Nitric Oxide Homeostasis Research Articles

Visualization of nitric oxide production and intracellular calcium in juxtamedullary afferent arteriolar endothelial cells.

Nitric oxide (NO) is an important signal transmitter with multiple haemodynamic functions in the kidney. Study of these is complicated by the difficulty in measuring NO directly or visualizing its production. Recently the synthesis of a group of new NO-sensitive fluorescent dyes, diaminofluoresceins (DAF), suitable for imaging applications has been reported. We attempted to use one DAF (DAF-2 DA) to investigate the relationship between endothelial calcium, NO production and afferent arteriolar reactivity. We used the isolated, perfused juxtamedullary nephron preparation (JMN) and loaded the afferent arteriolar endothelium with Fura-2 AM and DAF-2 DA (4,5-diaminofluorescein-2-diacetyl). After in vitro calibration of the imaging system, we measured Fura-2 and DAF-2 fluorescence in single endothelial cells of afferent arterioles (AA) perfused at a pressure of 100 mmHg. Carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (carboxy-PTIO) (10-3 m), a specific NO scavenger, decreased DAF-2 fluorescence in the endothelium by 16.1% and the mid-afferent arteriolar diameter by 10.2%, and increased endothelial calcium by 17.8%. Nomega-nitro-l-arginine methyl ester (l-NAME) (10-4 m) decreased fluorescence intensity of DAF-2 by 18.6%, increased cellular calcium level by 19.7% and constricted the vessels by 11.6%. Addition of carbachol (10-4 m) increased average DAF-2 fluorescence by 22.8% and endothelial calcium concentration by 28.9%, whereas the arteriolar diameter remained essentially unchanged. Carbachol failed to increase DAF-2 fluorescence when administered after l-NAME pre-treatment. We conclude that endothelial NO homeostasis is an important determinant of AA reactivity and suggest that DAF are suitable for real-time imaging of afferent arteriolar NO production in the isolated, perfused JMN and may be used in combination with calcium-sensitive fluorophores. We have found that NO reduction by carboxy-PTIO or l-NAME increases endothelial calcium, suggesting involvement of calcium signalling in an autocrine NO production feedback in the endothelium. This method should help to further clarify the role of endothelial NO in renal haemodynamics.

Spinal upregulation of the nitric oxide synthase-interacting protein NOSIP in a rat model of inflammatory pain

Nitric oxide and nitric oxide synthases are key players in synaptic plasticity events in the spinal cord, which underly the development of chronic pain states. To date, little is known about the molecular mechanisms regulating the activity of nitric oxide synthases in nociceptive systems. The present study was aimed at the immunohistochemical determination of the expression of a nitric oxide synthase-interacting protein (NOSIP) in the rat spinal cord and dorsal root ganglia and studying its regulation in states of nociceptive hypersensitivity in a rat model of post-inflammatory pain. NOSIP is predominantly expressed in nociceptive primary neurons and in neurons of the spinal dorsal horn and the number of NOSIP-positive spinal neurons increases significantly following induction of unilateral intraplantar injection of complete Freund's adjuvant. Thus, NOSIP may modulate nitric oxide homeostasis in physiological and pathological pain conditions.

Arginase and asthma: novel insights into nitric oxide homeostasis and airway hyperresponsiveness

For many years it has been supposed that the production of an excess of nitric oxide (NO) by inducible NO synthase (iNOS) plays a major role in inflammatory diseases, including asthma. However, recent studies indicate that a deficiency of beneficial, bronchodilating constitutive NOS (cNOS)-derived NO is important in allergen-induced airway hyperresponsiveness. Although several mechanisms are proposed to explain the reduction of cNOS activity, reduced substrate availability, caused by a combination of increased arginase activity and decreased cellular uptake of l-arginine, appears to play a key role. Recent evidence also indicates that iNOS-induced pathophysiological effects involve substrate deficiency. Thus, at low concentrations of l-arginine iNOS produces both NO and superoxide anions, which results in the increased synthesis of the highly reactive, detrimental oxidant peroxynitrite. Based on these observations, we propose that a relative deficiency of NO caused by increased arginase activity and altered l-arginine homeostasis is a major factor in the pathology of asthma.

Hypoxia-induced left ventricular dysfunction in myoglobin-deficient mice.

Myoglobin-deficient mice are viable and have preserved cardiac function due to their ability to mount a complex compensatory response involving increased vascularization and the induction of the hypoxia gene program (hypoxia-inducible factor-1alpha, endothelial PAS, heat shock protein27, etc.). To further define and explore functional roles for myoglobin, we challenged age- and gender-matched wild-type and myoglobin-null mice to chronic hypoxia (10% oxygen for 1 day to 3 wk). We observed a 30% reduction in cardiac systolic function in the myoglobin mutant mice exposed to chronic hypoxia with no changes observed in the wild-type control hearts. The cardiac dysfunction observed in the hypoxic myoglobin-null mice was reversible with reexposure to normoxic conditions and could be prevented with treatment of an inhibitor of nitric oxide (NO) synthases. These results support the conclusion that hypoxia-induced cardiac dysfunction in myoglobin-null mice occurs via a NO-mediated mechanism. Utilizing enzymatic assays for NO synthases and immunohistochemical analyses, we observed a marked induction of inducible NO synthase in the hypoxic myoglobin mutant ventricle compared with the wild-type hypoxic control ventricle. These new data establish that myoglobin is an important cytoplasmic cardiac hemoprotein that functions in regulating NO homeostasis within cardiomyocytes.

