Effects of nitric oxide on red blood cell deformability

In addition to its known action on vascular smooth muscle, nitric oxide (NO) has been suggested to have cardiovascular effects via regulation of red blood cell (RBC) deformability. The present study was designed to further explore this possibility. Human RBCs in autologous plasma were incubated for 1 h with NO synthase (NOS) inhibitors [N(omega)-nitro-l-arginine methyl ester (l-NAME) and S-methylisothiourea], NO donors [sodium nitroprusside (SNP) and diethylenetriamine (DETA)-NONOate], an NO precursor (l-arginine), soluble guanylate cyclase inhibitors (1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one and methylene blue), and a potassium channel blocker [triethylammonium (TEA)]. After incubation, RBC deformability at various shear stresses was determined by ektacytometry. Both NOS inhibitors significantly reduced RBC deformability above a threshold concentration, whereas the NO donors increased deformability at optimal concentrations. NO donors, as well as the NO precursor l-arginine and the potassium blocker TEA, were able to reverse the effects of NOS inhibitors. Guanylate cyclase inhibition reduced RBC deformation, with both SNP and DETA-NONOate able to reverse this effect. These results thus indicate the importance of NO as a determinant of RBC mechanical behavior and suggest its regulatory role for normal RBC deformability.     

Direct chemiluminescence detection of nitric oxide in aqueous solutions using the natural nitric oxide target soluble guanylyl cyclase

Nitric oxide (NO) is a free radical involved in many physiological processes including regulation of blood pressure, immune response, and neurotransmission. However, the measurement of extremely low, in some cases subnanomolar, physiological concentrations of nitric oxide presents an analytical challenge. The purpose of this methods article is to introduce a new highly sensitive chemiluminescence approach to direct NO detection in aqueous solutions using a natural nitric oxide target, soluble guanylyl cyclase (sGC), which catalyzes the conversion of guanosine triphosphate to guanosine 3′,5′-cyclic monophosphate and inorganic pyrophosphate. The suggested enzymatic assay uses the fact that the rate of the reaction increases by about 200 times when NO binds with sGC and, in so doing, provides a sensor for nitric oxide. Luminescence detection of the above reaction is accomplished by converting inorganic pyrophosphate into ATP with the help of ATP sulfurylase followed by light emission from the ATP-dependent luciferin–luciferase reaction. Detailed protocols for NO quantification in aqueous samples are provided. The examples of applications include measurement of NO generated by a nitric oxide donor (PAPA-NONOate), nitric oxide synthase, and NO gas dissolved in buffer. The method allows for the measurement of NO concentrations in the nanomolar range and NO generation rates as low as 100 pM/min.     

Modulation of endothelial nitric oxide synthase expression by red blood cell aggregation

The effects of enhanced red blood cell (RBC) aggregation on nitric oxide (NO)-dependent vascular control mechanisms have been investigated in a rat exchange transfusion model. RBC aggregation for cells in native plasma was increased via a novel method using RBCs covalently coated with a 13-kDa poloxamer copolymer (Pluronic F-98); control experiments used RBCs coated with a nonaggregating 8.4-kDa poloxamer (Pluronic F-68). Rats exchange transfused with aggregating RBC suspensions demonstrated significantly enhanced RBC aggregation throughout the 5-day follow-up period, with mean arterial blood pressure increasing gradually over this period. Arterial segments ( approximately 300 microm in diameter) were isolated from gracilis muscle on the fifth day and mounted between two glass micropipettes in a special chamber equipped with pressure servo-control system. Dose-dependent dilation by ACh and flow-mediated dilation of arterial segments pressurized to 30 mmHg and preconstricted to 45-55% of the original diameter by phenylephrine were significantly blunted in rats with enhanced RBC aggregation. Both responses were totally abolished by nonspecific NO synthase (NOS) inhibitor (Nomega-nitro-l-arginine methyl ester) treatment of arterial segments, indicating that the responses were NO related. Additionally, expression of endothelial NOS protein was found to be decreased in muscle samples obtained from rats exchanged with aggregating cell suspensions. These results imply that enhanced RBC aggregation results in suppressed expression of NO synthesizing mechanisms, thereby leading to altered vasomotor tonus; the mechanisms involved most likely relate to decreased wall shear stresses due to decreased blood flow and/or increased axial accumulation of RBCs.     

