Search

Abstract: It has been reported that l-glutamate (l-glu) microinjection into the hypothalamic paraventricular nucleus (PVN) evokes pressor and tachycardiac responses in unanesthetized rats. In the present study the hypothesis was tested that a local nitric oxide (NO)–guanylate cyclase interaction mediates cardiovascular effects of l-glu microinjection into the PVN of rats. The cardiovascular responses evoked by 10nmol/100nL of l-glu microinjected into the PVN were measured before and 10min after PVN treatment with vehicle, the selective neuronal NO-synthase (nNOS) inhibitor Nω-Propyl-l-arginine (N-Propyl, 0.04nmol or 4nmol/100nL), the NO scavenger carboxy-PTIO (C-PTIO, 1nmol/100nL) or the guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolol [4,3-a]quinoxalin-1-one (ODQ, 1nmol/100nL). In a final experiment, different doses of the NO donor sodium nitroprusside (SNP; 9, 27 or 45nmol/100nL) were microinjected into the PVN. Cardiovascular responses evoked by l-glu microinjection into the PVN were abolished by local pretreatment with N-Propyl in both anesthetized and unanesthetized rats. PVN treatment with either C-PTIO or ODQ also reduced l-glu cardiovascular responses. The microinjection of SNP into the PVN caused pressor and tachycardiac responses in unanesthetized rats, whereas depressor and bradycardiac responses were observed in anesthetized rats. The present results suggest that cardiovascular responses evoked by l-glu microinjection into the PVN involve a local production of NO and activation of guanylate cyclase. [Copyright &y& Elsevier]

Nitric oxide has been demonstrated to participate in β-cell damage during streptozotocin (STZ)-induced diabetes. STZ consists of 2-deoxy-D-glucose substituted by N-methyl-N-nitrosourea at C-2 and therefore can liberate ·NO. However, it has not been proven whether ·NO generation from STZ is responsible for the disease. We found that STZ treated in vitro with ultraviolet (UV) light liberated significantly more ·NO than non-irradiated STZ (1,134.4 ± 104 vs. 256.9 ± 240 nmol). Moreover, the diabetogenic effect of STZ was abolished by UV irradiation before its administration to experimental animals. In these animals the glucose and insulin values were significantly different from those of the diabetic group (151.3 ± 16.6 vs. 364.6 ± 63.4 mg/dl and 36.3 ± 17.9 vs. 0.08 ± 5.5 μIU/ml, respectively) and similar to those of the non-diabetic group (127.2 ± 34.1 mg/dl and 41.7 ± 13.9 μIU/ml, respectively). Carboxy-PTIO treatment returned glycemia to nearly normal levels in 60% of STZ-induced diabetic rats (157.5 ± 11.8 vs. 364.6 ± 63.6 mg/dl of the diabetic group). L-NAME and dexamethasone cannot return either glucose or insulin to normal levels. In conclusion, UV light increased ·NO liberation from STZ and suppressed its diabetogenic activity. It is possible that the diabetogenic activity of STZ is related to the liberation of nitric oxide from STZ, since carboxy-PTIO scavenger had a protective effect, while L-NAME and dexamethasone did not. It is possible that an increase in ·NO concentration into cell, independently of its endogenous or exogenous origin, can induce β-cell damage and diabetes. Copyright © 2004 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

We have developed a novel method that uses high performance liquid chromatography (HPLC)/multi-electrode electrochemical coulometric-array detection [ECD (coulometry)] for separating carboxy-PTIO and carboxy-PTI for nitric oxide (NO) determination. We found that the NO sensitivity of the proposed method using coulometric detection was 20 times those of HPLC/UV/electron spin resonance (ESR) and HPLC/ECD (amperometry), and the detection limits for 2-[4-carboxyphenyl]-4,4,5,5-tetramethylimidazoline-1-oxyl 3 oxide (carboxy-PTIO) and 2-[4-carboxyphenyl]-4,4,5,5-tetramethylimidazoline-1-oxyl (carboxy-PTI) were 3 and 50 nM, respectively. The calibration curves for carboxy-PTIO and carboxy-PTI, which were constructed by plotting concentration vs. signal intensity, showed good linearity in the 10 nM–200 µM range (r2 = 0.997). The proposed method was applied to rats sensitized with carrageenin and could determine NO generated by inflammation in the rats. [ABSTRACT FROM AUTHOR]

