Glucose scavenging of nitric oxide
Sergey V.
Brodsky1,
Albert Marcus
Morrishow2,
Nimish
Dharia1,
Steven S.
Gross2, and
Michael S.
Goligorsky1
1 Program in Biomedical Engineering, Departments of Medicine and
Physiology, State University of New York, Stony Brook 11794; and
2 Program in Biochemistry and Structural Biology, Department
of Pharmacology and Weill Medical College of Cornell University, New
York 10021
 |
ABSTRACT |
Endothelial dysfunction
accompanies suboptimal glucose control in patients with diabetes
mellitus. A hallmark of endothelial dysfunction is a deficiency in
production or bioavailability of vascular nitric oxide (NO). Here we
demonstrate that acute exposure of human endothelial cells to glucose,
at levels found in plasma of diabetic patients, results in a
significant blunting of NO responses to the endothelial nitric oxide
synthase (eNOS) agonists bradykinin and A-23187. Monitoring of NO
generation by purified recombinant bovine eNOS in vitro, using
amperometric electrochemical detection and an NO-selective porphyrinic
microelectrode, showed that glucose causes a progressive and
concentration-dependent attenuation of detectable NO. Addition of
glucose to pure NO solutions similarly elicited a sharp decrease in NO
concentration, indicating that glucose promotes NO loss. Electrospray
ionization-tandem mass spectrometry, using negative ion monitoring,
directly demonstrated the occurrence of a covalent reaction involving
unitary addition of NO (or a derived species) to glucose. Collectively,
our findings reveal that hyperglycemia promotes the chemical
inactivation of NO; this glucose-mediated NO loss may directly
contribute to hypertension and endothelial dysfunction in diabetic patients.
diabetes; endothelial dysfunction; vasculopathy; nitric oxide
synthase; electrospray ionization-mass spectrometry
 |
INTRODUCTION |
DEVELOPMENT OF
MICROVASCULAR and macrovascular complications in diabetic
patients has been linked to the quality of glycemic control (8,
22, 31). The finding that blood pressure is elevated in normal
subjects during hyperglycemic clamp (14) indicates that
elevated glucose can directly trigger vasoconstriction in diabetic
patients. Although a dysfunction in nitric oxide (NO) biosynthesis or
bioactivity is strongly implicated in the pathogenesis of diabetic
vasculopathy (4, 18, 25, 26), the mechanistic basis for NO
dysfunction is uncertain.
One possibility is that the chronic vasoconstrictive effect of
hyperglycemia results from accumulation of advanced glycation end
products (AGE) in the subendothelial compartment, leading to NO
quenching and a loss of NO-induced smooth muscle vasorelaxation (3); this scenario is, perhaps, not the only one. An
additional mode of glucose action is suggested by the observation that
endothelium-dependent vasodilatation is already impaired within 15 min
after the initiation of hyperglycemia (2), a period that
is far too brief to allow significant buildup of AGE.
Using an in vitro microassay for direct real-time electrochemical
monitoring of NO production by endothelial cells and purified recombinant endothelial nitric oxide synthase (eNOS), in conjunction with electrospray ionization (ESI)-mass spectrometry (MS) to identify chemical interactions that occur between glucose and NO, we demonstrate a direct NO-scavenging capacity of D-glucose. Thus rapid
chemical inactivation of NO by glucose may be an important contributor to the dampened NO bioactivity observed in blood vessels from diabetic patients.
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MATERIALS AND METHODS |
Cell culture.
Microvascular endothelial cells were previously established and
characterized by our laboratory; these SV-40-immortalized cells
established from explant cultures of microdissected rat renal
resistance arteries express receptors for acetylated low-density lipoprotein, immunodetectable von Willebrand antigen, and are capable
of capillary tube formation (30). Cells were grown on gelatin-coated dishes in medium 199 culture medium (Mediatech, Washington, DC) supplemented with 5% FBS (HyClone Laboratories, Logan,
Utah), 100 U/ml penicillin, and 100 µg/ml streptomycin (GIBCO-BRL,
Gaithersburg, MD).
