The synthetic peptide PnPP-19 potentiates erectile function via nNOS and iNOS.

PnPP-19 peptide has a primary sequence design based on molecular modeling studies of PnTx2-6 toxin. It comprises the amino acid residues that are potentially significant for the pharmacological action of PnTx2-6. Ex vivo and in vivo experiments in normotensive, hypertensive, or diabetic murine models have shown a significant improvement in penile erection after administration of PnPP-19. Given the potential use of PnPP-19 in pharmaceutical formulations to treat erectile dysfunction and the lack of information concerning its mode of action, the present work investigates its activities on the nitrergic system. PnPP-19 induced a significant increase in nitric oxide (NO) and cGMP levels in corpus cavernosum (cc). These effects were inhibited by l-NAME, a non-selective inhibitor of nitric oxide synthase (NOS); were partially inhibited by 7- Nitroindazole, a selective inhibitor of neuronal NOS (nNOS); and were abolished by L-NIL, a selective inhibitor of inducible NOS (iNOS). This potentiating effect was not affected by atropine. PnPP-19 also led to changes in mRNA levels, protein expression and phosphorylation at specific sites of NOS, in cc. Assays using cavernous tissue from knockout mice to endothelial NOS (eNOS), nNOS or iNOS showed that PnPP-19 potentiates relaxation only in eNOS-knockout mice, which suggests an essential role for nNOS. Surprisingly, iNOS enhanced the potentiation of erectile function evoked by PnPP-19. Our results demonstrate that this new synthetic peptide potentiates erectile function via nitric oxide activation and reinforce its role as a new pharmacological tool for the treatment of erectile dysfunction.

Perivascular Macrophages Regulate Blood Flow Following Tissue Damage.

[Figure: see text].

Nitric oxide displays a biphasic effect on calcium dynamics in microglia.

Calcium is a critical secondary messenger in microglia. In response to inflammation, microglia mobilize intracellular calcium and increase the expression of inducible nitric oxide synthase (iNOS), which produces nitric oxide (NO). This study set to explore whether NO regulates intracellular calcium dynamics through transient receptor potential (TRP) channels in primary wildtype (WT) and iNOS knockout (iNOS-/-) microglia, and the BV2 microglial cell line using calcium imaging and voltage-clamp recordings. Our results demonstrated that application of the NO-donor SNAP induced a biphasic calcium response in naïve murine microglia. Specifically, phase I was characterized by a rapid decline in calcium influx that was attenuated by pretreatment of the store operated calcium channel (SOCC) inhibitor 2APB, while phase II presented as a slow calcium influx that was abolished by pretreatment with the TRP vanilloid type 2 (TRPV2) channel inhibitor tranilast. Importantly, in the presence of a protein kinase G (PKG) inhibitor, the SNAP-mediated calcium decline in phase I persisted while the calcium influx in phase II was abolished. Application of thapsigargin to activate SOCCs caused a calcium influx through a nonselective cation conductance in BV2 microglia, which was abruptly attenuated by SNAP. Importantly, iNOS-/- microglia displayed a significantly larger calcium influx though SOCCs while expressing less stromal interaction molecule 1, Orai1, and TRP canonical type 1 and 3 mRNA, when compared to WT microglia. Together, these results demonstrate that NO signaling restricts calcium influx through SOCCs independent of PKG signaling and increases calcium influx through TRPV2 channels in a PKG-dependent mechanism in microglia.

Fatal Cytomegalovirus Infection in an Adult with Inherited NOS2 Deficiency.

