Quantitative Systems Pharmacology Model of NO Metabolome and Methemoglobin Following Long-Term Infusion of Sodium Nitrite in Humans.

A long-term sodium nitrite infusion is intended for the treatment of vascular disorders. Phase I data demonstrated a significant nonlinear dose-exposure-toxicity relationship within the therapeutic dosage range. This study aims to develop a quantitative systems pharmacology model characterizing nitric oxide (NO) metabolome and methemoglobin after sodium nitrite infusion. Nitrite, nitrate, and methemoglobin concentration-time profiles in plasma and RBC were used for model development. Following intravenous sodium nitrite administration, nitrite undergoes conversion in RBC and tissue. Nitrite sequestered by RBC interacts more extensively with deoxyhemoglobin, which contributes greatly to methemoglobin formation. Methemoglobin is formed less-than-proportionally at higher nitrite doses as characterized with facilitated methemoglobin removal. Nitrate-to-nitrite reduction occurs in tissue and via entero-salivary recirculation. The less-than-proportional increase in nitrite and nitrate exposure at higher nitrite doses is modeled with a dose-dependent increase in clearance. The model provides direct insight into NO metabolome disposition and is valuable for nitrite dosing selection in clinical trials.CPT: Pharmacometrics & Systems Pharmacology (2013) 2, e60; doi:10.1038/psp.2013.35; published online 31 July 2013.

Alzheimer's factors in postischemic dementia.

The way for explanation postischemic dementia processes has been one fraught with a wide range of complications and frequent revisions with a lack of a final clear solution. Data from animal models of brain ischemia and human ischemic brains studies have demonstrated an overexpression of amyloid precursor protein and increase production of a ß-amyloid peptide. Restoration brain activity following ischemic brain episode is delayed and not always complete due to an alteration related with increase in the level of the ß-amyloid peptide. In this paper, we will propose our idea about production of the ß-amyloid peptide from the amyloid precursor protein in ischemic brain lesions, and how this protein presents etiological and therapeutic targets that are now under consideration. Maturation of the ischemic brain tissue pathology may be caused not only by a neurodegeneration of selectively vulnerable neuronal cells destroyed following ischemia but also by acute and chronic pathology of resistant parts of the brain and chronic changes in the blood-brain barrier. We propose that in dementia following ischemia an initial ischemic episode precedes the brain tissue deposition of ß-amyloid peptide, which in turn amplifies the vascular dysfunction after first episode of ischemia triggering next focal ischemic episodes as vicious cycle preceding final ischemic degenerative changes and may gradually over a lifetime, progress to brain atrophy and finally to postischemic dementia with Alzheimer's phenotype.

One year follow up in ischemic brain injury and the role of Alzheimer factors.

Ongoing interest in brain ischemia research has provided data showing that ischemia may be involved in the pathogenesis of Alzheimer disease. Brain ischemia in the rat produces a stereotyped pattern of selective neuronal degeneration, which mimics early Alzheimer disease pathology. The objective of this study was to further develop and characterize cardiac arrest model in rats, which provides practical way to analyze Alzheimer-type neurodegeneration. Rats were made ischemic by cardiac arrest. Blood-brain barrier (BBB) insufficiency, accumulation of different parts of amyloid precursor protein (APP) and platelets inside and outside BBB vessels were investigated in ischemic brain up to 1-year survival. Ischemic brain tissue demonstrated haphazard BBB changes. Toxic fragments of APP deposits were associated with the BBB vessels. Moreover our study revealed platelet aggregates in- and outside BBB vessels. Toxic parts of APP and platelet aggregates correlated very well with BBB permeability. Progressive injury of the ischemic brain parenchyma may be caused not only by a degeneration of neurons destroyed during ischemia but also by chronic damage in BBB. Chronic ischemic BBB insufficiency with accumulation of toxic components of APP in the brain tissue perivascular space, may gradually over a lifetime, progress to brain atrophy and to full blown Alzheimer-type pathology.

