Release Of Nitric Oxide Metabolites Research Articles

Reduced nitric oxide synthesis in winter: A potential contributing factor to increased cardiovascular risk

BackgroundNitric oxide is a key signalling molecule that elicits a range of biological functions to maintain vascular homeostasis. A reduced availability of nitric oxide is implicated in the progression of cardiovascular diseases and increases the risk of pathogenic events. AimsTo compare the concentration of nitric oxide metabolites in healthy adults between winter and summer months. DesignAn observational study of healthy adults (age 32 ± 9 years) living in central Scotland. MethodsThirty-four healthy adults (13 females) were monitored for 7 days in summer and winter to record sunlight exposure (ultraviolet-A (UV-A) radiation), diet, and physical activity. At the end of each phase, blood pressure was measured, and samples of blood and saliva collected. The samples were analysed to determine the concentrations of plasma and salivary nitrate and nitrite and serum 25-hydroxyvitamin D (25(OH)D). ResultsThe participants maintained similar diets in each measurement phase but were exposed to more UV-A radiation (550%) and undertook more moderate-vigorous physical activity (23%) in the summer than in winter. Plasma nitrite (46%) and serum 25(OH)D (59%) were higher and blood pressure was lower in the summer compared to winter months. Plasma nitrite concentration was negatively associated with systolic, diastolic, and mean arterial blood pressure. ConclusionsPlasma nitrite, an established marker of nitric oxide synthesis, is higher in healthy adults during the summer than in winter. This may be mediated by a greater exposure to UV-A which stimulates the release of nitric oxide metabolites from skin stores. While it is possible that seasonal variation in nitric oxide availability may contribute to an increased blood pressure in the winter months, the overall impact on cardiovascular health remains to be determined.

Mechanisms underlying reduced P2Y1‐receptor‐mediated relaxation in superior mesenteric arteries from long‐term streptozotocin‐induced diabetic rats

Extracellular nucleotides activate cell-surface purinergic (P2) receptors, contribute to the local regulation of vascular tone and play important roles in pathophysiological states. However, little is known about the vasodilator effects of P2Y(1) -receptor activation in diabetic states. We hypothesized that in a model of established type 1 diabetes, long-term streptozotocin (STZ)-induced diabetic rats, the arterial relaxation elicited by a P2Y(1) -receptor agonist would be impaired. Relaxations to adenosine 5'-diphosphate sodium salt (ADP), 2-MeSADP (selective P2Y(1) -receptor agonist) and adenosine 5'-triphosphate disodium salt (ATP) were examined in superior mesenteric artery rings from long-term STZ-induced diabetic rats (at 50-57 weeks after STZ injection). ADP-stimulated nitric oxide (NO) production in the superior mesenteric artery was assessed by measuring the levels of NO metabolites. Mesenteric artery expressions of P2Y(1) receptor, and ADP-stimulated levels of phosphorylated endothelial NO synthase (eNOS) (at Ser(1177) and at Thr(495) ) and eNOS were detected by Western blotting. Arteries from diabetic rats exhibited (vs. those from age-matched control rats): (i) reduced ADP-induced relaxation, which was partly or completely inhibited by endothelial denudation, by NOS inhibitor treatment and by a selective P2Y(1) -receptor antagonist, (ii) reduced 2-MeSADP-induced relaxation, (iii) reduced ADP-stimulated release of NO metabolites and (iv) impaired ADP-induced stimulation of eNOS activity (as evidenced by reduced the fold increase in eNOS phosphorylation at Ser(1177) with no difference in fold increase in eNOS phosphorylation at Thr(495) ). The protein expression of P2Y(1) receptor did not differ between diabetic and control arteries. These results suggest that P2Y(1) -receptor-mediated vasodilatation is impaired in superior mesenteric arteries from long-term type 1 diabetic rats. This impairment is because of reduced P2Y(1) -receptor-mediated NO signalling, rather than to reduced P2Y(1) -receptor expression.

