Neuronal Nitric Oxide Synthase Knockout Research Articles

Endothelium-Derived Dopamine and 6-Nitrodopamine in the Cardiovascular System.

The review deals with the release of endothelium-derived dopamine and 6-nitrodopamine (6-ND) and its effects on isolated vascular tissues and isolated hearts. Basal release of both dopamine and 6-ND is present in human isolated umbilical cord vessels, human popliteal vessels, nonhuman primate vessels, and reptilia aortas. The 6-ND basal release was significantly reduced when the tissues were treated with Nω-nitro-l-arginine methyl ester and virtually abolished when the endothelium was mechanically removed. 6-Nitrodopamine is a potent vasodilator, and the mechanism of action responsible for this effect is the antagonism of dopamine D2-like receptors. As a vasodilator, 6-ND constitutes a novel mechanism by which nitric oxide modulates vascular tone. The basal release of 6-ND was substantially decreased in endothelial nitric oxide synthase knockout (eNOS-/-) mice and not altered in neuronal nitric oxide synthase knockout (nNOS-/-) mice, indicating a nonneurogenic source for 6-ND in the heart. Indeed, in rat isolated right atrium, the release of 6-ND was not affected when the atria were treated with tetrodotoxin. In the rat isolated right atrium, 6-ND is the most potent endogenous positive chronotropic agent, and in Langendorff's heart preparation, it is the most potent endogenous positive inotropic agent. The positive chronotropic and inotropic effects of 6-ND are antagonized by β1-adrenoceptor antagonists at concentrations that do not affect the effects induced by noradrenaline, adrenaline, and dopamine, indicating that blockade of the 6-ND receptor is the major modulator of heart chronotropism and inotropism. The review proposes that endothelium-derived catecholamines may constitute a major mechanism for control of vascular tone and heart functions, in contrast to the overrated role attributed to the autonomic nervous system.

The expression and function of glutamate aspartate transporters in Bergmann glia are decreased in neuronal nitric oxide synthase‐knockout mice during postnatal development

Bergmann glia (BG) predominantly use glutamate/aspartate transporters (GLAST) for glutamate uptake in the cerebellum. Recently, nitric oxide (NO) treatment has been shown to upregulate GLAST function and increase glutamate uptake in vitro. We previously discovered that neuronal nitric oxide synthase knockout (nNOS−/−) mice displayed structural and functional neuronal abnormalities in the cerebellum during development, in addition to previously reported motor deficits. Although these developmental deficits have been identified in the nNOS−/− cerebellum, it is unknown whether BG morphology and GLAST expression are also affected in the absence of nNOS in vivo. This study is the first to characterize BG morphology and GLAST expression during development in nNOS−/− mice using immunohistochemistry and western blotting across postnatal development. Results showed that BG in nNOS−/− mice exhibited abnormal morphology and decreased GLAST expression compared with wildtype (WT) mice across postnatal development. Treating ex vivo WT cerebellar slices with the NOS inhibitor L‐NAME decreased GLAST expression while treating nNOS−/− slices with the slow‐release NO‐donor NOC‐18 increased GLAST expression when compared with their respective controls. In addition, treating primary BG isolated from WT mice with the selective nNOS inhibitor 7N decreased the membrane expression of GLAST and influx of Ca2+/Na+, while treating nNOS−/− BG with SNAP increased the membrane expression of GLAST and Ca2+/Na+ influx. Moreover, the effects of SNAP on GLAST expression and Ca2+/Na+ influx in nNOS−/− BG were significantly reduced by a PKG inhibitor. Together, these results reveal a novel role for nNOS/NO signaling in BG development, regulated by a PKG‐mediated mechanism.