Dysfunction of nitric oxide synthases as a cause and therapeutic target in delayed cerebral vasospasm after SAH.

Nitric oxide (NO), also known as endothelium-derived relaxing factor, is produced by endothelial nitric oxide synthase (eNOS) in the intima and by neuronal nitric oxide synthase (nNOS) in the adventitia of cerebral vessels. It dilates the arteries in response to shear stress, metabolic demands, pterygopalatine ganglion stimulation, and chemoregulation. Subarachnoid haemorrhage (SAH) interrupts this regulation of cerebral blood flow. Hemoglobin, gradually released from erythrocytes in the subarachnoid space destroys nNOS-containing neurons in the conductive arteries. This deprives the arteries of NO, leading to the initiation of delayed vasospasm. But such vessel narrowing increases shear stress, which stimulates eNOS. This mechanism normally would lead to increased production of NO and dilation of arteries. However, a transient eNOS dysfunction evoked by an increase of the endogenous competitive nitric oxide synthase (NOS) inhibitor, asymmetric dimethyl-arginine (ADMA), prevents this vasodilation. eNOS dysfunction has been recently shown to be evoked by increased levels of ADMA in CSF in response to the presence of bilirubin-oxidized fragments (BOXes). A direct cause of the increased ADMA CSF level is most likely decreased ADMA elimination due to the disappearance of ADMA-hydrolyzing enzyme (DDAH II) immunoreactivity in the arteries in spasm. This eNOS dysfunction sustains vasospasm. CSF ADMA levels are closely associated with the degree and time-course of vasospasm; when CSF ADMA levels decrease, vasospasm resolves. Thus, the exogenous delivery of NO, inhibiting the L-arginine-methylating enzyme (IPRMT3) or stimulating DDAH II, may provide new therapeutic modalities to prevent and treat vasospasm. This paper will present results of preclinical studies supporting the NO-based hypothesis of delayed cerebral vasospasm development and its prevention by increased NO availability.

Correlation of end-tidal CO2 with transcranial Doppler flow velocity is decreased during chemoregulation in delayed cerebral vasospasm after subarachnoid haemorrhage--results of a pilot study.

BACKGROUND: Cerebrovascular responses to variations in blood pressure and CO2 are attenuated during delayed vasospasm after subarachnoid hemorrhage (SAH). Transcranial Doppler sonography (TCD) is routinely used to assess the presence of vasospasm, but cerebral blood flow velocities (CBF-V) measured by TCD do not necessarily reflect cerebral blood flow (CBF) or the severity of vasospasm. We hypothesized that the correlation of end-tidal pCO2 levels with CBF-V and CBF is equally decreased in subjects with cerebral vasospasm during variations in pCO2. METHODS: Four cynomolgus monkeys were assigned to the vasospasm group and eight animals to the control group. The animals in the vasospasm group underwent placement of an autologous subarachnoid blood clot and vasospasm was confirmed by angiography on day 7. In both groups, CBF and CBF-V were measured simultaneously while end-tidal pCO2 was altered. CBF was measured using a thermal probe placed on the cortical surface and CBF-V was measured using a commercial TCD device. RESULTS: Pearson's correlation coefficient between CBF-V values and pCO2 levels in the control group was strong (r = 0.94, p < 0.001) while it was moderate in the vasospasm group (r = 0.54, p = 0.04). The correlation of CBF values with pCO2 in healthy controls was equally strong (r = 0.87, p = 0.005), while there was no correlation in the vasospasm group (r = -0.09, p = 0.83). CONCLUSION: In this pilot study, correlations of CBF-V with pCO2 values during chemoregulation testing were lower in animals with vasospasm than in healthy ones. This correlation coefficient based on modifications in pCO2 may potentially facilitate the non-invasive assessment of vasospasm.

Micro-blood-brain barrier openings and cytotoxic fragments of amyloid precursor protein accumulation in white matter after ischemic brain injury in long-lived rats.