BACKGROUND: Cytomegalovirus (CMV) can cause severe disease in children and adults with a variety of inherited or acquired T-cell immunodeficiencies, who are prone to multiple infections. It can also rarely cause disease in otherwise healthy persons. The pathogenesis of idiopathic CMV disease is unknown. Inbred mice that lack the gene encoding nitric oxide synthase 2 (Nos2) are susceptible to the related murine CMV infection. METHODS: We studied a previously healthy 51-year-old man from Iran who after acute CMV infection had an onset of progressive CMV disease that led to his death 29 months later. We hypothesized that the patient may have had a novel type of inborn error of immunity. Thus, we performed whole-exome sequencing and tested candidate mutant alleles experimentally. RESULTS: We found a homozygous frameshift mutation in NOS2 encoding a truncated NOS2 protein that did not produce nitric oxide, which determined that the patient had autosomal recessive NOS2 deficiency. Moreover, all NOS2 variants that we found in homozygosity in public databases encoded functional proteins, as did all other variants with an allele frequency greater than 0.001. CONCLUSIONS: These findings suggest that inherited NOS2 deficiency was clinically silent in this patient until lethal infection with CMV. Moreover, NOS2 appeared to be redundant for control of other pathogens in this patient. (Funded by the National Center for Advancing Translational Sciences and others.).

Ablation of IL-17A leads to severe colitis in IL-10-deficient mice: implications of myeloid-derived suppressor cells and NO production.

IL-10 is an immune regulatory cytokine and its genetic defect leads to gastrointestinal inflammation in humans and mice. Moreover, the IL-23/Th17 axis is known to be involved in these inflammatory disorders. IL-17A, a representative cytokine produced by Th17 cells, has an important role for the pathological process of inflammatory diseases. However, the precise function of IL-17A in inflammatory bowel disease (IBD) remains controversial. In this study, we evaluated the effect of IL-17A on colitis in IL-10-deficient (Il10-/-) mice. Mice lacking both IL-10 and IL-17A (Il10-/-Il17a-/-) suffered from fatal wasting and manifested more severe colitis compared with Il10-/-Il17a+/- mice. Moreover, we found that CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs) accumulated in the bone marrow, spleen and peripheral blood of Il10-/-Il17a-/- mice. These MDSCs highly expressed inducible nitric oxide synthase (iNOS) (Nos2) and suppressed the T-cell response in vitro in a NOS-dependent manner. In correlation with these effects, the concentration of nitric oxide was elevated in the serum of Il10-/-Il17a-/- mice. Surprisingly, the severe colitis observed in Il10-/-Il17a-/- mice was ameliorated in Il10-/-Il17a-/-Nos2-/- mice. Our findings suggest that IL-17A plays suppressive roles against spontaneous colitis in Il10-/- mice in an iNOS-dependent manner and inhibits MDSC differentiation and/or proliferation.

Nitric oxide signaling inhibits microglia proliferation by activation of protein kinase-G.

Microglia population is primarily determined by a finely-regulated proliferation process during early development of the central nervous system (CNS). Nitric oxide (NO) is known to inhibit proliferation in numerous cell types. However, how NO signaling regulates microglia proliferation remains elusive. Using wildtype (WT) and inducible nitric oxide synthase knockout (iNOS-/-) mice, this study investigated the role and underlying mechanisms of iNOS/NO signaling in microglia proliferation. Here we reported that iNOS-/- mice displayed significantly more BrdU-labeled proliferating microglia in the cortex than that in WT mice at postnatal day 10. Compared to microglia isolated from WT mouse cortex, significantly more iNOS-/- microglia displayed the specific cell-cycle markers Ki67 and phospho-histone H3 (pH3) in their nuclei. In addition, treating WT microglia with the NOS inhibitor LNAME drastically increased the percentage of cells expressing Ki67 and pH3, whereas treating iNOS-/- microglia with NOC18, a slow-release NO-donor, significantly decreased the percentage of microglia expressing the two cell-cycle markers. Moreover, inhibition of protein kinase-G (PKG) in WT microglia increased the proportion of microglia expressing Ki67 and pH3, whereas activation of PKG signaling using 8Br-cGMP in iNOS-/- microglia significantly decreased the fraction of microglia displaying Ki67 and pH3. Interestingly, in the presence of a PKG inhibitor, NOC18 increased the quantity of iNOS-/- microglia expressing Ki67 and pH3. Together, these results indicate that basal activity of iNOS/NO signaling impedes microglial cell-cycle progression and attenuates proliferation through activation of the cGMP-PKG pathway. However, NO increases microglia cell-cycle progression in the absence of cGMP-PKG signaling.