Cervical nitric oxide release in Chlamydia trachomatis and high-risk human papillomavirus infection

To compare cervical fluid nitric oxide release in women with and without Chlamydia trachomatis and high-risk human papillomavirus infection (hrHPV). An open clinical study. University Hospital of Helsinki. Thirty-nine women with (n=21) and without C. trachomatis (n=18). Chlamydia trachomatis and/or hrHPV were studied by using specific RNA- and DNA-based tests. Levels of cervical fluid nitric oxide metabolite (NOx) were assessed by the Griess reaction. The difference in cervical fluid NOx between women with and without C. trachomatis and hrHPV. Fourteen (67%) C. trachomatis-infected women and three (17%) noninfected women had concomitant hrHPV. The level of cervical fluid NOx in women with C. trachomatis (median 37.5 μmol/l, 95% confidence interval 26.1-50.9) was higher (p=0.02) than that in C. trachomatis-noninfected women (median 19.7 μmol/L, 95% confidence interval 5.6-30.0). The presence of hrHPV did not associate with any difference in NOx levels between C. trachomatis-infected or -noninfected women. Chlamydia trachomatis was associated with increased release of nitric oxide metabolites in the uterine cervix. This stimulus was stronger than that of hrHPV, because no additional rise in NOx was seen in women with concomitant C. trachomatis and hrHPV infection.

Role of nitric oxide during rotavirus infection

The pathophysiological mechanisms behind rotavirus-induced diarrhoea still remain incomplete. Current views suggest that the non-structural protein 4 (NSP4) of rotavirus and the enteric nervous system (ENS) participate in water secretion and diarrhoea. In the present work the role of nitric oxide (NO) in rotavirus infection and disease has been studied in vitro, mice and humans. Incubation of human intestinal epithelial cells (HT-29) with purified NSP4 but not with infectious virus produced NO2/NO3 accumulation in the incubation media. The NSP4-induced release of NO metabolites occurred within the first minutes after the addition of the toxin. Mice infected with murine rotavirus (strain EDIM) accumulated NO2/NO3 in the urine at the onset for diarrhoea. Following rotavirus infection, inducible nitric oxide synthetase (iNOS) mRNA was upregulated in ileum, but not in duodenum or jejunum of newborn pups within 5 days post-infection. A prospective clinical study including 46 children with acute rotavirus infection and age-matched controls concluded that rotavirus infection stimulates NO production during the course of the disease (P < 0.001). These observations identify NO as an important mediator of host responses during rotavirus infection.

Inhibition of Nitric Oxide Synthase Reverses Permeability Changes in a Mouse Model of Acute Peritonitis

Acute peritonitis is the most frequent complication of peritoneal dialysis. Previous studies have suggested a major role for nitric oxide (NO) in the permeability changes and loss of ultrafiltration induced by acute peritonitis. In this study, we further investigated the potential role of NO in a mouse model of peritonitis induced by Escherichia coli Lipopolysaccharide (LPS). A 2-hour peritoneal equilibration test was performed in control and LPS-treated mice using 7% glucose dialysate supplemented or not with the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). The levels of NO metabolites in the dialysate were maximal 18 hours after LPS injection. At that time, acute peritonitis induced by LPS was reflected by an increased recruitment of leukocytes, an increased intraperitoneal release of interleukin-6, a significant increase in the peritoneal permeability for small solutes, a loss of sodium sieving, and a loss of ultrafiltration in comparison with controls. Addition of L-NAME in LPS-treated mice significantly reversed permeability modifications and prevented the release of NO metabolites into the dialysate. These results confirm that increased NO mediates permeability modifications during acute peritonitis, and illustrate the potential of mouse models to investigate the molecular mechanisms regulating peritoneal permeability.

Liposome-mediated transfer of nitric oxide synthase gene improves renal function in ureteral obstruction in rats

The protective effect of nitric oxide has been demonstrated in several renal disease models. We augmented renal nitric oxide production by transfer of the inducible nitric oxide synthase (iNOS) gene into rat kidney in controls and in unilateral ureteral obstruction (UUO). The human iNOS gene was inserted into a pcDNA 3.1-backbone plasmid with the FLAG epitope (FLAG-iNOS). In vitro, transduction of FLAG-iNOS was confirmed by Western blot and Griess reaction. In vivo, we transfected either FLAG-iNOS or control plasmid (CMV-LacZ), using cationic liposomes. Urinary nitric oxide metabolites and immunohistochemistry confirmed iNOS transduction. Renal function was also assessed. In vitro, increased iNOS expression was demonstrated in human embryonic kidney (HEK293) cells, along with increased release of nitric oxide metabolites, NO(2)/NO(3). In vivo, FLAG-iNOS was detected by polymerase chain reaction (PCR) up to 35 days after the transfection. Urine collection documented increased urinary NO(2)/NO(3). Immunohistochemistry localized iNOS to collecting ducts, distal tubules, and glomerulus of the injected kidney. Renal function measured up to 21 days after transfection in control animals was not significantly different between the two groups. In contrast, renal function after 24 hours of UUO was significantly improved in FLAG-iNOS-treated animals. This study demonstrates the feasibility of liposome-mediated iNOS gene transfer into the kidney. Furthermore, the improvement of renal function in UUO demonstrates that the transfected iNOS gene is active and suggests that decreased iNOS activity contributes to the decreased renal function in UUO. This iNOS construct may have therapeutic utility in the pathophysiologic sequelae of UUO and other renal diseases.