Measurement of eNOS activity with NO-selective microelectrodes in
vitro.
Recombinant bovine eNOS protein was purified from Escherichia
coli that had been transformed with independent vectors for expression of eNOS and Gro ELS, as previously described
(15). NO concentration ([NO]) was monitored with
porphyrin-electroplated, nafion-coated, carbon-fiber electrodes (30 µm OD) manufactured according to Bio-Logic Instruments instructions
(Grenoble, France; see Ref. 28). The electrode oxidation
current was low-pass-filtered at 0.5 Hz and sampled every 2 s.
Measurements were made using constant potential amperometry (0.7 volts)
and a highly sensitive potentiostat (InterMedical, Nagoya, Japan).
Calibration of the electrode was performed before each experiment using
dilutions of freshly prepared NO-saturated Krebs-Ringer solution.
The NO-selective and reference electrodes were equilibrated in a buffer
at room temperature with constant stirring until a stable baseline
current was obtained. The composition of the buffer was 50 mM
Tris · HCl, pH 7.4, 500 µM NADPH, 5 µM FAD, 5 µM FMN, 100 nM calmodulin, 10 µM CaCl2, and 20 µM
L-arginine. To avoid interference by high levels of
tetrahydrobiopterin (BH4), which is electroactive and
unstable at physiological pH, 1 µM BH4 was added to the
eNOS aliquots 12-24 h before the measurements. This permitted
binding of the cofactor to eNOS, which stabilizes bound BH4
while allowing unbound BH4 to decay away. After obtaining a
stable baseline, 25 pmol eNOS was pipetted to the cuvette, and the
electrochemical response was recorded continuously. For the determination of the effect of glucose, desired glucose concentrations were added to the cuvette before the administration of eNOS. When necessary, 0.2-2 mM
N
-methyl-L-arginine
(L-NMMA) was added to the solution to verify the NO
dependence of the recorded electrode current. At the completion of
experiments, electrode calibration was confirmed by evaluating responses to reference solutions of NO-saturated deionized water.
ESI-MS of D-(+)-glucose and
products formed upon reaction with acidified NaNO2.
Spectra were acquired on a Quattro II extended mass range (1-8,000
Da) triple quadrupole mass spectrometer (Micromass UK Limited, Manchester, UK) interfaced to a Windows NT workstation utilizing MassLynx version 2.3 software (Micromass UK Limited). The mass spectrometer was scanned in the negative ionization mode, with a source
temperature of 70°C and a 3.2-kV capillary potential. For tandem mass
spectrometry (MS/MS), a collision-induced dissociation energy of 10 volts was applied. Samples contained 10 mM D-(+)-glucose and 10 mM formic acid in the absence or presence of 10 mM
NaNO2. After incubation at 37°C for 30 min, a 100 µl
aliquot of each reaction mixture was diluted with 900 µl of
methanol-water-formic acid (94:4:2) and continuously infused into the
mass spectrometer's coaxial probe at a rate of 0.3 ml/h.
Statistics.
Statistical analyses were performed using a paired or unpaired
Student's t-test, with P < 0.05 considered
statistically significant. All values are presented as means ± SE.
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RESULTS |
NO production by cultured endothelial cells.
