, or epoxyeicosatrienoic acids. However, the antioxidant tempol efficiently prevented hypotension, bradycardia, hypothermia, and mortality, indicating the decisive involvement of reactive oxygen species (ROS) in these phenomena. Also, in classical TNF or lipopolysaccharide-induced shock models, tempol protected significantly. Experiments with (cell-permeable) superoxide dismutase or catalase, N-acetylcysteine and apocynin suggest that the ROS-dependent shock depends on intracellular \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ ^\bullet {\hbox{OH}} $$\end{document} radicals. Potassium channels activated by ATP (KATP) or calcium (KCa) are important mediators of vascular relaxation. While NO and PGI2-induced vasodilation involves KATP and large-conductance BKCa channels, small-conductance SKCa channels mediate vasodilation induced by EDHF. Interestingly, also SKCa inhibition completely prevented the ROS-dependent shock. Our data thus indicate that intracellular \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ ^\bullet {\hbox{OH}} $$\end{document} and SKCa channels represent interesting new therapeutic targets for inflammatory shock. Moreover, they may also explain why antioxidants other than tempol fail to provide survival benefit during shock.
PMCID: PMC2921058  PMID: 20496172
Shock; Hypotension; ROS; Nitric oxide; Potassium channels; Apamin
23.  Protective effects of nitric oxide synthase 3 and soluble guanylate cyclase on the outcome of cardiac arrest and cardiopulmonary resuscitation in mice 
Critical care medicine  2009;37(1):256-262.
Despite advances in resuscitation methods, survival after out-of-hospital cardiac arrest remains very low, at least in part due to post cardiac arrest circulatory and neurological failure. To elucidate the role of nitric oxide (NO) in the recovery from cardiac arrest and CPR, we studied the impact of NOS3/cGMP signaling on cardiac and neurological outcomes after cardiac arrest and cardiopulmonary resuscitation (CPR).
Prospective, randomized, controlled study
Animal research laboratory
Female wild-type mice (WT), NOS3-deficient mice (NOS3−/−), NOS3−/− mice with cardiomyocyte-specific overexpression of NOS3 (NOS3−/−CSTg), and mice deficient for soluble guanylate cyclase α1 (sGCα1−/−) were subjected to potassium-induced cardiac arrest (9 min) followed by CPR. Cardiac and neurological function and survival were assessed up to 24h post-CPR.
Measurements and Main Results
Cardiac arrest and CPR markedly depressed myocardial function in NOS3−/− and sGCα1−/− but not in WT and NOS3−/−CSTg. Neurological function score as well as 24h survival rate was lower in NOS3−/− and sGCα1−/− compared to WT and NOS3−/−CSTg. Detrimental effects of deficiency of NOS3 or sGCα1 were associated with enhanced inflammation of heart and liver and increased cell death in heart, liver, and brain that were largely prevented by cardiomyocyte-restricted NOS3 overexpression.
These results demonstrate an important salutary impact of NOS3/sGC signaling on the outcome of cardiac arrest. Myocardial NOS3 prevented post-cardiac arrest myocardial dysfunction, attenuated end-organ damage, and improved neurological outcome and survival. Our observations suggest that enhancement of cardiac NOS3 and/or sGC activity may improve outcome after cardiac arrest and CPR.
PMCID: PMC2862545  PMID: 19050616
cardiac arrest; cardiopulmonary resuscitation; nitric oxide; neurological dysfunction; myocardial dysfunction; apoptosis
24.  Nitric oxide synthase isoforms play distinct roles during acute peritonitis 
Background. Acute peritonitis is the most frequent complication of peritoneal dialysis (PD). Increased nitric oxide (NO) release by NO synthase (NOS) isoforms has been implicated in acute peritonitis, but the role played by the NOS isoforms expressed in the peritoneum is unknown.
Methods. We investigated the structural and functional consequences of acute peritonitis induced by LPS in wild-type (WT) mice versus knockout mice (KO) for the endothelial NOS (eNOS), the inducible NOS (iNOS) or the neuronal NOS (nNOS).
Results. The level of NO metabolites (NOx) in the dialysate was maximal 18 h after LPS injection. LPS induced a significant increase in the transport of small solutes and decreased ultrafiltration in WT mice. These changes, which occurred without vascular proliferation, were paralleled by the upregulation of nNOS and eNOS, and the induction of iNOS. The transport modifications induced by LPS were significantly reversed in eNOS KO mice, but not modified in mice lacking iNOS or nNOS. In contrast, the increase of dialysate NOx was abolished in iNOS KO mice and significantly reduced in eNOS KO mice, but left unchanged in mice lacking nNOS. Mice lacking iNOS also showed more severe inflammatory changes, and a trend towards increased mortality following LPS.
Conclusion. These data demonstrate specific roles for NOS isoforms in the peritoneal membrane and suggest that selective eNOS inhibition may improve peritoneal transport during acute peritonitis.
PMCID: PMC2796899  PMID: 19706695
acute peritonitis; knockout mice; LPS; NO synthases; peritoneal dialysis

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