Stimulation of cystine uptake by nitric oxide: regulation of endothelial cell glutathione levels

Nitric oxide (NO)is known to produce some of its biological activity throughmodification of cellular thiols. Return of cellular thiols to theirbasal state requires the activity of the GSH redox cycle, suggestingimportant interactions between NO signaling and regulation of cellularredox status. Because continuous exposure to NO may lead to adaptiveresponses in cellular redox systems, we investigated the effects of NOon cellular GSH levels in vascular endothelial cells. Acute exposure (1 h) of cells to >1 mMS-nitroso-N-acetyl-penicillamine (SNAP) led to depletion of GSH. On the other hand, chronic exposure tolower concentrations of SNAP (1 mM) led to a progressive increase incytosolic GSH, reaching fourfold above basal by 16 h. The mechanism mayinvolve an increase in GSH biosynthesis through effects on biosyntheticenzymes or through increased supply of cysteine, the limitingsubstrate. In this regard, we report that chronic exposure to SNAP ledto a concentration-dependent increase in cystine uptake over a timecourse similar to that seen for elevation of GSH. The effect of SNAP oncystine uptake was inhibitable by either cycloheximide or actinomycinD, suggesting a requirement for both RNA and protein synthesis.Furthermore, uptake was Na⁺independent and was blocked by extracellular glutamate. Extracellular glutamate also blocked SNAP-mediated elevation of cytosolic GSH. Finally, in a coculture model, NO produced by cytokine-pretreated RAW264.7 cells increased both GSH levels and cystine uptake in naiveendothelial cells. These findings strongly suggest that NO leads toadaptive induction of thex_c amino acidtransport system, increased cystine uptake, and elevation ofintracellular GSH levels.     

Active nitric oxide produced in the red cell under hypoxic conditions by deoxyhemoglobin-mediated nitrite reduction

Recent studies have generated a great deal of interest in a possible role for red blood cells in the transport of nitric oxide (NO) to the microcirculation and the vascular effect of this nitric oxide in facilitating the flow of blood through the microcirculation. Many questions have, however, been raised regarding such a mechanism. We have instead identified a completely new mechanism to explain the role of red cells in the delivery of NO to the microcirculation. This new mechanism results in the production of NO in the microcirculation where it is needed. Nitrite produced when NO reacts with oxygen in arterial blood is reutilized in the arterioles when the partial pressure of oxygen decreases and the deoxygenated hemoglobin formed reduces the nitrite regenerating NO. Nitrite reduction by hemoglobin results in a major fraction of the NO generated retained in the intermediate state where NO is bound to Hb(III) and in equilibrium with the nitrosonium cation bound to Hb(II). This pool of NO, unlike Hb(II)NO, is weakly bound and can be released from the heme. The instability of Hb(III)NO in oxygen and its displacement when flushed with argon requires that reliable determinations of red blood cell NO must be performed on freshly lysed samples without permitting the sample to be oxygenated. In fresh blood samples Hb(III)NO accounts for 75% of the red cell NO with appreciably higher values in venous blood than arterial blood. These findings confirm that nitrite reduction at reduced oxygen pressures is a major source for red cell NO. The formation and potential release from the red cell of this NO could have a major impact in regulating the flow of blood through the microcirculation.     

Androgen-dependent nitric oxide release in rat penis correlates with levels of constitutive nitric oxide synthase isoenzymes.