Diverse effects of NOS inhibition on mitochondrial bioenergetics in rat primary cortical neuronal cells under normoxia and hypoxia

ObjectiveIschemic brain injury has been shown to be increased in endothelial nitric oxide synthase (eNOS) knockout mice but reduced in neuronal nitric oxide synthase (nNOS) knockout mice compared to the wild‐type mice. It has been proposed that NO derived from nNOS in neurons is detrimental whereas NO derived from eNOS in endothelial cells is protective after stroke. However, the functional mechanism underlying this protection or damage has never been studied. Mitochondria play an important role in a wide range of physiological and pathophysiological processes. Besides the generation of cellular energy, mitochondria are also involved in reactive oxygen species (ROS) production and the activation of cell death pathways. Therefore, the effect of NOS inhibitors on mitochondrial respiration is important to investigate as mitochondria determine the fate of cell survival after stroke or ischemic injury. Our objective is to determine the effects of NOS inhibition on mitochondrial bioenergetics in primary rat cortical neurons under normoxic and oxygen‐glucose deprivation‐reoxygenation (OGD‐R) conditions.MethodsCortical neurons were isolated from brain and exposed to normoxic and OGD‐R conditions with and without non‐specific NOS inhibitor N‐ω‐propyl‐L‐arginine (L‐NAME). Oxygen consumption rates (OCR) and respiratory parameters were measured using Seahorse XFe24 cell mito‐stress test (Agilent technologies). Cell viability was measured using CCK‐8 kit to determine the effect of NOS inhibition under hypoxic and normoxic conditions. Electron spin resonance (ESR) spectroscopy (Bruker Bio‐Spin spectrometer, Germany) was used to measure ROS using spin probe CMH.ResultsIn neurons, OCR showed a decrease in basal respiration, maximal respiration, spare respiratory capacity, proton leak and non‐mitochondrial respiration under hypoxic condition compared to normoxic control. Interestingly, NOS inhibition under normoxic conditions increased basal respiration, maximal respiration, spare respiratory capacity, and non‐mitochondrial respiration but not proton leak whereas, NOS inhibition decreased basal respiration, maximal respiration, spare respiratory capacity, proton leak and non‐mitochondrial respiration under hypoxic condition. Thus, NOS inhibition has opposite effects on mitochondrial respiratory parameters under normoxic and hypoxic conditions. Viability studies showed NOS inhibition has improved cell viability under hypoxic conditions. In contrast, NOS inhibition under normoxic conditions showed a decrease in cell viability. ROS measurements by ESR have shown that L‐NAME treatment increased ROS levels under normoxic conditions, however, NOS inhibition decreased ROS levels under hypoxic conditions.ConclusionsNOS inhibition has opposite effects on mitochondrial bioenergetics, viability and superoxide levels under hypoxia compared to normoxia. nNOS appears to inhibit ROS generation under hypoxia but promotes the ROS generation following hypoxia possibly due to uncoupling of nNOS. Thus, the change in the generation of free radicals may underlie the diverse effects of nNOS in neurons following exposure to normoxia and hypoxiaSupport or Funding InformationAmerican Heart Association (PVG: 14SDG20490359 and VNS: 16PRE31450006), and National Institute of Health: (PVK: R01NS094834).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect.

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., β-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

Lack of neuronal nitric oxide synthase results in attention deficit hyperactivity disorder-like behaviors in mice.

Nitric oxide (NO) is an important molecule for the proper development and function of the central nervous system. In this study, we investigated the behavioral alterations in the neuronal NO synthase knockout mice (NOS1 KO) with a deficient NO production mechanism in the brain, characterizing it as a potential rodent model for attention deficit hyperactivity disorder (ADHD). NOS1 KO exhibited higher locomotor activity than their wildtype counterparts in a novel environment, as measured by open field (OF) test. In a 2-way active avoidance paradigm (TWAA), we found sex-dependent effects, where male KO displayed deficits in avoidance and escape behavior, sustained higher incidences of shuttle crossings, and higher incidences of intertrial interval crossings, suggesting learning, and/or performance impairments. On the other hand, female KO demonstrated few deficits in TWAA. Molsidomine (MSD), a NO donor, rescued TWAA deficits in male KO when acutely administered before training. In a passive avoidance paradigm, KO of both sexes displayed significantly shorter step-through latencies after training. Further, abnormal spontaneous motor activity rhythms were found in the KO during the dark phase of the day, indicating dysregulation of rhythmic activities. These data indicate that NOS1 KO mimics certain ADHD-like behaviors and could potentially serve as a novel rodent model for ADHD.