Our study demonstrates that ischemia-reperfusion brain injury induces an increase in blood-brain barrier (BBB) permeability in the periventricular white matter. This chronic insufficiency of BBB may allow entry of neurotoxic fragments of amyloid precursor protein (APP) and other blood components such as platelets into the perineurovascular white matter tissue. These components may have secondary and chronic harmful effects on the ischemic myelin and axons and can intensify the phagocytic activity of microglial cells. Pathological accumulation of toxic fragments of APP in myelinated axons and oligodendrocytes appears after ischemic BBB injury and seem to be concomitant with, but independent of neuronal injury. It seems that ischemia-reperfusion disturbances may play important roles, both directly and indirectly, in the pathogenesis of white matter lesions. This pathology appears to have distribution similar to that of sporadic Alzheimer's disease. We noted micro-BBB openings in ischemic white matter lesions that probably would act as seeds of future Alzheimer's-type pathology.

Blood-brain barrier dysfunction and amyloid precursor protein accumulation in microvascular compartment following ischemia-reperfusion brain injury with 1-year survival.

This study examined the late microvascular consequences of brain ischemia due to cardiac arrest in rats. In reacted vibratome sections scattered foci of extravasated horseradish peroxidase were noted throughout the brain and did not appear to be restricted to any specific area of brain. Ultrastructural investigation of leaky sites frequently presented platelets adhering to the endothelium of venules and capillaries. Endothelial cells demonstrated pathological changes with evidence of perivascular astrocytic swelling. At the same time, we noted C-terminal of amyloid precursor protein/beta-amyloid peptide (CAPP/betaA) deposits in cerebral blood vessels, with a halo of CAPP/betaA immunoreactivity in the surrounding parenchyma suggested diffusion of CAPP/betaA out of the vascular compartment. Changes predominated in the hippocampus, cerebral and entorhinal cortex, corpus callosum, thalamus, basal ganglia and around the lateral ventricles. These data implicate delayed abnormal endothelial function of vessels following ischemia-reperfusion brain injury as a primary event in the pathogenesis of the recurrent cerebral infarction.

Mechanisms of the antioxidant effects of nitric oxide.

The Janus face of nitric oxide (NO) has prompted a debate as to whether NO plays a deleterious or protective role in tissue injury. There are a number of reactive nitrogen oxide species, such as N2O3 and ONOO-, that can alter critical cellular components under high local concentrations of NO. However, NO can also abate the oxidation chemistry mediated by reactive oxygen species such as H2O2 and O2- that occurs at physiological levels of NO. In addition to the antioxidant chemistry, NO protects against cell death mediated by H2O2, alkylhydroperoxides, and xanthine oxidase. The attenuation of metal/peroxide oxidative chemistry, as well as lipid peroxidation, appears to be the major chemical mechanisms by which NO may limit oxidative injury to mammalian cells. In addition to these chemical and biochemical properties, NO can modulate cellular and physiological processes to limit oxidative injury, limiting processes such as leukocyte adhesion. This review will address these aspects of the chemical biology of this multifaceted free radical and explore the beneficial effect of NO against oxidative stress.

Effects of nitric oxide on reactive oxygen species production and infarction size after brain reperfusion injury.