Androgens are known to influence penile erection and nitric oxide synthase (NOS) activity in cavernosal tissue homogenates. The present study was an assessment of the effects of castration and androgen replacement on the in vivo release of nitric oxide (NO), and of the simultaneously recorded intracavernosal pressure (ICP) changes elicited by electrostimulation of the cavernosal nerves (SCN) in the anesthetized rat. The extracellular levels of NO in the corpora were monitored electrochemically using porphyrin microsensors. The content of NOS isoenzymes in corporal homogenates was determined by immunoblotting. The responses of castrated rats with or without testosterone (T) implants were compared to those of intact animals. Castration virtually abolished both the NO and the ICP responses to SCN. There was a concomitant significant decrease in the content of both the neuronal (nNOS) and the endothelial (eNOS) isoenzymes in the cavernosal tissue. All these effects of castration were prevented by T replacement. The NO response to SCN was positively correlated with the levels of nNOS and eNOS, especially when the values of the two isoforms were added (r = 0.71, P < 0.001). These data suggest that the facilitatory action of androgens on penile erection involves the up-regulation of both constitutive NOS isoenzymes in the corpora cavernosa.     

Role of hemoglobin oxygenation in the modulation of red blood cell mechanical properties by nitric oxide

It has been previously demonstrated that both externally generated and internally synthesized nitric oxide (NO) can affect red blood cell (RBC) deformability. Further studies have shown that the RBC has active NO synthesizing mechanisms and that these mechanisms may play role in maintaining normal RBC mechanical properties. However, hemoglobin within the RBC is known to be a potent scavenger of NO; oxy-hemoglobin scavenges NO faster than deoxy-hemoglobin via the dioxygenation reaction to nitrate. The present study aimed at investigating the role of hemoglobin oxygenation in the modulation of RBC rheologic behavior by NO. Human blood was obtained from healthy volunteers, anticoagulated with sodium heparin (15 IU/mL), and the hematocrit was adjusted to 0.4 L/L by adding or removing autologous plasma. Several two mL aliquots of blood were equilibrated at room temperature (22 ± 2 °C) with moisturized air or 100% nitrogen by a membrane gas exchanger, The NO donor sodium nitroprusside (SNP), at a concentration range of 10^?7–10^?4 M, was added to the equilibrated aliquots which were maintained under the same conditions for an additional 60 min. The effect of the non-specific NOS inhibitor l-NAME was also tested at a concentration of 10^?3 M. RBC deformability was measured using an ektacytometer with an environment corresponding to that used for the prior incubation (i.e., oxygenated or deoxygenated). Our results indicate an improvement of RBC deformability with the NO donor SNP that was much more pronounced in the deoxygenated aliquots. SNP also had a more pronounced effect on RBC aggregation for deoxygenated RBC. Conversely, l-NAME had no effect on deoxygenated blood but resulted in impaired deformability, with no change in aggregation for oxygenated blood. These findings can be explained by a differential behavior of hemoglobin under oxygenated and deoxygenated conditions; the influence of oxygen partial pressure on NOS activity may also play a role. It is therefore critical to consider the oxygenation state of intracellular hemoglobin while studying the role of NO as a regulator of RBC mechanical properties.     

Detection of human red blood cell-bound nitric oxide

Major disparities in reported levels of basal human nitric oxide metabolites have resulted in a recent literature focusing almost exclusively on methods. We chose to analyze triiodide chemiluminescence, drawn by the prospect of identifying why the most commonly employed assay in nitric oxide biology typically yielded lower metabolite values, compared with several other techniques. We found that the sensitivity of triiodide was greatly affected by the auto-capture of nitric oxide by deoxygenated cell-free heme in the reaction chamber. Potential contaminants and signal losses were also associated with standard sample purification procedures and the chemistry involved in nitrite removal. To inhibit heme nitric oxide auto-capture, we added potassium ferricyanide to the triiodide reagent, reasoning this would provide a more complete detection of any liberated nitric oxide. From human venous blood samples, we established nitric oxide levels ranging from 0.000178 to 0.00024 mol nitric oxide/mol hemoglobin. We went on to find significantly elevated nitric oxide levels in venous blood taken from diabetic patients in comparison to healthy controls (p < 0.0001). We concluded that the lack of signals reported of late by several groups using triiodide chemiluminescence for the detection of hemoglobin-bound nitric oxide may not represent levels on the border of assay sensitivity but rather underestimated values because of methodological limitations. We therefore stress the need for assay systems to be developed that differentiate between individual nitric oxide metabolite species and overcome the limitations we outline, allowing accurate conclusions to be drawn regarding physiological nitric oxide metabolite levels.     