The role of gaseous neurotransmitters in the antinociceptive effects of morphine during acute thermal pain

Treatment with a carbon monoxide-releasing molecule (tricarbonyldichlororuthenium(II) dimer, CORM-2) or a classical inducible heme oxygenase (HO-1) inducer (cobalt protoporphyrin IX, CoPP) enhanced the antinociceptive effects of morphine during chronic pain but the role played by these compounds in acute thermal nociception was not evaluated. The effects of CORM-2 and CoPP treatments on the local antinociceptive actions of morphine and their interactions with nitric oxide during acute pain were evaluated by using wild type (WT), neuronal (nNOS-KO) or inducible (iNOS-KO) nitric oxide synthase knockout mice and assessing their thermal nociception to a hot stimulus with the hot plate test. Our results showed that the absence of nNOS or iNOS genes did not alter licking and jumping responses nor the antinociceptive effects produced by morphine indicating that the local thermal inhibitory effects produced by this drug in the absence of inflammation or injury are not mediated by the nitric oxide pathway triggered by nNOS or iNOS enzymes. Moreover, while the systemic administration of CORM-2 or CoPP inhibited licking and jumping latencies in all genotypes, these treatments only enhanced the local inhibition of jumping latencies produced by morphine in WT and nNOS-KO mice which effects were reversed by the peripheral administration of an HO-1 inhibitor. These data indicate that the co-administration of morphine with CORM-2 or CoPP produced remarkable local antinociceptive effects in WT and nNOS-KO mice and reveal that a significant interaction between carbon monoxide and nitric oxide systems occurs on the local antinociceptive effects produced by morphine during acute thermal nociception.

Oxidative stress reduces the expression of neuronal NOS in cardiac tissue and cell (545.3)

Obesity is a major global health concern and greatly increases the risk of developing insulin resistance and type 2 diabetes. Diabetic patients are also predisposed to various cardiovascular diseases; however, the underlying mechanism of the effects of diabetes on cardiomyocyte viability and thus, overall heart function remains to be established. Increased fatty acid oxidation during diabetes results in increased oxidative stress, which likely contributes to the adverse changes in the myocardium. Nitric oxide (NO) is involved in the regulation of cardiovascular homeostasis in experimental animal models and humans. Neuronal nitric oxide synthase (nNOS) knockout in mice resulted in increased oxidative stress in heart. In cardiac myocytes. nNOS was shown to coimmunoprecipitate with xanthine oxidoreductase, a critical enzyme for superoxide production, and to inhibit its production by the formation of peroxynitrite, all of which suggest a critical role of nNOS‐derived NO in regulating oxidative stress in heart. To investigate the effect of oxidative stress or fatty acids on nNOS expression in cardiomyocytes, we treated HL‐1 cardiomyocyte cells with hydrogen peroxide(H2O2) or palmitate. We show here for the first time that H2O2 or palmitate decreases the expression of nNOS in the cardiomyocytes, as assessed by immunoblot analysis. Furthermore, nNOS expression is also reduced in the heart lysates from thioredoxin 2 (TRX 2) knockout mice, which are prone to oxidative stress. The effect of such a decrease in nNOS expression by oxidative stress or saturated fatty acid treatment on cardiomyocyte viability, overall cardiac hypertrophy and cardiac function is under investigation.Grant Funding Source: Supported by NIH #HL07446 and #GM052419

Nitric-Oxide Synthase Knockout Modulates Ca2+-Sensing Receptor Expression and Signaling in Mouse Mesenteric Arteries