Diabetic ketosis activates lymphomonocyte-inducible nitric oxide synthase.

Inappropriate production of nitric oxide (NO) may be responsible for the haemodynamic disturbances of diabetic ketoacidosis. We investigated whether this metabolic condition is associated with increased plasma nitrate (the stable oxidation product of NO) levels and NO synthase gene expression in lymphomonocytes. Plasma nitrate concentrations, lymphomonocyte-inducible nitric oxide synthase (iNOS) gene expression, tumour necrosis factor-alpha (TNF-alpha) and soluble thrombomodulin were measured in 11 Type 1 diabetic patients at baseline, during mild ketosis and after euglycaemia was re-established. During diabetic ketosis plasma nitrate concentrations were higher (18 (16-21) vs. 9 (7-11) micro mol/l; (95% lower-upper confidence interval) P < 0.05) than at baseline. At baseline lymphomonocyte iNOS mRNA expression and iNOS protein levels were undetectable, but in ketosis both were increased (both at P < 0.0001). After recovery from ketosis, NO3 concentration, iNOS mRNA, and iNOS expression (270 +/- 36%, mean +/- sd) decreased but not significantly. No significant changes were observed in either TNF-alpha or soluble thrombomodulin levels between the three conditions. Diabetic ketosis is associated with increased nitrate levels and the activation of iNOS expression in circulating lymphomonocytes, but it does not affect either the proinflammatory cytokine TNF-alpha or a marker of endothelial dysfunction such as thrombomodulin. Our data support the hypothesis that, during diabetic ketosis, alterations in NO homeostasis are present in circulating lymphomonocytes.

Complementary distribution of NADPH-diaphorase and l-arginine in the snail nervous system.

Since the interneuronal messenger nitric oxide (NO) can not be stored in neurones, the regulation of the NO-producing enzyme nitric oxide synthase (NOS) is crucial. Neuronal NOS metabolises L-arginine to nitric oxide (NO) and L-citrulline in a Ca(2+)-dependent manner. Thus, availability of L-arginine to NOS may modulate NO production. In this study, we examined the cellular distribution of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase, L-arginine and L-citrulline. Using NADPH-diaphorase histochemistry to visualise putative NO-producing cells and immunocytochemistry to localise L-arginine, we showed that the distribution of L-arginine-immunoreactive neurones correlates well with those of NADPH-diaphorase-positive neurones in cerebral ganglia of the pulmonate Helix pomatia. However, substrate and enzyme were visualised in separate but adjacent neurones. We further examined whether NADPH-diaphorase-labelled cells contain the L-citrulline. Following elevation of intracellular Ca(2+) by the Ca(2+) ionophore, ionomycin, or by a high-K(+) solution, the number of L-citrulline-immunoreactive neurones in mesocerebrum and pedal lobe increased up to tenfold. Preincubation of ganglia with the NOS inhibitor N(G)-nitro-L-arginine prevented ionomycin or high-K(+) solution-induced L-citrulline synthesis. Most L-citrulline-immunoreactive neurones contain NADPH-diaphorase activity. In conclusion, these experiments indicate a complementary distribution of NOS and L-arginine and suggest an unknown signalling pathway between neurones to maintain L-arginine and NO homeostasis.

Neuroprotective MK801 is associated with nitric oxide synthase during hypoxia/reoxygenation in rat cortical cell cultures.

The neuroprotective effect of MK801 against hypoxia and/or reoxygenation-induced neuronal cell injury and its relationship to neuronal nitric oxide synthetase (nNOS) expression were examined in cultured rat cortical cells. Treatment of cortical neuronal cells with hypoxia (95% N(2)/5% CO(2)) for 2 h followed by reoxygenation for 24 h induced a release of lactate dehydrogenase (LDH) into the medium, and reduced the protein level of MAP-2 as well. MK801 attenuated the release of LDH and the reduction of the MAP-2 protein by hypoxia, suggesting a neuroprotective role of MK801. MK801 also diminished the number of nuclear condensation by hypoxia/reoxygenation. The NOS inhibitors 7-nitroindazole (7-NI) and N (G)-nitro-L-arginine methyl ester (L-NAME), as well as the Ca(2+) channel blocker nimodipine, reduced hypoxia-induced LDH, suggesting that nitric oxide (NO) and calcium homeostasis contribute to hypoxia and/or the reoxygenation-induced cell injury. The levels of nNOS immunoactivities and mRNA by RT-PCR were enhanced by hypoxia with time and, down regulated following 24 h reoxygenation after hypoxia, and were attenuated by MK801. In addition, the reduction of nNOS mRNA levels by hypoxia/reoxygenation was also diminished by MK801. Further delineation of the mechanisms of NO production and nNOS regulation are needed and may lead to additional strategies to protect neuronal cells against hypoxic/reoxygenation insults.