Measurement of acetylcholine-induced endothelium-derived nitric oxide in aorta using a newly developed catheter-type nitric oxide sensor

Intra-aortic measurement of nitric oxide (NO) would provide valuable insights into NO bioavailability in systemic circulation and vascular endothelial function. In the present study, we thus developed a catheter-type NO sensor to measure intra-aortic NO concentration in vivo. An NO sensor was encased and fixed in a 4-Fr catheter. The sensor was then located in the thoracic aorta via the femoral artery through a 7-Fr catheter to measure intra-aortic plasma NO concentration in vivo in anesthetized dogs. Infusion of acetylcholine (10 microg/kg) increased base-to-peak plasma NO level in the aorta by 2.4+/-0.4 nM (n=7). After 20-min infusion of N(G)-methyl-L-arginine (NO synthase inhibitor), changes in plasma NO concentration in response to acetylcholine were attenuated significantly (1.8+/-0.4 nM, P<0.003, n=7). In conclusion, the newly developed catheter-type NO sensor successfully measured acetylcholine-induced changes in intra-aortic plasma concentration of endothelium-derived NO in vivo and demonstrated applicability to direct evaluation of intravascular NO bioavailability.     

Endothelial nitric oxide synthase gene haplotypes and circulating nitric oxide levels significantly associate with risk of essential hypertension

Nitric oxide (NO), a potent vasodilator, plays a pivotal role in blood pressure regulation. Endothelial NO synthase gene (NOS3) polymorphisms influence NO levels. Here, we investigated the role of the – 922A/G, – 786T/C, 4b/4a, and 894G/T polymorphisms of the NOS3 and NO_x levels in 800 consecutive unrelated subjects comprising 455 patients of essential hypertension and 345 controls. The polymorphisms were investigated independently and as haplotypes. Plasma NO_x levels (nitrate and nitrite) were estimated by the Griess method. Genotype frequencies for the –786T/C, 4b/4a, and 894G/T polymorphisms differed significantly (P < 0.001) between patients and controls and were associated with an increased risk of hypertension (OR = 2.0, OR = 3.8, OR = 1.6, respectively). The 4-locus haplotypes ATaG (H1), ATaT (H2), and GCaG (H3) were significantly associated with essential hypertension and served as susceptible haplotypes (P ≤ 0.0001). On the other hand, haplotypes ATbG (H4) and GTbG (H5) were negatively associated with hypertension and served as protective haplotypes (P < 0.0001). NO_x levels were significantly lower in patients than controls (P < 0.0001). The individual polymorphisms showed marginal association with NO_x level; however, the susceptible haplotype H2 associated significantly with lower NO_x levels in patients (P < 0.001) and conversely the haplotype H4 with higher NO_x levels in controls (P < 0.001). In conclusion, the 4b/4a and likely – 786T/C polymorphisms were identified as the determinants modifying the risk of hypertension. This study identifies the NOS3 variants and haplotypes as genetic risk factors and as useful markers of increased susceptibility to the risk of essential hypertension.     

Evaluation of nitric oxide synthase activity and inhibition kinetics by chemiluminescence.

The binding affinity (K(I)) and inactivation rate (k(inact)) parameters of nitric oxide synthase (NOS) inhibitors are typically estimated by kinetic activity studies. Methods currently used in the estimation of these parameters frequently employ radiolabeled materials and require intensive sample preparation. We have devised a simple, reproducible, and sensitive method for the kinetic analysis of NOS activity and inhibition kinetics using chemiluminescence. We have used this method to characterize enzyme activity for purified murine macrophage nitric oxide synthase (NOS II). Using this method, we have also estimated the inhibitory parameters for a series of competitive antagonists and mechanism-based inactivators of NOS II. The estimated parameters are in agreement with those reported using other methods. We conclude that the chemiluminescence method can be used for kinetic studies of NOS activity and inhibition. This method represents a more efficient means for conducting kinetic studies of NOS inhibition.     