Extracellular calcium (Ca²⁺(e))-induced relaxation of isolated, phenylephrine (PE)-contracted mesenteric arteries is dependent on an intact perivascular sensory nerve network that expresses the Ca²⁺-sensing receptor (CaSR). Activation of the receptor stimulates an endocannabinoid vasodilator pathway, which is dependent on cytochrome P450 and phospholipase A₂ but largely independent of the endothelium. In the present study, we determined the role of nitric oxide (NO) in perivascular nerve CaSR-mediated relaxation of PE-contracted mesenteric resistance arteries isolated from mice. Using automated wire myography, we studied the effects of NO synthase (NOS) gene knockout (NOS(-/-)) and pharmacologic inhibition of NOS on Ca²⁺(e)-induced relaxation of PE-contracted arteries. Endothelial NOS knockout (eNOS(-/-)) upregulates but neuronal NOS knockout (nNOS(-/-)) downregulates CaSR expression. NOS(-/-) reduced maximum Ca²⁺(e)-induced relaxation with no change in EC₅₀ values, with eNOS(-/-) having the largest effect. The responses of vessels to calindol and Calhex 231 indicate that the CaSR mediates relaxation. L-N⁵-(1-iminoethyl)-ornithine reduced Ca²⁺(e)-induced relaxation of PE-contracted arteries from C57BL/6 control mice by ≈38% but had a smaller effect in vessels from eNOS(-/-) mice. 7-Nitroindazole had no significant effect on relaxation of arteries from NOS(-/-) mice, but both N(G)-nitro-L-arginine methylester and N(G)-monomethyl-L-arginine significantly reduced the relaxation maxima in all groups. Interestingly, the nNOS-selective inhibitor S-methyl-L-thiocitrulline significantly increased the EC₅₀ value by ≈60% in tissues from C57BL/6 mice but reduced the maximum response by ≈80% in those from nNOS(-/-) mice. Ca²⁺-activated big potassium channels play a major role in the process, as demonstrated by the effect of iberiotoxin. We conclude that CaSR signaling in mesenteric arteries stimulates eNOS and NO production that regulates Ca²⁺(e)-induced relaxation.

Effects of increased systolic Ca2+ and β-adrenergic stimulation on Ca2+ transient decline in NOS1 knockout cardiac myocytes

We have previously shown that the main factor responsible for the faster [Ca2+]i decline rate with β-adrenergic (β-AR) stimulation is the phosphorylation of phospholamban (PLB) rather than the increase in systolic Ca2+ levels. The purpose of this study was to correlate the extent of augmentation of PLB Serine16 phosphorylation to the rate of [Ca2+]i decline. Thus, ventricular myocytes were isolated from neuronal nitric oxide synthase knockout (NOS1−/−) mice, which we observed had lower basal PLB Serine16 phosphorylation levels, but equal levels during β-AR stimulation. Ca2+ transients (Fluo-4) were measured in myocytes superfused with 3mM extracellular Ca2+ ([Ca2+]o) and a non-specific β-AR agonist isoproterenol (ISO, 1μM) with 1mM [Ca2+]o. This allowed us to get matched Ca2+ transient amplitudes in the same myocyte. Similar to our previous work, Ca2+ transient decline was significantly faster with ISO compared to 3mM [Ca2+]o, even with matched Ca2+ transient amplitudes. Interestingly, when we compared the effects of ISO on Ca2+ transient decline between NOS1−/− and WT myocytes, ISO had a larger effect in NOS1−/− myocytes, which resulted in a greater percent decrease in the Ca2+ transient RT50. We believe this is due to a greater augmentation of PLB Serine16 phosphorylation in these myocytes. Thus, our results suggest that not only the amount but the extent of augmentation of PLB Serine16 phosphorylation are the major determinants for the Ca2+ decline rate. Furthermore, our data suggest that the molecular mechanisms of Ca2+ transient decline is normal in NOS1−/− myocytes and that the slow basal Ca2+ transient decline is predominantly due to decreased PLB phosphorylation.

Histone acetylation rescues contextual fear conditioning in nNOS KO mice and accelerates extinction of cued fear conditioning in wild type mice