Rat renal microvascular endothelial cells (RMVEC) were incubated in
Krebs-Henseleit-HEPES buffer supplemented with 100 µM L-arginine and 5 mM D-glucose. Cells were
repeatedly stimulated with 1 µM bradykinin at 30-min intervals to
elicit NO production; once a maximal NO response was obtained, the
agonist was removed by changing the bath. These treatments resulted in
a series of NO responses of consistent amplitude (not significantly
diminished compared with the initial response; see Fig. 1). The same
time course of bradykinin stimulation was repeated under identical conditions, except that the buffer glucose concentration was increased sixfold to 30 mM. As shown in Fig. 1, the
amplitude of consecutive responses to bradykinin was reduced by ~30%
in high-glucose buffer. These data indicate that free [NO] is
diminished in a high-glucose environment. Furthermore, NO production by
RMVEC stimulated by the calcium ionophore A-23187 was compared in the
following two groups of cells: those bathed in control Krebs' buffer
(5 mM glucose) and those bathed in buffer with 30 mM glucose. In accord
with observations using bradykinin as stimulus, NO responses to A-23187 were significantly less in the high-glucose buffer. Figure
2 summarizes dose-response relations
between the concentration of D-glucose and the
A-23187-induced NO release. Although some decline in NO release was
observed at 20 mM glucose, a statistically significant decrease was
documented at 30 mM. Furthermore, 30 mM L-glucose exhibited
the same effect on A-23187-induced NO release in contrast to the
unperturbed NO production in the medium made equiosmolar with the
addition of NaCl. The dampened NO detection observed with high glucose
could potentially arise from inhibition of endothelial NO synthesis or
abbreviation of the NO life time.

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Fig. 1.
Nitric oxide (NO) release from cultured endothelial cells.
A: consecutive responses of endothelial cells to bradykinin.
After acquisition of NO responses to 1 µM bradykinin by endothelial
cells bathed in Krebs buffer with 5 mM glucose, the bath was exchanged
to Krebs buffer with 5 or 30 mM glucose, and stimulation by bradykinin
was repeated. Thirty-minute interval between stimuli was sufficient to
restore the amplitude of NO release in Krebs buffer with 5 mM glucose
(tracings on top). The same time course resulted in a
blunted response to bradykinin by cells incubated in 30 mM glucose
(tracings on bottom). Inset: examples of actual
recordings, with washes indicated by long arrows and application of
buffers with differing glucose concentration shown by short arrows.
A, bottom, is a diagrammatic summary of changes
in NO responses to bradykinin. [NO], NO concentration. B:
A-23187-stimulated NO release from endothelial cells bathed in Krebs
buffer containing 5 or 30 mM glucose. *Significant difference from
control (P < 0.05).
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Fig. 2.
NO release by endothelial cells stimulated with A-23187.
A: dose-response of NO production in the presence of
increasing concentrations of D-glucose. B:
modulation of NO production in the presence of D-glucose
(D-Gl), L-glucose (L-Gl), or
equiosmolar NaCl. *P < 0.05 vs. control (5 mM
D-glucose).
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In vitro NO generation by recombinant eNOS.
In vitro studies were performed where the activity of purified
recombinant bovine eNOS was continuously monitored using an NO-selective porphyrinic microelectrode. As shown in Fig.
3, addition of eNOS to the reaction
buffer resulted in immediate detection of an NO signal that was
attenuated upon addition of the prototypic NOS inhibitor
L-NMMA. Detection of NO was optimal with the electrode holding potential of 0.7 mV that was employed for these measurements; when the electrode holding potential was reduced to 0.4 mV, electrode current was not detectably affected by either eNOS-derived NO or
solutions of purified NO gas (data not shown). These findings demonstrate selectivity of NO detection by the amperometric technique and validate the method as an effective means to directly measure levels of eNOS-derived NO. Addition of D-glucose to
NO-forming enzyme reactions resulted in a rapid and dose-dependent
decrease in steady-state electrode current, consistent with a
diminished NO life time or synthesis rate (Fig. 3). Notably, glucose on
its own did not affect the electrode current (Fig.
4). To distinguish whether the apparent
glucose-induced lowering of [NO] resulted from accelerated NO loss,
in a next series of experiments we studied the effect of glucose on
[NO] in solutions of pure NO gas.

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Fig. 3.
NO-microelectrode monitoring [NO] in in vitro endothelial nitric
oxide synthase (eNOS) assay. Steady-state release of NO by recombinant
eNOS is inhibitable by 1 mM
N -methyl-L-arginine
(L-NMMA) and shows gradual decline upon repeated
administration of 25 mM glucose. Inset, example of actual
tracing.