The hypotensive effect and the acute toxicity of trans-[Ru(NH3)4P(OEt)3(NO)](PF6)3 (RuNO) were investigated in conscious animals. The approximate lethal dose of RuNO is 257.5 μmol/kg in mice i.p. and the IC50 values evaluated for V79 culture cell cytotoxicity were higher than 2.0 mM, suggesting that the ruthenium species are significantly less toxic than Na2[Fe(CN)5(NO)] (SNP) species. The RuNO hypotensive effect measured through in-bolus intravenous administration in chronically instrumented normotensive and hypotensive adult male Wistar rats is similar to that exhibited by equivalent doses of SNP. The hypotensive effect of the ruthenium complex is fully inhibited by methylene blue and PTIO, suggesting that the RuNO effect is likely to be primarily dependent on the NO–[cGMP] pathway in the smooth muscle cells. [Copyright &y& Elsevier]

Ever since it was found to mediate the endothelium-dependent dilation of blood vessels, nitric oxide (NO) has generated enormous research interest throughout the biological sciences. Over thirty years of research has identified NO as a ubiquitous and versatile regulatory factor utilized by both vertebrates and invertebrates. The short lifetime and low concentration of NO make quantitation difficult. Here we report a method for measuring NO using the selective reaction with 2-​(4-​carboxyphenyl)-​4,​5-​dihydro-​4,​4,​5,​5-​tetramethyl-1H-​imidazolyl-​1-​oxy-​3-​oxide (carboxy-PTIO) to form carboxy-PTI. We used tandem mass spectrometry to verify the validity of this reaction, and liquid chromatography - mass spectrometry to quantitate the amount of carboxy-PTI formed. Using diethylamine nonoate as a NO donor we demonstrate this method can quantitate NO concentrations with a detection limit of 5 nM. We successfully determined the amount of NO generated endogenously by frog heart/aorta when stimulated by carbachol, a non-selective acetylcholine receptor agonist. Based on these results, we suggest that this technique can be useful for the quantitative determination of NO in biological samples.•We report a method to measure NO by reacting it with carboxy-PTIO to form carboxy-PTI.•The carboxy-PTI is quantified by liquid chromatography mass spectrometry (LCMS).•This method can quantitate NO concentrations ranging from 5 nM to 1 µM., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors. Published by Elsevier B.V.)

Adoptive immunotherapy with cytotoxic T lymphocytes (CTLs) can result in robust and durable antitumor responses. Tumor-infiltrating CTLs produce IFNγ and mediate antitumor activity, but they simultaneously induce counter-regulatory immunosuppressive mechanisms in the tumor by recruiting monocytic myeloid-derived suppressor cells (MDSCs) that limit their proliferation and effector function. Using a murine model of adoptive immunotherapy for B16 melanoma, we developed a strategy to augment CTL activity by downregulating immunosuppression by MDSCs. Intravenous injection of transgenic pmel-1 CTLs into tumor-bearing mice, resulted in their infiltration into the tumor, but this was accompanied by the accumulation of large numbers of monocytic MDSCs (M-MDSCs). These cells hampered CTL function and reduced their numbers in the tumor. We determined that one mechanism responsible for this immunosuppression was the production of nitric oxide (NO) by MDSCs in the tumor. Therefore, mice were given the NO scavenger carboxy-PTIO (C-PTIO) on the day after CTL transfer. This led to the restoration of impaired proliferative capacity and function of the CTLs, resulting in sustained suppression of tumor growth. Thus, we conclude that CTL therapy can be improved by counter-acting immunosuppression. Targeting NO, one mediator of the immunosuppressive activity of M-MDSCs, may be an appropriate strategy to restore impaired CTL function and improve the efficacy of immunotherapy. [ABSTRACT FROM PUBLISHER]