Epigenetic regulation of chromatin structure is an essential molecular mechanism that contributes to the formation of synaptic plasticity and long-term memory (LTM). An important regulatory process of chromatin structure is acetylation and deacetylation of histone proteins. Inhibition of histone deacetylase (HDAC) increases acetylation of histone proteins and facilitate learning and memory. Nitric oxide (NO) signaling pathway has a role in synaptic plasticity, LTM and regulation of histone acetylation. We have previously shown that NO signaling pathway is required for contextual fear conditioning. The present study investigated the effects of systemic administration of the HDAC inhibitor sodium butyrate (NaB) on fear conditioning in neuronal nitric oxide synthase (nNOS) knockout (KO) and wild type (WT) mice. The effect of single administration of NaB on total H3 and H4 histone acetylation in hippocampus and amygdala was also investigated. A single administration of NaB prior to fear conditioning (a) rescued contextual fear conditioning of nNOS KO mice and (b) had long-term (weeks) facilitatory effect on the extinction of cued fear memory of WT mice. The facilitatory effect of NaB on extinction of cued fear memory of WT mice was confirmed in a study whereupon NaB was administered during extinction. Results suggest that (a) the rescue of contextual fear conditioning in nNOS KO mice is associated with NaB-induced increase in H3 histone acetylation and (b) the accelerated extinction of cued fear memory in WT mice is associated with NaB-induced increase in H4 histone acetylation. Hence, a single administration of HDAC inhibitor may rescue NO-dependent cognitive deficits and afford a long-term accelerating effect on extinction of fear memory of WT mice.

Acetaminophen-Induced Hepatotoxicity and Protein Nitration in Neuronal Nitric-Oxide Synthase Knockout Mice

In overdose acetaminophen (APAP) is hepatotoxic. Toxicity occurs by metabolism to N-acetyl-p-benzoquinone imine, which depletes GSH and covalently binds to proteins followed by protein nitration. Nitration can occur via the strong oxidant and nitrating agent peroxynitrite, formed from superoxide and nitric oxide (NO). In hepatocyte suspensions we reported that an inhibitor of neuronal nitric-oxide synthase (nNOS; NOS1), which has been reported to be in mitochondria, inhibited toxicity and protein nitration. We recently showed that manganese superoxide dismutase (MnSOD; SOD2) was nitrated and inactivated in APAP-treated mice. To understand the role of nNOS in APAP toxicity and MnSOD nitration, nNOS knockout (KO) and wild-type (WT) mice were administered APAP (300 mg/kg). In WT mice serum alanine aminotransferase (ALT) significantly increased at 6 and 8 h, and serum aspartate aminotransferase (AST) significantly increased at 4, 6 and 8 h; however, in KO mice neither ALT nor AST significantly increased until 8 h. There were no significant differences in hepatic GSH depletion, APAP protein binding, hydroxynonenal covalent binding, or histopathological assessment of toxicity. The activity of hepatic MnSOD was significantly lower at 1 to 2 h in WT mice and subsequently increased at 8 h. MnSOD activity was not altered at 0 to 6 h in KO mice but was significantly decreased at 8 h. There were significant increases in MnSOD nitration at 1 to 8 h in WT mice and 6 to 8 h in KO mice. Significantly more nitration occurred at 1 to 6 h in WT than in KO mice. MnSOD was the only observed nitrated protein after APAP treatment. These data indicate a role for nNOS with inactivation of MnSOD and ALT release during APAP toxicity.

Working memory deficits in neuronal nitric oxide synthase knockout mice: Potential impairments in prefrontal cortex mediated cognitive function

Neuronal nitric oxide synthase (nNOS) forms nitric oxide (NO), which functions as a signaling molecule via S-nitrosylation of various proteins and regulation of soluble guanylate cyclase (cGC)/cyclic guanosine monophosphate (cGMP) pathway in the central nervous system. nNOS signaling regulates diverse cellular processes during brain development and molecular mechanisms required for higher brain function. Human genetics have identified nNOS and several downstream effectors of nNOS as risk genes for schizophrenia. Besides the disease itself, nNOS has also been associated with prefrontal cortical functioning, including cognition, of which disturbances are a core feature of schizophrenia. Although mice with genetic deletion of nNOS display various behavioral deficits, no studies have investigated prefrontal cortex-associated behaviors. Here, we report that nNOS knockout (KO) mice exhibit hyperactivity and impairments in contextual fear conditioning, results consistent with previous reports. nNOS KO mice also display mild impairments in object recognition memory. Most importantly, we report for the first time working memory deficits, potential impairments in prefrontal cortex mediated cognitive function in nNOS KO mice. Furthermore, we demonstrate Disrupted-in-Schizophrenia 1 (DISC1), another genetic risk factor for schizophrenia that plays roles for cortical development and prefrontal cortex functioning, including working memory, is a novel protein binding partner of nNOS in the developing cerebral cortex. Of note, genetic deletion of nNOS appears to increase the binding of DISC1 to NDEL1, regulating neurite outgrowth as previously reported. These results suggest that nNOS KO mice are useful tools in studying the role of nNOS signaling in cortical development and prefrontal cortical functioning.