Podokinesis in endothelial cell migration: role of nitric oxide

Previously, we demonstrated the role of nitric oxide (NO) intransforming epithelial cells from a stationary to locomoting phenotype[E. Noiri, T. Peresleni, N. Srivastava, P. Weber, W. F. Bahou, N. Peunova, and M. S. Goligorsky. Am. J. Physiol. 270 (CellPhysiol. 39): C794-C802, 1996] and itspermissive function in endothelin-1-stimulated endothelial cellmigration (E. Noiri, Y. Hu, W. F. Bahou, C. Keese, I. Giaever, and M. S. Goligorsky. J. Biol. Chem. 272:1747-1753, 1997). In the present study, the role of functional NOsynthase in executing the vascular endothelial growth factor(VEGF)-guided program of endothelial cell migration and angiogenesiswas studied in two independent experimental settings. First, VEGF,shown to stimulate NO release from simian virus 40-immortalized microvascular endothelial cells, induced endothelial cell transwell migration, whereasN^G-nitro-L-arginine methyl ester(L-NAME) or antisenseoligonucleotides to endothelial NO synthase suppressed this effect ofVEGF. Second, in a series of experiments on endothelial cell woundhealing, the rate of VEGF-stimulated cell migration was significantlyblunted by the inhibition of NO synthesis. To gain insight into thepossible mode of NO action, we next addressed the possibility that NOmodulates cell matrix adhesion by performing impedance analysis ofendothelial cell monolayers subjected to NO. The data showed thepresence of spontaneous fluctuations of the resistance in ostensiblystationary endothelial cells. Spontaneous oscillations were induced byNO, which also inhibited cell matrix adhesion. This process we propose to term "podokinesis" to emphasize a scalar form ofmicromotion that, in the presence of guidance cues, e.g., VEGF, istransformed to a vectorial movement. In conclusion, execution of theprogram for directional endothelial cell migration requires twocoexisting messages: NO-induced podokinesis (scalar motion) andguidance cues, e.g., VEGF, which imparts a vectorial component to themovement. Such a requirement for the dual signaling may explain amismatch in the demand and supply with newly formed vessels indifferent pathological states accompanied by the inhibition of NOsynthase.

     

Visible chemiluminescence induced by t-butyl hydroperoxide in red blood cell suspensions

Red blood cells from Wistar rats were exposed to milimolar concentrations of t-butyl hydroperoxide. Extensive hemoglobin oxidation (methemoglobin formation), t-butyl hydroperoxide cleavage (t-butanol formation) and peroxidation (measured by oxygen consumption and thiobarbituric acid reactive substances) was observed. Significant chemiluminescence was emitted by the system. Hemoglobin oxidation and t-butanol production were independent of oxygen pressure and free radical scavengers, however, luminescence was enhanced as oxygen pressure increased and it was reduced by addition of free radical scavengers. The spectral distribution of the light emitted suggests that the luminescence detected is not due to singlet oxygen dimol emission. The results are in agreement with a lipid peroxidative mechanism initiated by t-butoxy radicals produced in the interaction of hemoglobin and t-butyl hydroperoxide.     

Upregulation of neuronal nitric oxide synthase, edema and cell injury following heat stress are reduced by pretreatment with EGB-761 in the rat

Rats exposed to 4 h heat stress (HS) at 38°C exhibited marked upregulation of neuronal nitric oxide synthase (nNOS) in the brain regions exhibiting blood–brain barrier (BBB) breakdown, brain edema and cell damage. Pretreatment with an anti-oxidant compound EGB-761 (an extract of Gingko biloba) administered 50 mg/kg, per os for 5 days, significantly attenuated nNOS expression, BBB disruption, brain edema and cell injury. These results suggest that EGB-761 is neuroprotective in heat stress and this effect of the compound is related with the inhibition of NOS expression, not reported earlier.     