Inducible Nitric Oxide Synthase is required for parasite restriction and inflammatory modulation during Neospora caninum infection.

Neospora caninum infection is an important cause of neuromuscular disease in dogs and abortion in cattle, leading to significant economic losses in beef and dairy industries. The protective immunity against apicomplexan parasites, specifically Toxoplasma gondii and N. caninum, is typically achieved by inducing an IL-12-driven Th1 immune response. IL-12 stimulates IFN-γ production, which activates Inducible Nitric Oxide Synthase (iNOS) and promotes consequent Nitric Oxide (NO) synthesis, classically described as one of the main effector mechanisms for parasite elimination. Here, we aimed to evaluate the role played by iNOS during N. caninum infection. Our results show that N. caninum infection in C57BL/6 wild type (WT) mice induce NO production in vivo and in vitro. In agreement, iNOS deficient mice, as well as WT mice treated with iNOS inhibitor aminoguanidine, succumbed during acute infection with a dose lethal to 50 % of the WT mice, and presented significant increase in parasite load when submitted to sub-lethal infection protocols. Interestingly, the lack of control of parasite proliferation observed in iNOS-/- mice was associated with notable CNS inflammation and increased production of the main systemic proinflammatory cytokines (IL-12, IFN-γ, IL-6, TNF and IL-17A). Taken together, our findings show that iNOS plays an important role in restricting N. caninum replication, while also modulates the inflammatory process induced by the infection.

Nitric oxide upregulates microglia phagocytosis and increases transient receptor potential vanilloid type 2 channel expression on the plasma membrane.

Microglia phagocytosis is critical for central nervous system development, and dysregulation of phagocytosis may contribute to a variety of neurological disorders. During initial stages of phagocytosis, microglia display increased nitric oxide (NO) production via inducible nitric oxide synthase (iNOS) activity and amplified calcium entry through transient receptor potential vanilloid type 2 (TRPV2) channels. The present study investigated the regulatory role of iNOS/NO signaling in microglial phagocytosis and TRPV2 channel activation using phagocytosis assay, calcium imaging, patch clamp electrophysiology, immunocytochemistry, and immunoblot assays. Results showed that primary microglia from iNOS-knockout (iNOS-/- ) mice exhibited substantial deficits in phagocytic capacity and TRPV2 channel activity relative to wild-type (WT) controls. Specifically, iNOS-/- microglia displayed a lower level of TRPV2 protein localized on the plasma membrane (PM) without any significant change in the mRNA levels of Fc-gamma receptors and TRPV2. In addition, iNOS-/- microglia, unlike their WT controls, failed to elicit a calcium influx in response to application of the TRPV2-agonist 2-aminoethoxydiphenyl borate (2APB). Importantly, the phagocytic capacity and the PM expression and activity of TRPV2 in iNOS-/- microglia were largely corrected by pretreatment with NO-donors. Accordingly, the 2APB-evoked calcium influx and the PM expression of TRPV2 in WT microglia were significantly decreased by selective inhibition of iNOS, protein kinase-G (PKG), or phosphoinositide-3-kinase (PI3K), respectively. Together, results from this study indicated that iNOS/NO signaling upregulates microglial phagocytosis and increases TRPV2 trafficking to the PM via PKG/PI3K dependent pathway(s).

Vasoreactivity of isolated aortic rings from dyslipidemic and insulin resistant inducible nitric oxide synthase knockout mice.