Acetaminophen (APAP)‐Induced Hepatotoxicity and Protein Nitration in neuronal Nitric Oxide Synthase (nNOS) Knockout Mice

In overdose APAP is hepatotoxic. Toxicity occurs by metabolism to N‐acetyl‐p‐benzoquinone imine which covalently binds to proteins followed by protein nitration. Nitration is by peroxynitrite, formed from superoxide and nitric oxide (NO). In freshly isolated hepatocytes we reported that the NO is from nNOS which is believed to be in mitochondria. MnSOD is nitrated by 1 hr in APAP toxicity in mice resulting in MnSOD inactivation. This leads to increased peroxynitrite. To understand the role of nNOS in APAP toxicity and MnSOD nitration, nNOS knockout mice (KO) and wildtype mice (WT) were administered APAP (300 mg/kg). Serum ALT significantly increased at 6 and 8 h in WT and at 8 h in KO. ALT levels in WT were significantly higher than in KO at 6 h but not 8 h. There were no significant differences in hepatic GSH depletion, APAP protein covalent binding, or histopathological evaluation of hepatic necrosis. Hepatic MnSOD activity was significantly lower at 1–4 h in WT and 8 h in KO. MnSOD activity in WT was significantly higher than in KO at 8 h. Western blot analysis for MnSOD nitration indicated a significant increase at 1–8 h in WT and at 6 and 8 h in KO. There was dramatically more nitration of MnSOD in WT than in KO. Nitration in WT was significantly different from KO at 1–6 h. These data indicate that nNOS is important in protein nitration but the role of nNOS and nitration in in vivo toxicity is unclear. Supported by 1 R01 DK079008.

Immunohistochemical study on the expression of calcium binding proteins (calbindin-D28k, calretinin, and parvalbumin) in the cerebral cortex and in the hippocampal region of nNOS knock-out(-/-) mice

Nitric oxide (NO) modulates the activities of various channels and receptors to participate in the regulation of neuronal intracellular Ca2+ levels. Ca2+ binding protein (CaBP) expression may also be altered by NO. Accordingly, we examined expression changes in calbindin-D28k, calretinin, and parvalbumin in the cerebral cortex and hippocampal region of neuronal NO synthase knockout(-/-) (nNOS-/-) mice using immunohistochemistry. For the first time, we demonstrate that the expression of CaBPs is specifically altered in the cerebral cortex and hippocampal region of nNOS-/- mice and that their expression changed according to neuronal type. As changes in CaBP expression can influence temporal and spatial intracellular Ca2+ levels, it appears that NO may be involved in various functions, such as modulating neuronal Ca2+ homeostasis, regulating synaptic transmission, and neuroprotection, by influencing the expression of CaBPs. Therefore, these results suggest another mechanism by which NO participates in the regulation of neuronal Ca2+ homeostasis. However, the exact mechanisms of this regulation and its functional significance require further investigation.

Effects of Metformin in Experimental Stroke

Adenosine 5'-monophosphate-activated protein kinase (AMPK) is an important sensor of energy balance. Stroke-induced AMPK activation is deleterious because both pharmacological inhibition and genetic deletion of AMPK are neuroprotective. Metformin is a known AMPK activator but reduces stroke incidence in clinical populations. We investigated the effect of acute and chronic metformin treatment on infarct volume and AMPK activation in experimental stroke. Male mice were subjected to middle cerebral artery occlusion after acute (3 days) or chronic (3 weeks) administration of metformin. Infarct volumes, AMPK activation, lactate accumulation, and behavioral outcomes were assessed. The roles of neuronal nitric oxide synthase and AMPK were examined using mice with targeted deletion of AMPK or neuronal nitric oxide synthase. Acute metformin exacerbated stroke damage, enhanced AMPK activation, and led to metabolic dysfunction. This effect was lost in AMPK and neuronal nitric oxide synthase knockout mice. In contrast, chronic metformin given prestroke was neuroprotective, improved stroke-induced lactate generation, and ameliorated stroke-induced activation of AMPK. Similarly, the neuroprotective effect of chronic prestroke metformin was lost in neuronal nitric oxide synthase knockout mice. AMPK is an important potential target for stroke treatment and prevention. These studies show that the timing, duration, and amount of AMPK activation are key factors in determining the ultimate downstream effects of AMPK on the ischemic brain.