OBJECTIVE: Deleterious effects of strokes may be ameliorated when thrombolysis (i.e., with recombinant tissue plasminogen activator) restores circulation. However, reperfusion injury, mediated by oxygen free radicals (reactive oxygen species [ROS]), may limit the benefits of recombinant tissue plasminogen activator treatment. We hypothesized that, during reperfusion, exogenous nitric oxide (NO) would reduce stroke size by quenching ROS. METHODS: To investigate this hypothesis, we used two in vivo ischemia-reperfusion models, i.e., autologous cerebral embolism in rabbits and filament middle cerebral artery occlusion in rats. Using these models, we measured ROS levels (rabbit model) and stroke volumes (rat model) in response to transient ischemia, with and without intracarotid administration of ultrafast NO donor proline NO (proliNO). RESULTS: In the rabbit cerebral embolism model, intracarotid administration of proliNO (10(-6) mol/L) (n = 6) during reperfusion decreased free radical levels from 538 +/- 86 nmol/L in the vehicle-treated group (n = 7) to 186 +/- 31 nmol/L (2,3'-dihydroxybenzoic acid; P < 0.001) and from 521 +/- 86 nmol/L (n = 7) to 201 +/- 39 nmol/L (2,5'-dihydroxybenzoic acid; P < 0.002). In the rat middle cerebral artery occlusion model, intracarotid administration of proliNO (10(-5) mol/L) (n = 10) during reperfusion reduced the brain infarction volume from 256 +/- 48 mm3 in the vehicle-treated group (n = 8) to 187 +/- 41 mm3 (P < 0.005). In both experimental groups, intracarotid infusion of proliNO did not affect regional cerebral blood flow, mean arterial blood pressure, or brain and body temperatures. CONCLUSION: The beneficial effects of early restoration of cerebral circulation after cerebral ischemia were enhanced by intracarotid infusion of proliNO, most likely because of ROS scavenging by NO. These findings suggest the possibility of preventive treatment of reperfusion injury using NO donors.

Amyloid beta(1-42) and its beta(25-35) fragment induce in vitro phosphatidylcholine hydrolysis in bovine retina capillary pericytes.

We describe the inhibitory effect of full-length Abeta(1-42) and Abeta(25-35) fragment of amyloid-beta peptide on phosphatidylcholine (PtdCho) metabolism in bovine retina capillary pericytes. Cell cultures were incubated with Abetas for 24 h. Peroxidation indices (malondialdehyde and lactate dehydrogenase release) significantly increased after 20-50 microM Abeta(1-42) or Abeta(25-35) treatment. In addition, [Me-3H]choline incorporation into PtdCho strongly decreased while either 3H-choline or 14C-arachidonic acid release from prelabeled cells increased, indicating PtdCho hydrolysis. The effect was very likely due to prooxidant action of both Abeta peptides. Reversed-sequence Abeta(35-25) peptide did not depress 3H-choline incorporation nor stimulate PtdCho breakdown. With addition of Abetas at low concentrations (2-20 microM) to pericytes, marked ultrastructural changes, well connected to metabolic alterations, emerged including shrinkage of cell bodies, retraction of processes, disruption of the intracellular actin network. Cells treated with higher concentrations (50-200 microM) displayed characteristics of necrotic cell death. The data suggest that: (a) Abeta(1-42) and Abeta(25-35) peptides may modulate phospholipid turnover in microvessel pericytes; (b) together with endothelial cells, pericytes could be the target of vascular damage during processes involving amyloid accumulation.

Production of reactive oxygen species after reperfusion in vitro and in vivo: protective effect of nitric oxide.

OBJECT: Thrombolytic treatments for ischemic stroke can restore circulation, but reperfusion injury, mediated by oxygen free radicals, can limit their utility. The authors hypothesized that, during reperfusion, nitric oxide (NO) provides cytoprotection against oxygen free radical species. METHODS: Levels of NO and oxygen free radicals were determined in both reoxygenation in vitro and reperfusion in vivo models using an NO electrochemical probe and high-performance liquid chromatography with the 2,3- and 2,5-dihydroxybenzoic acid trapping method, before and after addition of the NO donor diethanolamine nitric oxide (DEA/NO). Reoxygenation after anoxia produced a twofold increase in NO release by human fetal astrocytes and cerebral endothelial cells (p < 0.005). In both cell lines, there was also a two- to threefold increase in oxygen free radical production (p < 0.005). In human fetal astrocytes and cerebral endothelial cells given a single dose of DEA/NO, free radical production dropped fivefold compared with peak ischemic levels (p < 0.001). In a study in which a rat global cerebral ischemia model was used, NO production in a vehicle-treated group increased 48 +/- 16% above baseline levels after reperfusion. After intravenous DEA/NO infusion, NO reached 1.6 times the concentration of the postischemic peak in vehicle-treated animals. In vehicle-treated animals during reperfusion, free radical production increased 4.5-fold over basal levels (p < 0.01). After intravenous DEA/NO infusion, free radical production dropped nearly 10-fold compared with peak levels in vehicle-treated animals (p < 0.006). The infarct volume in the vehicle-treated animals was 111 +/- 16.9 mm3; after DEA/NO infusion it was 64.8 +/- 23.4 mm3 (p < 0.01). CONCLUSIONS: The beneficial effect of early restoration of cerebral circulation after cerebral ischemia is limited by reperfusion injury. These results indicate that NO release and oxygen free radical production increase during reperfusion, and suggest a possible early treatment of reperfusion injury using NO donors.