Recent study from this lab indicated enhanced susceptibility of iNOS KO mice for diet induced obesity (DIO) and systemic insulin resistance (IR) as compared to C57BL/6 (WT) mice. The present study investigates aortic vasoreactivity in high fat diet (HFD) induced insulin resistant iNOS KO mice. WT and iNOS KO mice were fed with 45% HFD/10% LFD for ten weeks. Systemic IR was assessed via measurement of circulating lipids, glucose, and insulin; while phenylephrine (PE)/acetylcholine (ACh) induced responses were monitored in the isolated aortic rings. To understand the mechanism, qPCR or Western blotting experiments were performed in aorta and Ea.hy926 cells. After 10 weeks of HFD feeding, significant increase in the body weight/fat mass, augmented circulating lipids, glucose, insulin and inflammatory cytokines along with impaired acetylcholine induced aortic vasorelaxation and enhanced iNOS expression was observed in the aortic tissue of WT mice. In the aminoguanidine (AG, 20 mg/kg for 4 weeks) treated WT mice and also in iNOS KO mice, acetylcholine induced vasorelaxation was significantly preserved. Further, acetylcholine mediated vasorelaxation correlated with increased eNOS phosphorylation at Ser1177 residue in the iNOS KO mice and same was also observed in the iNOS silenced Ea.hy926 cells. Moreover, treatment of Ea.hy926 cells with palmitic acid or TNFα also caused a significant decrease in eNOS activity, which was reversed in iNOS silenced Ea.hy926 cells suggesting the role of iNOS in the reduction of eNOS activity. The study thus implies a critical role of iNOS in vascular diseases associated with dyslipidemia/IR.

Nitrosative stress drives heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is a common syndrome with high morbidity and mortality for which there are no evidence-based therapies. Here we report that concomitant metabolic and hypertensive stress in mice-elicited by a combination of high-fat diet and inhibition of constitutive nitric oxide synthase using Nω-nitro-L-arginine methyl ester (L-NAME)-recapitulates the numerous systemic and cardiovascular features of HFpEF in humans. Expression of one of the unfolded protein response effectors, the spliced form of X-box-binding protein 1 (XBP1s), was reduced in the myocardium of our rodent model and in humans with HFpEF. Mechanistically, the decrease in XBP1s resulted from increased activity of inducible nitric oxide synthase (iNOS) and S-nitrosylation of the endonuclease inositol-requiring protein 1α (IRE1α), culminating in defective XBP1 splicing. Pharmacological or genetic suppression of iNOS, or cardiomyocyte-restricted overexpression of XBP1s, each ameliorated the HFpEF phenotype. We report that iNOS-driven dysregulation of the IRE1α-XBP1 pathway is a crucial mechanism of cardiomyocyte dysfunction in HFpEF.

The iNOS Activity During an Immune Response Controls the CNS Pathology in Experimental Autoimmune Encephalomyelitis.

Inducible nitric oxide synthase (iNOS) plays a critical role in the regulation of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). Previous studies have shown that iNOS plays pathogenic as well as regulatory roles in MS and EAE. However, how does iNOS alters the pathophysiology of the central nervous system (CNS) in neuronal autoimmunity is not clearly understood. In the present work, we show that treatment of mice with L-NAME, an iNOS inhibitor, during the antigen-priming phase primarily alters brain pathology, while in the subsequent effector phase of the immune response, the spinal cord is involved. Inhibition of iNOS during the priming phase of the immune response promotes the infiltration of pathogenic CD11b+F4/80-Gr-1+ cells, but there is low recruitment of regulatory CD11b+F4/80+ cells in the brain. Inhibition of iNOS during the effector phase shows similar pathogenic alterations in the spinal cord, instead of in the brain. Treatment of wild-type mice with L-NAME or mice having genetic deficiency of iNOS show lower MHC-II expression on the dendritic cells, but not on macrophages. Our data suggest that iNOS has a critical regulatory role during antigen-priming as well as in the effector phase of EAE, and inhibition iNOS at different stages of the immune response can differentially alter either the brain or spinal cord pathology. Understanding the cellular and molecular mechanisms through which iNOS functions could help to design a better strategies for the clinical management of neuroinflammation and neuronal autoimmunity.