A functional NOS1 promoter polymorphism interacts with adverse environment on functional and dysfunctional impulsivity

Neuronal nitric oxide synthase (NOS1) knockout results in increased impulsive aggression in mice under adverse housing conditions. In line with this, we have previously shown that a functional promoter polymorphism of NOS1, termed NOS1 ex1f-VNTR, is associated with impulsivity-related traits and related disorders. This study aims to examine whether adverse environment interacts with the risk allele on impulsivity-related measures. We here studied a population-based cohort of Estonian pupils, recruited at the age of 9 years and followed up for another 9 years. For 435 subjects, measures on impulsivity (Adaptive and Maladaptive Impulsivity Scale, BIS-11, Stop Signal data, and Visual Comparison Test, VCT), environmental conditions (stressful life events and family environment), and NOS1 ex1f-VNTR genotype were available. We found a genotype main effect in that presence of a short NOS1 ex1f-VNTR allele was associated with higher levels of adaptive impulsivity, especially in males, but also worse performance in the VCT and the Stop Signal test. Both stressful life events as well as adverse family environment interacted with the risk genotype to increase maladaptive impulsivity. This study provides further evidence that short alleles of NOS1 ex1f-VNTR go along with impulsive behavior. In the absence of adverse environmental conditions, this may lead to a beneficial effect as functional forms of impulsivity are affected. This however is reversed under negative conditions, as dysfunctional impulsivity is increased under these circumstances. This data provides evidence that NOS1 ex1f-VNTR is subject to balancing selection potentially explaining persistence of the risk allele in the population.

Neuronal Nitric Oxide Synthase in Epidermis Is Involved in Cutaneous Circulatory Response to Mechanical Stimulation

The source of nitric oxide (NO) in the cutaneous circulation remains controversial. We hypothesized that epidermis might generate NO in response to mechanical stimulation. In hairless mouse (HR-1) skin organ culture, mechanical stimulation resulted in NO release, which declined within 30 minutes after cessation. A similar NO release occurred in a reconstructed skin model containing only keratinocytes and fibroblasts and was suppressed after detachment of the epidermal layer. Moreover, the stimulation-induced NO release was significantly lower in skin organ culture from neuronal NO synthase knockout (nNOS-KO) mice, compared with wild-type (WT) mice. Mechanical stimulation of skin organ cultures from HR-1, nNOS-KO, endothelial NOS-KO (eNOS-KO), and WT mice caused an enlargement of cutaneous lymphatic vessels. The enlargement was significantly lower after detachment of the epidermal layer than in normal skin samples and was significantly lower for nNOS-KO than for WT mice. Skin blood flow in nNOS-KO mice after stimulation was significantly lower than in WT mice. eNOS-KO mice also showed lower responses than WT mice, and the difference was similar to that in the case of nNOS-KO mice. These results are consistent with the idea that NO generated by epidermal nNOS has a significant role in the cutaneous circulatory response to mechanical stimulation.

P2Y1 Receptors Mediate Apamin-Sensitive and -Insensitive Inhibitory Junction Potentials in Murine Colonic Circular Smooth Muscle