The role of apolipoprotein E in the deposition of beta-amyloid peptide during ischemia-reperfusion brain injury. A model of early Alzheimer's disease.

Transient brain ischemia in the rat produces a stereotyped pattern of selective neuronal degeneration which simulates early Alzheimer's disease (AD) pathology. The aim of the present study was to determine if apolipoprotein E (ApoE) variables are related to alterations in other proteins which play a central role in the pathogenesis of AD; amyloid precursor protein (APP) and beta-amyloid peptide (A beta). The postischemic time course of ApoE and APP and A beta immunoreactivity in brain was examined at survival time from 2 days to 1 year in rats subjected to 10 min cardiac arrest. These data indicate that there are long lasting alterations of ApoE and A beta after brain ischemia. The most likely stimulus for promoting increase of both ApoE and A beta expression are ischemic-reperfusion processes. Our data suggest that ApoE modulates the outcome following cerebral ischemia via molecular events in common with AD pathogenesis. We propose that ischemic-reperfusion processes in brain are the fountain-head of a cycle of molecular and cellular events that have neurodegenerative consequences which finally lead to AD.

Neuroprotection by the stable nitroxide Tempol during reperfusion in a rat model of transient focal ischemia.

OBJECT: The use of thrombolytic agents in the treatment of stroke has yielded surprisingly modest success, possibly because of reperfusion injury mediated by reactive oxygen species (ROS). Therefore, scavenging ROS may be of therapeutic value in the treatment of stroke. Nitroxides are low-weight superoxide dismutase mimics, which allows them to act as cell-permeable antioxidants. In this study the nitroxide 4-hydroxy-2,2,6,6,-tetramethylpiperidine-1-oxyl (Tempol) is investigated to determine its ability to reduce reperfusion injury. METHODS: Male Sprague-Dawley rats weighing between 280 g and 350 g underwent middle cerebral artery occlusion with an intraluminal suture for 60 minutes. Regional cerebral blood flow, blood pressure, cerebral temperature, and rectal temperature were monitored during the procedure. After reperfusion, the animals were randomized to groups receiving blinded intravenous administration of either Tempol (10 mg/kg; eight animals) or vehicle (eight animals) over the first 20 minutes of reperfusion (Study I). In a second study to determine dose dependency, animals were randomized to groups receiving Tempol (20 mg/kg; eight animals), low-dose Tempol (5 mg/kg; eight animals), or vehicle (eight animals; Study II). The rats were killed after 4 hours of reperfusion, and brain sections were stained with 2,3,5 triphenyltetrazolium chloride. Infarct volumes were measured using digital imaging. Animals receiving Tempol had significantly reduced infarct volumes at doses of 20 mg/kg and 10 mg/kg compared with controls (49.01+/-18.22% reduction [p = 0.003] and 47.47+/-34.57 [p = 0.02], respectively). No significant differences in the physiological variables measured were observed between groups. CONCLUSIONS: Tempol provides significant neuroprotection after reperfusion in a rat model of transient focal ischemia. These results support the importance of ROS in reperfusion injury and encourage further study of this molecule as a therapeutic agent following thrombolysis.