Immunometabolic phenotype of BV-2 microglia cells upon murine cytomegalovirus infection.

Microglia are resident brain macrophages with key roles in development and brain homeostasis. Cytomegalovirus (CMV) readily infects microglia cells, even as a possible primary target of infection in development. Effects of CMV infection on a cellular level in microglia are still unclear; therefore, the aim of this research was to assess the immunometabolic changes of BV-2 microglia cells following the murine cytomegalovirus (MCMV) infection. In light of that aim, we established an in vitro model of ramified BV-2 microglia (BV-2∅FCS, inducible nitric oxide synthase (iNOSlow), arginase-1 (Arg-1high), mannose receptor CD206high, and hypoxia-inducible factor 1α (HIF-1αlow)) to better replicate the in vivo conditions by removing FCS from the cultivation media, while the cells cultivated in 10% FCS DMEM displayed an ameboid morphology (BV-2FCS high, iNOShigh, Arg-1low, CD206low, and HIF-1αhigh). Experiments were performed using both ramified and ameboid microglia, and both of them were permissive to productive viral infection. Our results indicate that MCMV significantly alters the immunometabolic phenotypic properties of BV-2 microglia cells through the manipulation of iNOS and Arg-1 expression patterns, along with an induction of a glycolytic shift in the infected cell cultures.

Mice lacking Casp1, Ifngr and Nos2 genes exhibit altered depressive- and anxiety-like behaviour, and gut microbiome composition.

Converging evidence supports the involvement of pro-inflammatory pathways and the gut microbiome in major depressive disorder (MDD). Pre-clinical and clinical studies suggest that decreasing pro-inflammatory signaling may provide clinical benefit in MDD. In this study, we used the chronic unpredictable stress (CUS) paradigm to assess whether mice lacking the pro-inflammatory caspase 1, interferon gamma-receptor, and nitric oxide synthase (Casp1, Ifngr, Nos2)-/- present altered depressive- and anxiety-like behaviour at baseline and in response to CUS. In comparison to wild-type (wt) mice, (Casp1, Ifngr, Nos2)-/- mice displayed decreased depressive- and anxiety-like behaviour, and increased hedonic-like behaviour and locomotor activity at baseline, and resistance to developing anhedonic-like behaviour and a heightened emotional state following stress. Plasma levels of ACTH and CORT did not differ between the triple knockout and wt mice following stress. The faecal microbiome of (Casp1, Ifngr, Nos2)-/- mice differed from that of wt mice at baseline and displayed reduced changes in response to chronic stress. Our results demonstrate that simultaneous deficit in multiple pro-inflammatory pathways has antidepressant-like effects at baseline, and confers resilience to stress-induced anhedonic-like behaviour. Moreover, accompanying changes in the gut microbiome composition suggest that CASP1, IFNGR and NOS2 play a role in maintaining microbiome homeostasis.

Breathing hydrogen sulfide prevents delayed paraplegia in mice.

Delayed paraplegia complicates the recovery from spinal cord ischemia or traumatic spinal cord injury. While delayed motor neuron apoptosis is implicated in the pathogenesis, no effective treatment or preventive measures is available for delayed paraplegia. Hydrogen sulfide exerts anti-apoptotic effects. Here, we examined effects of hydrogen sulfide breathing on the recovery from transient spinal cord ischemia. Breathing hydrogen sulfide starting 23 h after reperfusion for 5 h prevented delayed paraplegia after 5 min of spinal cord ischemia. Beneficial effects of hydrogen sulfide were mediated by upregulation of anti-apoptotic Bcl-XL and abolished by nitric oxide synthase 2 deficiency. S-nitrosylation of NFkB p65 subunit, which is induced by nitric oxide derived from nitric oxide synthase 2, facilitated subsequent sulfide-induced persulfidation of p65 and transcription of anti-apoptotic genes. These results uncover the molecular mechanism of the anti-apoptotic effects of sulfide based on the interaction between nitric oxide and sulfide. Exploitation of the anti-apoptotic effects of delayed hydrogen sulfide breathing may provide a new therapeutic approach for delayed paraplegia.