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Fig. 4.
NO availability depends on ambient glucose concentration.
A: addition of D-glucose does not interfere with
electrode current. B: influence of cumulative glucose
addition on amperometrically detected [NO] (thick tracing). Similar
decrements in [NO] were observed with additions of 30 mM
L-glucose or 30 mM mannitol (interrupted tracings).
Steady-state levels of NO were measured in a 1:1,000 dilution of
NO-saturated solution before and after cumulative additions of glucose.
Note that decrements in the electrochemically detectable NO occurring
with incremental increases in ambient glucose concentration are similar
whether NO derives from eNOS (shown in Fig. 3) or gaseous NO in
solution. C: changes in [NO] accompanying incremental rise
in D-glucose levels.
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As shown in Fig. 4, increasing ambient D-glucose resulted
in a progressive and concentration-dependent decline in the level of
free NO detected in solutions of pure NO gas. Remarkably similar decrements in electrochemically detectable [NO] were observed after
addition of 30 mM L-glucose or 30 mM mannitol.
Interestingly, the observed decrement in [NO] occurring with each
incremental elevation in D-glucose concentration was
similar regardless of the source of NO, either eNOS-derived or an NO
gas tank. These findings suggested that glucose abbreviates the life
time of NO, raising the possibility of a covalent reaction between NO
and glucose. To test this prospect, we investigated whether a
glucose-NO adduct could be detected by ESI-MS.
ESI-MS/MS of D-(+)-glucose, before
and after treatment with acidified NaNO2.
Only charged species can be identified by MS; accordingly, a neutral
carbohydrate such as D-(+)-glucose is poorly detected. Nonetheless, negative ion monitoring ESI-MS revealed a prominent peak
for D-glucose at 179 Da, reflecting the deprotonated
species (native glucose = 180.2 Da). Because NO has a mass of 30 Da, the addition of NO to deprotonated glucose would predictably yield a species with mass 209 Da. Negative ion scanning ESI-MS of acidified glucose solution failed to identify any ions in the mass range of 206 to 213 Da (Fig. 5A). In
contrast, addition of nitrite to acidified glucose resulted in the
rapid appearance of two novel negative ion species at 208.5 and 210.4 Da (Fig. 5B). These ions are attributed to the product of NO
addition to deprotonated D-glucose, followed by loss or
gain of a hydrogen atom.

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Fig. 5.
Electrospray ionization (ESI)-mass spectrometry (MS) negative ion
scan of D-(+)-glucose before and after exposure to
acidified nitrite. Concentrations of reactants and MS conditions are as
detailed in METHODS AND MATERIALS. Tracings depict 206- to
213-Da ion scans unreacted with D-glucose (A)
and D-glucose after exposure to acidified NaNO2
(B).
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In formic acid, D-glucose is predominantly detected by
negative ion monitoring ESI-MS as a formate adduct with mass 225 Da (formate ion mass is 45 Da). Addition of nitrite to
D-glucose/formate resulted in the appearance of a novel
product ion with mass 255 Da, consistent with monoaddition of NO (30 Da) to glucose/formate. A similar peak with mass of 255 Da was observed
when L-glucose was added to acidified nitrite (data not
shown). Ions predicted to result from addition of two or more NO
molecules to glucose or glucose/formate were not observed. Argon
collision gas-induced fragmentation patterns of
D-glucose/formate and D-glucose-NO/formate ions
are depicted in Fig. 6. ESI-MS/MS of
D-glucose/formate (Fig. 6) revealed several prominent
collision-induced dissociation negative ion peaks from the 225-Da
parent ion; these are ascribed to deprotonized glucose (178.5 Da), free
formate (45.3 Da), and ions derived from 3, 4, and 5 carbon-containing
glucose fragmentation products (88.8, 118.8, and 148.8 Da,
respectively). Dehydration products of intact D-glucose and
its 3, 4, and 5 carbon fragments were also identified (160.4, 70.4, 130.4, and 100.4 Da, respectively). In contrast, a more minimal
pattern of negative ion fragmentation products derived from the 225-Da
glucose-NO/formate parent ion (Fig. 6B). Predominant species
are ascribed to the loss of NO (223 Da), loss of formate (209 Da), and
loss of both NO and formate (177 Da). Loss of NO from
D-glucose or D-glucose/formate was associated with a 31-Da decrement in mass, suggesting the additional abstraction of a hydrogen atom. Evidence for a NO-formate adduct is suggested by
negative ion species of mass 75 Da. Taken together, ESI-MS/MS findings
affirm the formation of one or more covalent monoaddition products of
NO and glucose.