Increased cerebral blood flow but no reversal or prevention of vasospasm in response to L-arginine infusion after subarachnoid hemorrhage.

OBJECT: The reduction in the level of nitric oxide (NO) is a purported mechanism of delayed vasospasm after subarachnoid hemorrhage (SAH). Evidence in support of a causative role for NO includes the disappearance of nitric oxide synthase (NOS) from the adventitia of vessels in spasm, the destruction of NO by hemoglobin released from the clot into the subarachnoid space, and reversal of vasospasm by intracarotid NO. The authors sought to establish whether administration of L-arginine, the substrate of the NO-producing enzyme NOS, would reverse and/or prevent vasospasm in a primate model of SAH. METHODS: The study was composed of two sets of experiments: one in which L-arginine was infused over a brief period into the carotid artery of monkeys with vasospasm, and the other in which L-arginine was intravenously infused into monkeys over a longer period of time starting at onset of SAH. In the short-term infusion experiment, the effect of a 3-minute intracarotid infusion of L-arginine (intracarotid concentration 10(-6) M) on the degree of vasospasm of the right middle cerebral artery (MCA) and on regional cerebral blood flow (rCBF) was examined in five cynomolgus monkeys. In the long-term infusion experiment, the effect of a 14-day intravenous infusion of saline (control group, five animals) or L-arginine (10(-3) M; six animals) on the occurrence and degree of cerebral vasospasm was examined in monkeys. The degree of vasospasm in all experiments was assessed by cerebral arteriography, which was performed preoperatively and on postoperative Days 7 (short and long-term infusion experiments) and 14 (long-term infusion experiment). In the long-term infusion experiment, plasma levels of L-arginine were measured at these times in the monkeys to confirm L-arginine availability. Vasospasm was not affected by the intracarotid infusion of L-arginine (shown by the reduction in the right MCA area on an anteroposterior arteriogram compared with preoperative values). However, intracarotid L-arginine infusion increased rCBF by 21% (p < 0.015; PCO2 38-42 mm Hg) in all vasospastic monkeys compared with rCBF measured during the saline infusions. In the long-term infusion experiment, vasospasm of the right MCA occurred with similar intensity with or without continuous intravenous administration of L-arginine on Day 7 and had resolved by Day 14. The mean plasma L-arginine level increased during infusion from 12.7+/-4 microg/ml on Day 0 to 21.9+/-13.1 microg/ml on Day 7 and was 18.5+/-3.1 microg/ml on Day 14 (p < 0.05). CONCLUSIONS: Brief intracarotid and continuous intravenous infusion of L-arginine did not influence the incidence or degree of cerebral vasospasm. After SAH, intracarotid infusion of L-arginine markedly increased rCBF in a primate model of SAH. These findings discourage the use of L-arginine as a treatment for vasospasm after SAH.

Possible reverse transport of beta-amyloid peptide across the blood-brain barrier.

Our experiments were performed to test the hypothesis that human beta-amyloid peptide 42 (beta A) is able to enter and exit the brain parenchyma through the blood-brain barrier. In an effort to determine the effect of beta A in an animal model, we have injected beta A i.v. into rats following single and repeated brain ischemia. Rats were sacrificed at 3 and 12 months after injection and beta A was localized by monoclonal antibody (mAb) 4G8. The present observations revealed an abundant presence of beta A in the extracellular space of the brain, which appeared to be dilated, and a vigorous uptake of beta A into the cytoplasm of endothelial and ependymal cells, pericytes, astrocytes and neurons. Some of the beta A deposits were associated and/or had migrated to the vessels and to the ventricles, and by 3 months a significant amount of beta A was directly associated with the vessels and was observed inside the ventricular space. Virtually no soluble and aggregating beta A was found in brain tissue 1 year later. This suggests that phagocytic pericytes and astrocytes take up exogenous beta A in an attempt to clear the peptide from the brain extracellular space and deliver it to the circulation. Further, direct removal of beta A from the ventricles by the bloodstream is also possible. These observations suggest that a reverse transport of beta A across endothelial cells of microvessels represents one of the possible mechanisms responsible for removal of extravasated beta A. The findings of the present study indicate that in normal conditions beta A is rapidly cleared from the cerebrospinal fluid and brain parenchyma, suggesting that irreversible changes in the physico-chemical properties of the cerebrovascular endothelial cell surface are involved in beta A deposition in the brain in Alzheimer's disease (AD).