Primary Role of Suppressor of Cytokine Signaling 1 in Mycobacterium bovis BCG Infection.

Suppressor of cytokine signaling 1 (SOCS1) is a negative regulator of JAK/STAT signaling and is induced by mycobacterial infection. To understand the major function of SOCS1 during infection, we established a novel system in which recombinant Mycobacterium bovis bacillus Calmette-Guérin expressed dominant-negative SOCS1 (rBCG-SOCS1DN) because it would not affect the function of SOCS1 in uninfected cells. When C57BL/6 mice and RAG1-/- mice were intratracheally inoculated with rBCG-SOCS1DN, the amount of rBCG-SOCS1DN in the lungs was significantly reduced compared to that in the lungs of mice inoculated with a vector control counterpart and wild-type BCG. However, these significant differences were not observed in NOS2-/- mice and RAG1-/- NOS2-/- double-knockout mice. These findings demonstrated that SOCS1 inhibits nitric oxide (NO) production to establish mycobacterial infection and that rBCG-SOCS1DN has the potential to be a powerful tool for studying the primary function of SOCS1 in mycobacterial infection.

Nrf2 Deficiency Unmasks the Significance of Nitric Oxide Synthase Activity for Cardioprotection.

The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key master switch that controls the expression of antioxidant and cytoprotective enzymes, including enzymes catalyzing glutathione de novo synthesis. In this study, we aimed to analyze whether Nrf2 deficiency influences antioxidative capacity, redox state, NO metabolites, and outcome of myocardial ischemia reperfusion (I/R) injury. In Nrf2 knockout (Nrf2 KO) mice, we found elevated eNOS expression and preserved NO metabolite concentrations in the aorta and heart as compared to wild types (WT). Unexpectedly, Nrf2 KO mice have a smaller infarct size following myocardial ischemia/reperfusion injury than WT mice and show fully preserved left ventricular systolic function. Inhibition of NO synthesis at onset of ischemia and during early reperfusion increased myocardial damage and systolic dysfunction in Nrf2 KO mice, but not in WT mice. Consistent with this, infarct size and diastolic function were unaffected in eNOS knockout (eNOS KO) mice after ischemia/reperfusion. Taken together, these data suggest that eNOS upregulation under conditions of decreased antioxidant capacity might play an important role in cardioprotection against I/R. Due to the redundancy in cytoprotective mechanisms, this fundamental antioxidant property of eNOS is not evident upon acute NOS inhibition in WT mice or in eNOS KO mice until Nrf2-related signaling is abrogated.

Chronic hyperinsulinemia promotes meta-inflammation and extracellular matrix deposition in adipose tissue: Implications of nitric oxide.

Various imperative studies support the notion that hyperinsulinemia (HI) itself serves as the common link between adipose tissue inflammation (ATI) and metabolic syndrome. However, the contribution of HI mediated ATI and its metabolic consequences are yet to be explored. We induced chronic HI per se in mice by administration of exogenous insulin for 8 weeks through mini-osmotic pumps. For the reduction of circulating insulin in response to excess calorie intake, we have partially ablated ß-cells by using streptozotocin (STZ) in the diet-induced obesity (DIO) and genetic mice models (db/db). Flow cytometry analysis was performed for the quantification of immune cells in stromal vascular fraction (SVF) isolated from epididymal white adipose tissue (eWAT). Our studies demonstrated that chronic HI augmented ATI in terms of elevated pro-inflammatory cells (M1 macrophages and NK-cells) and suppressed anti-inflammatory cells (M2 macrophages, eosinophils and regulatory T-cells). These results were correlated with altered obesity-associated metabolic phenotype. Partial reduction of circulating insulin level attenuated excess calorie-induced ATI and improved insulin sensitivity. Mechanistically, an imbalance in M1 and M2 macrophage proportions in eWAT promoted iNOS (inducible nitric oxide synthase): arginase-1 imbalance that resulted into extracellular matrix (ECM) deposition and insulin resistance (IR) development. However, iNOS-/- mice were protected from HI-induced M1:M2 macrophage imbalance, ECM deposition and IR in adipose tissue. Overall, we conclude that chronic HI per se contributed in ATI and iNOS corroborated ECM deposition.