Mitochondrial nitric oxide in the signaling of cell integrated responses

Mitochondria are the specialized organelles for energy metabolism, but, as a typical example of system biology, they also activate a multiplicity of pathways that modulate cell proliferation and mitochondrial biogenesis or oppositely promote cell arrest and programmed cell death by a limited number of oxidative or nitrosative reactions. These reactions are influenced by matrix nitric oxide (NO) steady-state concentration, either from local production or by gas diffusion to mitochondria from the canonical sources. Likewise, in a range of 30–200 nM, NO turns mitochondrial O₂ utilization down by binding to cytochrome oxidase and elicits a burst of superoxide anion and hydrogen peroxide that diffuses outside mitochondria. Depending on NO levels and antioxidant defenses, more or less H₂O₂ accumulates in cytosol and nucleus, and the resulting redox grading contributes to dual activation of proliferating and proapoptotic cascades, like ERK1/2 or p38 MAPK. Moreover, these sequential activating pathways participate in rat liver and brain development and in thyroid modulation of mitochondrial metabolism and contribute to hypothyroid phenotype through complex I nitration. On the contrary, lack of NO disrupts pathways like S-nitrosylation or H₂O₂ production and likewise is a gateway to disease in amyotrophic lateral sclerosis with superoxide dismutase 1 mutations or to cancer proliferation. peroxynitrite; hydrogen peroxide; mitochondrial nitric oxide synthase; mitogen-activated protein kinase     

Activation of the human red cell calcium ATPase by calcium pretreatment

Some kinetic parameters of the human red cell Ca²⁺-ATPase were studied on calmodulin-free membrane fragments following preincubation at 37°C. After 30 min treatment with EGTA(1 mm) plus dithioerythritol (1 mm), a V _max of about 0.4 μmol P_i/mg × hr and a K _s of 0.3 μm Ca²⁺ were found. When Mg²⁺ (10 mm) or Ca²⁺(10 μm) were also added during preincubation, V _maxbut not Kwas altered. Ca²⁺ was more effective than Mg²⁺, thus increasing V _max to about 1.3 μmol P_i/mg × hr. The presence of both Ca²⁺ and Mg²⁺ during pretreatment decreasedKto 0.15 μm, while having no apparent effect on V _max. Conversely, addition of ATP (2 mm) with either Ca²⁺ or Ca²⁺ plus Mg²⁺increased V_max without affecting K. Preincubation with Ca²⁺ for periods longer than 30 min further increased V_maxand reduced Kto levels as low as found with calmodulin treatment. The Ca²⁺ activation was not prevented by adding proteinase inhibitors (iodoacetamide, 10 mm; leupeptin, 200 μm; pepstatinA, 100 μm; phenylmethanesulfonyl fluoride, 100 μm). The electrophoretic pattern of membranes preincubated with or without Mg²⁺, Ca²⁺ or Ca²⁺ plus Mg²⁺ did not differ significantly from each other. Moreover, immunodetection of Ca²⁺-ATPase by means of polyclonal antibodiesrevealed no mobility change after the various treatments. The above stimulation was not altered by neomycin (200 μm), washing with EGTA (5 mm) or by both incubating and washing with delipidized serum albumin (1 mg/ml), or omitting dithioerythritol from the preincubation medium. On the other hand, the activation elicited by Ca²⁺ plus ATP in the presence of Mg²⁺ was reduced 25–30% by acridine orange (100 μm), compound 48/80 (100 μm) or leupeptin (200 μm) but not by dithio-bis-nitrobenzoic acid (1 mm). The fluorescence depolarization of 1,6-diphenyl-and l-(4-trimethylammonium phenyl)-6-phenyl 1,3,5-hexatriene incorporated into membrane fragments was not affected after preincubating under the different conditions. The results show that proteolysis, fatty acid production, an increased phospholipid metabolism or alteration of membrane fluidity are not involved in the Ca²⁺ effect. Ca²⁺ preincubation may stimulate the Ca²⁺-ATPase activity by stabilizing or promoting the E₁ conformation.     