Evidence that nitric oxide-glutamate cascade modulates spinal antinociceptive effect of morphine: a behavioural and microdialysis study in rats

We evaluated the ability of spinally administered nitric oxide (NO) synthase inhibitor to modulate antinociceptive action of intrathecal (i.t.) morphine in rats by measuring the early and late phases of flinching and licking/biting in the formalin test. To determine the contribution of spinal NO and glutamate, we measured the release of NO metabolites (nitrite/nitrate) and glutamate from the spinal cord in rats, using a microdialysis probe placed in the lumbar space. The i.t. administration of N G-nitro l-arginine methyl ester ( l-NAME) produced a dose-dependent reduction in the number of flinches during the late phase, whereas there were no significant alterations in the late phase licking/biting, and early phase flinching and licking/biting. Spinal administration of morphine at low doses produced a significant antinociceptive activity in the early and late phases of the flinching behaviour, whereas higher doses of morphine were required to obtain a significant effect in the licking/biting behaviour during both phases. Combination of l-NAME with morphine resulted in an enhanced reduction in the early and late phase flinching. Enhanced antinociceptive activity was observed in the late phase licking/biting by i.t. combined administration of l-NAME (400 nmol) and morphine (1.25 nmol). In the present study, we have confirmed our prior results that injection of formalin (5.0%) into the plantar surface of the paw evoked a biphasic spinal release of nitrite/nitrate and a transient release of glutamate. Formalin-evoked release of nitrite/nitrate and glutamate was also reduced markedly by i.t. combined administration of l-NAME and morphine. These behavioural and biochemical results suggest that i.t. administered l-NAME may enhance morphine-induced antinociception through an increased inhibition of nitrite/nitrate and glutamate releases evoked by formalin injection at the spinal cord level.

Characterization of nociceptive responses and spinal releases of nitric oxide metabolites and glutamate evoked by different concentrations of formalin in rats

A comparison was made of spontaneous nociceptive behaviors elicited by subcutaneous injection of formalin (0.5–10.0%) into the plantar or dorsal surface of the right hindpaw in rats. In the present study, we also examined the effect of paw formalin injection on the release of nitric oxide (NO) metabolites (nitrite/nitrate) and glutamate from the spinal cord in anesthetized rats using a dialysis probe placed in the lumbar subarachnoid space. Two distinct quantifiable behaviors indicative of pain were identified by formalin injected into both regions of the paw. There were no significant alterations in the number of flinches during the early and late phases induced by different regions of formalin injection. However, the early phase licking/biting activity evoked by formalin injection into the plantar surface of the paw was significantly higher than that evoked by formalin injected into the dorsal region. The maximum effect in the early and late phases was produced by 5.0% formalin injection into the dorsal and plantar paw. At a higher concentration (10.0%) of formalin, nociceptive behavioral responses were decreased except for the late phase flinching when injected into the dorsal paw. Injections of formalin (5.0%) into both regions of the paw evoked a biphasic spinal release of nitrite/nitrate with a significant increase during the early phase (0–10 min) and the late phase (30–80 or 90 min). A higher concentration of formalin (10.0%) failed to produce a clear-cut release of nitrite/nitrate. A significant increase of glutamate was observed in the 0–10 min samples obtained after injection of formalin (5.0%) into the plantar and dorsal surface of the paw, whereas 0.5 and 10.0% formalin induced no substantial release. These results suggest that 5.0% formalin should be used when studying antinociceptive activity of NO- and N-methyl- d-aspartate-related compounds in the formalin test in rats. Formalin injection into the plantar surface of the paw might prove to be useful for evoking the licking/biting response, particularly in the early phase.