Differential behavioral and glial responses induced by dopaminergic mechanisms in the iNOS knockout mice.

The interaction between distinctive nitric oxide synthase (NOS) isoforms and the dopamine system provides new avenues to the development of pharmacological tools for the pathophysiological conditions of the dopaminergic system. Our aim was to investigate the influences of dopamine-induced effects in inducible NOS knockout (iNOS KO) mice. In order to characterize iNOS KO mice phenotype, the animals were submitted to the basal analyses of motor, sensorimotor and sensorial abilities. Pharmacological challenging of the dopaminergic system included the investigation of amphetamine-induced prepulse inhibition (PPI) disruption, haloperidol-induced catalepsy, reserpine-induced oral involuntary movements and hyperlocomotion induced by amphetamine in reserpine treated mice. The iNOS KO mice showed significant reduction of spontaneous motor activity, but there was no significant difference in sensorimotor or sensorial responses of iNOS KO mice compared to wild type (WT). Regarding the dopaminergic system, iNOS KO mice showed a significant increase of haloperidol-induced catalepsy. This effect was confirmed through an iNOS pharmacological inhibitor (1400 W) in WT mice. In addition, iNOS KO reserpine treated mice showed reduced oral involuntary movements and amphetamine-induced hyperlocomotion. Knowing that iNOS is mainly expressed in glial cells we analyzed the immunoreactivity (ir) for GFAP (astrocyte marker) and IBA-1 (microglial marker) in the striatum, an area enrolled in motor planning among other functions. iNOS KO presented reduced GFAP-ir and IBA-1-ir compared with WT. Reserpine treatment increased GFAP-ir in both WT and iNOS KO. However, these effects were slighter in iNOS KO. Activated state of microglia was increased by reserpine only in WT mice. Our results further demonstrated that the absence of iNOS interfered with dopamine-mediated behavioral and molecular responses. These results increase the understanding of the dopamine and NO system interaction, which is useful for the management of the dopamine-related pathologies.

A heterogeneous Ly-6B2+ leukocyte population consists of yet undescribed iNOS-expressing cell types in murine skin wounds.

The gaseous mediator nitric oxide (NO) is a central regulatory molecule during the inflammatory phase of cutaneous tissue repair. The inducible NO-synthase (iNOS) represents the main isoform of the three NO producing enzymes at the wound site. In particular, keratinocytes and macrophages are described as main sources of iNOS-derived NO in skin wounds. Here we provide experimental evidence that Ly-6B2+ leukocytes are an additional cellular source of iNOS-derived NO in wounds. As wound iNOS protein expression temporally coincides with both macrophage and neutrophil infiltration, we used immunohistochemistry (IHC) and fluorescence-activated cell sorting (FACS) to address iNOS expression in both macrophages and neutrophil subsets. IHC analyses excluded F4/80+ macrophages as iNOS producers, but indicated Ly-6G/C (Gr-1)+ neutrophils to express iNOS in wound granulation tissue. A subsequent FACS-based analysis from cellular wound tissue preparations revealed an iNOS-expressing fraction of Ly-6B2-determined leukocytes that consisted of Ly-6G+ and Ly-6G- cells, meaning that mainly mature neutrophils (Ly-6B2+/Ly-6G+) as well as inflammatory monocytes (Ly-6B2+/Ly-6G-) are dominant iNOS-expressing cell types in the developing granulation tissue of acute wounds.