Compromised proteasome degradation elevates neuronal nitric oxide synthase levels and induces apoptotic cell death

The significance of impairment of proteasome activity in PC12 cells was examined in connection with nitrative/nitrosative stress and apoptotic cell death. Treatment of differentiated PC12 cells with MG132, a proteasome inhibitor, elicited a dose- and time-dependent increase in neuronal nitric oxide synthase (nNOS) protein levels, decreased cell viability, and increased cytotoxicity. Viability and cytotoxicity were ameliorated by L-NAME (a broad NOS inhibitor). Nitric oxide/peroxynitrite formation was increased upon treatment of PC12 cells with MG132 and decreased upon treatment with the combination of MG132 and 7-NI (a specific inhibitor of nNOS). The decreases in cell viability appeared to be effected by an activation of JNK and its effect on mitochondrial Bcl-x_L phosphorylation. These effects are strengthened by the activation of caspase-9 along with increased caspase-3 activity upon treatment of PC12 cells with MG132. These results suggest that impairment of proteasome activity and consequent increases in nNOS levels lead to a nitrative stress that involves the coordinated response of JNK cytosolic signaling and mitochondrion-driven apoptotic pathways.     

Myristoylation of endothelial cell nitric oxide synthase is important for extracellular release of nitric oxide

Endothelial cell nitric oxide synthase (NOS) is known to have a N-myristoylation consensus sequence. Such a consensus sequence is not evident in the macrophage, smooth muscle and neuronal NOS. A functional role for this N-terminal myristoylation is not clear yet. In the present study, we examined the effect of N-terminal myristoylation on the NOS activity determined by the conversion of L-[³H]arginine to L-[³H]citrulline and extracellular NO release determined by nitrite production in the conditioned medium from the COS-7 cells transfected with wild type bovine aortic endothelial cell (BAEC) NOS cDNA or nonmyristoylated BAEC-NOS mutant cDNA. NOS activity of wild type BAEC-NOS in COS-7 cells was localized in the particulate fraction and that of mutant NOS was in the cytosolic fraction. In contrast, nitrite production from COS-7 cells transfected with wild type BAEC-NOS cDNA was greater than that of mutant cDNA in a time dependent and a concentration dependent manner. These results suggest that membrane localization of NOS with myristoylation facilitates extracellular transport of NO and leads to enhanced NO signaling on the vascular smooth muscle cells and the intravascular blood cells including neutrophils, macrophages and platelets.     

Humic acid induces the generation of nitric oxide in human umbilical vein endothelial cells: stimulation of nitric oxide synthase during cell injury

Humic acid (HA) has been implicated as an etiological factor in the peripheral vasculopathy of blackfoot disease (BFD). In this study, we examined the effects of HA upon the generation of nitric oxide (NO) during the process of lethal cell injury in cultured human umbilical vein endothelial cells (HUVECs). NO production was measured by the formation of nitrite (NO(2)(-)), the stable end-metabolite of NO. Cell death was assessed by measuring the release of intracellular lactate dehydrogenase (LDH). Treatment HUVECs with HA at a concentration of 50, 100, and 200 microg/ml concentration-dependently increased nitrite levels, reaching a peak at 12 h subsequent to HA treatment, with a maximal response of approximately 400 pmole nitrite (from 1 x 10(4) cells). HA-induced nitrite formation was blocked completely by N(G)-nitro-L-arginine methyl ester (L-NAME) and also by N(G)-methyl-L-arginine (L-NMA), both being specific inhibitors of NO synthase. The LDH released from endothelial cells was evoked at from 24 h after the addition of HA (50, 100, 200 microg/ml) in a concentration- and time-dependent manner. The HA-induced LDH release was also reduced by the presence of both L-NAME and L-NMA. The addition of Ca(2+) chelator (BAPTA) inhibited both nitrite formation and LDH release by HA. Moreover, the antioxidants (superoxide dismutase, vitamin C, vitamin E) and protein kinase inhibitor (H7) effectively suppressed HA-induced nitrite formation. These results suggest that HA treatment of endothelial cells stimulates NO production, which can elicit cell injury via the stimulation of Ca(2+)-dependent NO synthase activity by increasing cytosolic Ca(2+) levels. Because the destruction of endothelial cells has been implicated in triggering the onset of BFD, the induction of excessive levels of NO and consequent endothelial-cell injury may be important to the etiology of HA-induced vascular disorders associated with BFD for humans.