Minocycline, a Tetracycline Derivative, Is Neuroprotective against Excitotoxicity by Inhibiting Activation and Proliferation of Microglia

Minocycline, a semisynthetic tetracycline derivative, protects brain against global and focal ischemia in rodents. We examined whether minocycline reduces excitotoxicity in primary neuronal cultures. Minocycline (0.02 microm) significantly increased neuronal survival in mixed spinal cord (SC) cultures treated with 500 microm glutamate or 100 microm kainate for 24 hr. Treatment with these excitotoxins induced a dose-dependent proliferation of microglia that was associated with increased release of interleukin-1beta (IL-1beta) and was followed by increased lactate dehydrogenase (LDH) release. The excitotoxicity was enhanced when microglial cells were cultured on top of SC cultures. Minocycline prevented excitotoxin-induced microglial proliferation and the increased release of nitric oxide (NO) metabolites and IL-1beta. Excitotoxins induced microglial proliferation and increased the release of NO metabolites and IL-1beta also in pure microglia cultures, and these responses were inhibited by minocycline. In both SC and pure microglia cultures, excitotoxins activated p38 mitogen-activated protein kinase (p38 MAPK) exclusively in microglia. Minocycline inhibited p38 MAPK activation in SC cultures, and treatment with SB203580, a p38 MAPK inhibitor, but not with PD98059, a p44/42 MAPK inhibitor, increased neuronal survival. In pure microglia cultures, glutamate induced transient activation of p38 MAPK, and this was inhibited by minocycline. These findings indicate that the proliferation and activation of microglia contributes to excitotoxicity, which is inhibited by minocycline, an antibiotic used in severe human infections.

Role of endogenous nitric oxide and prostaglandin in duodenal bicarbonate response induced by mucosal acidification in rats.

Duodenal HCO3- secretion increases in response to mucosal acidification by luminal acid. Although this process is known to be mediated by endogenous prostaglandins (PGs), the role of nitric oxide (NO) in this response has been little studied. We examined the effects of indomethacin and N(G)-nitro-L-arginine methyl ester (L-NAME) on the acid-induced HCO3- secretion in the rat duodenum, together with those on PGE2 generation as well as luminal release of NO metabolites (NOx). A proximal duodenal loop was perfused with saline, and the HCO3- secretion was measured at pH 7.0 using a pH-stat method and by adding 10 mM HCl. Mucosal acidification was performed by exposing the loop to 10 or 100 mM HCl for 10 min. Acidification of the duodenal mucosa stimulated the HCO3- secretion, with concomitant increase of mucosal PGE2 contents and luminal release of NOx, the response being much greater in case of 100 mM HCl. Indomethacin significantly inhibited the acid-induced HCO3- secretion as well as the PGE2 biosynthetic response, without influence on the NOx release. Pretreatment of the animals with L-NAME attenuated both the increase of mucosal PGE2 contents and luminal release of NOx following the acidification, resulting in a marked inhibition of the acid-induced HCO3- response, and these effects were significantly antagonized by coadministration of L-arginine. Duodenal HCO3- secretion was also increased by mucosal exposure to NOR-3 (a NO donor), with concomitant increase of PGE2 generation, but these effects were mitigated in the presence of indomethacin. In addition, the duodenal damage caused by mucosal perfusion with 100 mM HCl for 4 hr was markedly aggravated by pretreatment with L-NAME as well as indomethacin. These results suggest that both endogenous NO and PGs are involved in the mechanism for the acid-induced duodenal HCO3- secretion, and that NO may increase the HCO3- secretion by stimulating PG generation.

Subacute but not acute generation of nitric oxide in focal cerebral ischemia.

Excessive release of nitric oxide (NO) has been implicated in the pathophysiology of neurodegeneration in ischemic stroke. We compared intracerebral release of indicators of NO generation at the acute and subacute stages of transient focal cerebral ischemia. In vivo microdialysis in the rat striatum was performed at the acute (first hours) and subacute (after 24 or 48 hours) stages of cerebral ischemia or sham operation to monitor intracerebral release of the stable NO metabolites nitrite and nitrate. Whereas only a nonsignificant trend toward increased release of these NO metabolites was evidenced in acute cerebral ischemia, a significant NO generation was observed subacutely, 48 hours after induction of cerebral ischemia. Aminoguanidine, a selective inhibitor of inducible NO synthase, suppressed this delayed release of nitrite and nitrate. Whereas these observations do not support a major NO generation in acute cerebral ischemia, they indicate an inducible NO synthase-dependent NO generation predominantly at the subacute phase of ischemic neurodegeneration. Therefore, NO generation may play a pathophysiological role in delayed ischemic neurodegeneration.