Migraine, but not subarachnoid hemorrhage, is associated with differentially increased NPY-like immunoreactivity in the CSF

To test whether migraine and subarachnoid hemorrhage (SAH) are associated with increased sympathetic tone, we compared the neuropeptide Y-like (NPY-LI) and chromogranin A-like immunoreactivities (LI) of cerebrospinal fluid (CSF) from migraneurs and SAH patients with those from control subjects. Increased sympathetic tone was expected to produce higher co-release of these co-stored peptides and concordant changes in their CSF levels. In addition, we investigated a possible disturbed nitric oxide homeostasis by measuring CSF nitrites (NO). More than 70% of CSF NPY-LI corresponded to the chromatographic peak (HPLC) for the intact molecule in all three groups. Migraneurs had 64% higher CSF NPY-LI, but no significant difference in CSF chromogranin A-LI, as compared to controls. In contrast, SAH patients had 74% less CSF chromogranin A-LI and a trend to lower NPY-LI, as compared to controls. No differences in CSF NO were detected among groups. These results argue against an increased sympathetic tone in patients with either migraine or SAH, and suggest that the higher CSF NPY-LI of migraneurs probably originates from central neurons. Furthermore, our findings in SAH patients argue in favor of a decreased sympathetic tone; this could be a homeostatic response to counterbalance vasoconstriction mediated by other mechanisms.

Non-Enzymatic Production of Nitric Oxide (NO) from NO Synthase Inhibitors

The gaseous signal molecule, nitric oxide (NO•), is generated enzymatically by NO synthase (NOS) from L-arginine. Overproduction of NO contributes to cell and tissue damage assequelaeof infection and stroke. Strategies to suppress NO synthesis rely heavily on guanidino-substituted L-arginine analogs (L-NAME, L-NA, L-NMMA, L-NIO) as competitive inhibitors of NOS, which are often used in high doses to compete with millimolar concentrations of intracellular arginine. We show that these analogs are also a source for non-enzymatically produced NO. Enzyme-independent NO release occurs in the presence of NADPH, glutathione, L-cysteine, dithiothreitol and ascorbate. This non-enzymatic synthesis of NO can produce potentially toxic, micromolar concentrations of NO and can oppose the effects of NOS inhibition. NO production driven by NOS inhibitors was demonstratedex vivoin the central nervous and peripheral tissues of gastropod molluscsAplysiaandPleurobranchaeausing electron paramagnetic resonance and spin-trapping techniques. These results have important implications for therapeutic regulation of NO homeostasis.

Assessment of the correlation of spectral diagnostics cardiac status CK-MB/ myoglobin rapid test to Dade Stratus CK-MB and myoglobin

The physiological role of myoglobin (Mb) within the heart depends on its oxygenation state. The myocardium exhibits a broad oxygen partial pressure (pO2) spectrum with a transmural gradient from the epicardial to the subendocardial layer, ranging from arterial values to an average of 19.3 mm Hg down to 0 mm Hg. The function of Mb as an O2 storage depot is well appreciated, especially during systolic compression. In addition, Mb controls myocardial nitric oxide (NO) homeostasis and thus modulates mitochondrial respiration under physiological and pathological conditions. We recently discovered the role of Mb as a myocardial O2 sensor; in its oxygenated state Mb scavenges NO, protecting the heart from the deleterious effects of excessive NO. Under hypoxia, however, deoxygenated Mb changes its role from an NO scavenger to an NO producer. The NO produced protects the cell from short phases of hypoxia and from myocardial ischemia/reperfusion injury. In this review we summarize the traditional and novel aspects of Mb and its (patho)physiological role in the heart.

Drugs that alter nitric oxide homeostasis: a reply

Drugs that alter nitric oxide homeostasis: a reply

Therapeutic value of drugs which modulate nitric oxide homeostasis

Therapeutic value of drugs which modulate nitric oxide homeostasis

Drugs which alter nitric oxide homeostasis are unlikely to provide therapeutic benefit in cardiovascular disease

Journal Article Drugs which alter nitric oxide homeostasis are unlikely to provide therapeutic benefit in cardiovascular disease Get access David C Lefroy David C Lefroy Department of Cardiac Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY: Dr D C Lefroy. Search for other works by this author on: Oxford Academic PubMed Google Scholar Cardiovascular Research, Volume 27, Issue 12, December 1993, Page 2105, https://doi.org/10.1093/cvr/27.12.2105 Published: 01 December 1993

Drugs which modulate nitric oxide homeostasis are likely to provide therapeutic benefit in cardiovascular disease.

Drugs which modulate nitric oxide homeostasis are likely to provide therapeutic benefit in cardiovascular disease.