Purinergic inhibitory neuromuscular transmission plays an important role in the control of intestinal motility. In most tissues this neurotransmission is apamin-sensitive, but recent studies in human colonic circular smooth muscle (CSM) suggest the presence of apamin-insensitive purinergic inhibitory junction potentials (IJPs). The current studies used conventional intracellular recordings on colonic CSM strips to characterize the purinergic IJPs in murine colonic CSM. P2Y1 receptor expression was examined by using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. The IJP induced by nerve stimulation (NS) of one and four pulses in neuronal nitric-oxide synthase knockout mice consists of an apamin-sensitive and a dominant apamin-resistant component. These are identical to the IJPs in wild-type and CD1 mice in the presence of N(omega)-nitro-l-arginine methyl ester (200 microM) and were significantly inhibited by alpha,beta-methylene ATP (50 microM), an analog of ATP. IJPs were not affected by the P2X receptor antagonist 2',3'-o-(2,4,6-trinitrophenyl)-ATP (10 microM). Furthermore, apamin-resistant IJPs induced by single-pulse NS were abolished by pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (100 microM), a P2 receptor antagonist; 2'-deoxy-N6-methyl adenosine 3,5-diphosphate (MRS-2179; 10 microM), a selective P2Y1 receptor antagonist; and tetrodotoxin (1 microM). Aboral NS induced apamin-sensitive purinergic IJPs, whereas oral and circumferential NS produced apamin-sensitive and -resistant IJPs, with the latter predominating. RT-PCR and immunohistochemistry confirmed the presence of P2Y1 receptors on smooth muscle and in the myenteric plexus. These data suggest that, depending on stimulus location, activation of P2Y1 receptors produces both apamin-sensitive and apamin-resistant IJPs in murine colonic CSM.

Up-Regulation of the RhoA/Rho-Kinase Signaling Pathway in Corpus Cavernosum from Endothelial Nitric-Oxide Synthase (NOS), but Not Neuronal NOS, Null Mice

We tested the hypothesis that the basal release of nitric oxide (NO) from endothelial cells modulates contractile activity in the corpus cavernosum (CC) via inhibition of the RhoA/Rho-kinase signaling pathway. Cavernosal strips from wild-type (WT), endothelial nitric-oxide synthase knockout [eNOS(-/-)], and neuronal nitric-oxide synthase knockout [nNOS(-/-)] mice were mounted in myographs, and isometric force was recorded. mRNA and protein expression of key molecules in the RhoA/Rho-kinase pathway were analyzed by real-time polymerase chain reaction and Western blot, respectively. The cGMP levels were determined. The Rho-kinase inhibitors (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) and (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl] homopiperazine (H-1152) reduced cavernosal contractions evoked by phenylephrine or electrical field stimulation (EFS) in a concentration-dependent manner, although this inhibition was less effective in tissues from eNOS(-/-) mice. Y-27632 enhanced relaxations induced by sodium nitroprusside, EFS, and NO (administered as acidified NaNO2) without affecting the cGMP content of the cavernosal strips. This enhancement was less prominent in CC from eNOS(-/-). The protein expression of RhoA, Rho-guanine dissociation inhibitor, and Rho-kinase beta did not differ among the strains. However, in eNOS(-/-) CC, the protein expression of Rho-kinase alpha and both mRNA and protein expression of p115-Rho-associated guanine exchange factor (RhoGEF), PDZ-RhoGEF, and leukemia-associated RhoGEF were up-regulated. Phosphorylation of MYPT1 at Thr696 was higher in tissues from eNOS(-/-) mice. A high concentration of Y-27632 significantly enhanced NO release in CC stimulated by EFS. These results suggest a basal release of NO from endothelial cells, which inhibits contractions mediated by the RhoA/Rho-kinase pathway and modulates the expression of proteins related to this pathway in mouse CC. It indicates that endothelial integrity is essential to the maintenance of erectile function.

Immunohistochemical study on the expression of calcium binding proteins (calbindin-D28k, calretinin, and parvalbumin) in the cerebellum of the nNOS knock-out(-/-) mice

Nitric Oxide (NO) actively participates in the regulation of neuronal intracellular Ca2+ levels by modulating the activity of various channels and receptors. To test the possibility that modulation of Ca2+ buffer protein expression level by NO participates in this regulatory effect, we examined expression of calbindin-D28k, calretinin, and parvalbumin in the cerebellum of neuronal NO synthase knock-out (nNOS(-/-)) mice using immunohistochemistry. We observed that in the cerebellar cortex of the nNOS(-/-) mice, expression of calbindin-D28k and parvalbumin were significantly increased while expression of calretinin was significantly decreased. These results suggest another mechanism by which NO can participate in the regulation of Ca2+ homeostasis.