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Fig. 6.
ESI-MS/MS negative ion scan of D-(+)-glucose before and
after exposure to acidified nitrite. Concentrations of reactants and MS
conditions are as detailed in MATERIALS AND METHODS.
A: daughter ion scan of the 224.6-Da formate adduct of
D-glucose. B: daughter ion scan of the 254.4-Da
formate adduct of D-glucose-NO. In both cases,
fragmentation was elicited using a collision energy of 10 volts.
Similar results were obtained with L-glucose (data not
shown).
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DISCUSSION |
The rapidity of blood pressure elevation after establishment of
hyperglycemia in normal volunteers (14) indicates that
high glucose levels trigger functional alterations in vasomotor
control. Three NO-based mechanisms have been invoked to explain this
phenomenon: 1) inhibition of eNOS activity, 2)
abbreviation of NO bioavailabilty, and 3) diminished
capacity of NO to activate guanylyl cyclase (9, 6, 21).
Studies presented herein demonstrate a direct NO-scavenging effect of
glucose, providing a molecular explanation for abbreviated NO
bioavailabilty. Specifically, the data obtained with cultured cells
showed that elevated NO levels, in response to both receptor-dependent
(bradykinin) and receptor-independent (calcium ionophore) activators of
eNOS, were consistently diminished in high-glucose Krebs buffer
compared with control buffer. Together with in vitro findings that
elevated glucose diminished steady-state levels of NO derived from
purified recombinant eNOS, it appeared that glucose could suppress eNOS
activity. However, a similar diminution in NO levels was observed in
the absence of eNOS, when glucose was added to solutions of purified NO
gas. This latter finding strongly suggested that glucose abbreviates
the NO life time, and this phenomenon can explain the observed lowering
of [NO] after an acute elevation in glucose concentration. To examine whether NO can chemically react with glucose, mass spectrometric analyses were performed.
ESI-MS/MS analysis directly revealed that glucose undergoes addition of
30 Da upon exposure to a nitrosating environment (acidified nitrite).
Because 30 Da is the mass of NO, this finding suggests that a single
molecule of NO adds to glucose, presumably to a hydroxy-oxygen atom.
Specification of the precise site of NO addition to glucose, based on
molecular fragmentation analysis of this adduct, was complicated by the
equal masses of NO and HCOH fragmentation units of glucose.
Accordingly, we are unable to assign a precise structure to the
NO-glucose product. We are nonetheless able to confirm by MS that
glucose can capture NO (at low yield with regard to glucose) to form a
yet unspecified addition product. The disappearance of NO in the form
of this reaction product may be sufficient to explain the observed
ability of high glucose (30 vs. 5 mM) to lower [NO] in biological solutions.