Impaired platelet responsiveness to nitric oxide (NO resistance) is a common characteristic of many cardiovascular disease states and represents an independent risk factor for cardiac events and mortality. NO resistance reflects both scavenging of NO by superoxide (O2(-)), and impairment of the NO receptor, soluble guanylate cyclase (sGC). There is thus an urgent need for circumvention of NO resistance in order to improve clinical outcomes. Nitroxyl (HNO), like NO, produces vasodilator and anti-aggregatory effects, largely via sGC activation, but is not inactivated by O2(-). We tested the hypothesis that HNO circumvents NO resistance in human platelets. In 57 subjects with or without ischemic heart disease, platelet responses to the HNO donor isopropylamine NONOate (IPA/NO) and the NO donor sodium nitroprusside (SNP) were compared. While SNP (10μM) induced 29±3% (p<0.001) inhibition of platelet aggregation, IPA/NO (10μM) caused 75±4% inhibition (p<0.001). In NO-resistant subjects (n=28), the IPA/NO:SNP response ratio was markedly increased (p<0.01), consistent with partial circumvention of NO resistance. Similarly, cGMP accumulation in platelets was greater (p<0.001) with IPA/NO than with SNP stimulation. The NO scavenger carboxy-PTIO (CPTIO, 200μM) inhibited SNP and IPA/NO responses by 92±7% and 17±4% respectively (p<0.001 for differential inhibition), suggesting that effects of IPA/NO are only partially NO-mediated. ODQ (10μM) inhibited IPA/NO responses by 36±8% (p<0.001), consistent with a contribution of sGC/haem to IPA/NO inhibition of aggregation. There was no significant relationship between whole blood ROS content and IPA/NO responses. Thus the HNO donor IPA/NO substantially circumvents platelet NO resistance while acting, at least partially, as a haem-mediated sGC activator., (Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.)

In contrast to the well known immunostimulatory roles of IL-12, little has been known about its immunosuppressive roles. In the present study, IL-12-activated lymphocyte-mediated macrophage apoptosis was investigated by employing murine lymphocyte/macrophage cocultures. IL-12-activated lymphocytes and their culture supernatants induced an inducible nitric oxide synthase (iNOS)-mediated nitric oxide (NO) synthesis in macrophages. The NO synthesis was markedly inhibited by blocking antibodies to IFN-γ and TNF-α, suggesting the key role of these lymphocyte cytokines in mediating the NO synthesis. The endogenously produced NO inhibited macrophage proliferation, and induced apoptosis in concordance with the accumulation of p53, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and DR5, and the activation of caspase-3, processes that were inhibited by N(G)-monomethyl-l-arginine, aminoguanidine (NO synthase inhibitors) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (an NO scavenger). These results were further supported by the findings obtained from the experiments employing IFN-γ-knockout and iNOS-knockout mice. Our study demonstrated a novel, non-contact-dependent mechanism of macrophage suppression by IL-12-activated lymphocytes: induction of growth inhibition and apoptosis of macrophages due to endogenous NO synthesis induced by cytokines secreted from IL-12-activated lymphocytes., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Sulfhydryl groups on protein Cys residues undergo an array of oxidative reactions and modifications, giving rise to a virtual redox zip code with physiological and pathophysiological relevance for modulation of protein structure and functions. While over two decades of studies have established NO-dependent S-nitrosylation as ubiquitous and fundamental for the regulation of diverse protein activities, proteomic methods for studying H2S-dependent S-sulfhydration have only recently been described and now suggest that this is also an abundant modification with potential for global physiological importance. Notably, protein S-sulfhydration and S-nitrosylation bear striking similarities in terms of their chemical and biological determinants, as well as reversal of these modifications via group-transfer to glutathione, followed by the removal from glutathione by enzymes that have apparently evolved to selectively catalyze denitrosylation and desulfhydration. Here we review determinants of protein and low-molecular-weight thiol S-sulfhydration/desulfhydration, similarities with S-nitrosylation/denitrosylation, and methods that are being employed to investigate and quantify these gasotransmitter-mediated cell signaling systems., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)