No effect of anti-oxidative therapy on cerebral amyloidosis following ischemia-reperfusion brain injury.

The distribution patterns of amyloid precursor protein (APP) fragments were studied immunocytochemically in the rat brain before, after 10 min ischemia and following treatment by idebenone. Six months after brain ischemia intense staining for APP appeared in extra- and intracellular space. These findings indicate that APP is involved in the degeneration process of brain neuronal and glial cells following ischemia-reperfusion injury and anti-oxidative therapy did not prevent and/or stop this phenomenon.

Nitrosative capacity of macrophages is dependent on nitric-oxide synthase induction signals.

Nitrosative stress can occur when reactive nitric oxide (NO) species compromise the function of biomolecules via formation of NO adducts on critical amine and thiol residues. The capacity of inducible nitric-oxide synthase (iNOS) to generate nitrosative stress was investigated in the murine macrophage line ANA-1. Sequential activation with the cytokines IFN-gamma and either tumor necrosis factor-alpha or interleukin-1beta resulted in the induction of iNOS and production of nitrite (20 nM/min) but failed to elicit nitrosation of extracellular 2,3-diaminonapthalene. Stimulation with IFN-gamma and bacterial lipopolysaccharide increased the relative level of iNOS protein and nitrite production of ANA-1 cells 2-fold; however, a substantial level of NO in the media was also observed, and nitrosation of 2,3-diaminonapthalene was increased greater than 30-fold. Selective scavenger compounds suggested that the salient nitrosating mechanism was the NO/O(2) reaction leading to N(2)O(3) formation. These data mimicked the pattern observed with a 5 microM concentration of the synthetic NO donor (Z)-1-[N-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium -1,2-diolate (PAPA/NO). The NO profiles derived from iNOS can be distinct and depend on the inductive signal cascades. The diverse consequences of NO production in macrophages may reside in the cellular mechanisms that control the ability of iNOS to form N(2)O(3) and elicit nitrosative stress.

Hypoxia modulates nitric oxide-induced regulation of NMDA receptor currents and neuronal cell death.

Nitric oxide (NO) released from a new chemical class of donors enhances N-methyl-D-aspartate (NMDA) channel activity. Using whole cell and single-channel patch-clamp techniques, we have shown that (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]-NO (PAPA-NO) and diethylamine NO, commonly termed NONOates, potentiate the glutamate-mediated response of recombinant rat NMDA receptors (NR1/NR2A) expressed in HEK-293 cells. The overall effect is an increase in both peak and steady-state whole cell currents induced by glutamate. Single-channel studies demonstrate a significant increase in open probability but no change in the mean single-channel open time or mean channel conductance. Reduction in oxygen levels increased and prolonged the PAPA-NO-induced change in both peak and steady-state glutamate currents in transfected HEK cells. PAPA-NO also enhanced cell death in primary cultures of rodent cortical neurons deprived of oxygen and glucose. This potentiation of neuronal injury was blocked by MK-801, indicating a critical involvement of NMDA receptor activation. The NO-induced increase in NMDA channel activity as well as NMDA receptor-mediated cell death provide firm evidence that NO modulates the NMDA channel in a manner consistent with both a physiological role under normoxic conditions and a pathophysiological role under hypoxic conditions.