Glucose scavenging of NO can explain a host of earlier observations in
which hyperglycemia has been linked to a diminished availability of NO
in blood vessels. It has long been appreciated that isolated vascular
rings, incubated in media with elevated glucose concentration, show
impaired relaxation in response to NO-dependent vasodilators such as
ACh (26). Exposure of endothelial cells to elevated
glucose concentration has also been associated with reduced
bradykinin-stimulated cGMP formation in RFL-6 reporter cells
(17). Similarly, it has been demonstrated that acute
hyperglycemia enhances NO degradation by endothelial cells, resulting
in decreased endothelium-derived relaxing factor bioactivity
(10). In normal human subjects, elevation of plasma
glucose to 270 mg/dl caused a marked reduction in NO-mediated
vasoactivity, leading to increased systolic and diastolic blood
pressure and decreased leg blood flow within 30 min after initiation of
the hyperglycemic clamp (9). It should be emphasized that
these acute effects of glucose occur on the background of the insulin
or insulin-like growth factor I-induced chronic vasodilation
characteristic of uncomplicated diabetes mellitus (Ref. 28
and references therein). Furthermore, the observed acute effects of
high glucose levels are independent of its chronic effects mediated via
kinases or inducible NO synthase (1, 11, 29).
In addition to promoting endothelial cell dysfunction, the observed
reaction of NO with glucose may have far-reaching sequelae for other NO
targets. For instance, elevated ambient glucose levels reduced
NO-dependent cGMP production with an apparent IC50 of 30 mM
in human neuroblastoma cells, suggesting that the proposed mechanism of
NO inactivation by glucose plays a role in the development of diabetic
neuropathy (21). J774 macrophages incubated in media with
elevated glucose concentration showed significantly reduced NO
production in response to stimulation with lipopolysaccharide (29). Furthermore, the NO dependence of early phases of
glucose-stimulated insulin secretion should alert to the possibility
that decreased availability of NO may result in defective insulin
secretion (20, 22). In addition, reduced bioavailability
of NO can compromise insulin-stimulated glucose uptake in skeletal
muscles, a process that requires NO (19). Hence, the
reaction of NO with glucose, described herein, may be implicated in a
host of pathophysiological processes resulting in the impaired
macrophage and neuronal cell functions, insulin secretion, and glucose
utilization, thus not only exacerbating glucose control but also
promoting neurological and immunological complications. The aggregate
effect of NO scavenging by glucose on vascular dysfunction and
carbohydrate metabolism needs further investigation.
In summary, endothelial cells incubated in high-glucose buffer exhibit
blunted NO responses to two eNOS agonists, bradykinin and A-23187. In
vitro monitoring of NO production by eNOS suggested that glucose
addition decreases either NO production or life time. Experiments with
glucose additions to aqueous solutions of pure NO supported the latter
possibility, namely, a glucose-mediated decline in NO life time.
Finally, electrospray MS/MS analysis directly demonstrated that glucose
can form a chemical adduct with NO. Based on these data, we conclude
that glucose is capable of scavenging NO and predict important in vivo
consequences for such reactions. Specifically, we hypothesize that
acute hyperglycemia may elevate arterial blood pressure by diverting NO
to glucose and away from smooth muscle cell targets of vasorelaxation.
Loss of NO to glucose may similarly attenuate other important actions of endothelium-derived NO in the blood vessel wall; these include inhibition of smooth muscle proliferation, preventing platelet adhesion
and aggregation, and limiting diapedesis of polymorphonuclear cells and
monocytes (5, 7, 12, 13, 16, 24, 27). The extent to which
glucose/NO adducts accumulate in plasma of diabetic patients, the
precise chemical identities of these products, sites of formation
(intra- or extracellular), metabolic fates, and potential to serve as
precursor molecules for subsequent release of NO or other bioactive
species all remain to be explored.
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ACKNOWLEDGEMENTS |
These studies were supported in part by National Institutes of
Health Grants DK-45462, DK-54602 (M. S. Goligorsky), HL-50656, HL-44603, and RR-11360 (S. S. Gross).
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FOOTNOTES |
Address for reprint requests and other correspondence: M. S. Goligorsky, Dept. of Medicine, State Univ. of New York, Stony Brook,
NY 11794-8152 (E-mail: mgoligorsky{at}mail.som.sunysb.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10 July 2000; accepted in final form 7 November 2000.
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