Prsentation PowerPoint

1
ELECTROCHEMICAL CHARACTERIZATION OF CELLULAR
PRODUCTION OF NITRIC OXIDE AND SUPEROXIDE RADICALS
Fethi BEDIOUI
Laboratoire dÉlectrochimie et Chimie Analytique,
UMR n7575 CNRS-ENSCP, École Nationale Supérieure
de Chimie de Paris, 11 rue Pierre et Marie
Curie, 75231 Paris cedex 05, France.
2
NO PHYSIOLOGICAL FUNCTIONS
NO can be synthesized by mammalian cells and acts
as physiological messenger and cytotoxic
agent. NO is a signaling molecule in the
cardiovascular system (R. F. Furchgott, L. J.
Ignarro and F. Murad, Medecine Nobel Price
1998) NO also plays numerous physiological roles
and functions in the nervous and immune systems.
3
NO BIOSYNTHESIS
NO is synthesized by NO-synthase, NOS, from
L-arginine, in several cell types endothelial,
neuronal, neutrophils, platelets,
leukocytes L-arginine 2 NADPH 2 O2 2 H ?
NO citrulline 2 NADP 2 H2O Three NOS
isoforms have been identified - constitutive
neuronal cNOS calcium dependent - constitutive
endothelial eNOS calcium dependent - inducible
iNOS calcium independent
4
VASCULAR PRODUCTION OF NO
Platelets (antiaggregatory effetcs) Leucocytes
(anti activation adhesion effects)
Bradykinin
Ca2
Endothelial cell
Red corpuscles
platelets
adventice
Ca2/Calmoduline
endothelium
NOS
L-Arginine ? L-Citrulline NO
Vaso mover nerve
Inhibition of the adhesion activation of
leucocytes
relaxation of the smooth muscular cells
GC
GTP ? GMPc
Smooth muscular cell
VASODILATATION
5
REGULATORY, PROTECTIVE AND DELETERIOUS
BIOLOGICAL EFFECTS OF NO
Regulatory Vascular tone Cellular
adhesion Vascular permeability Inhibits
platelets adhesion
Deleterious Inhibits enzyme function Promotes
DNA damage Induces lipid peroxidation Depletes
antioxidant stores Increases succeptibility
to radiation, alkylation agent, toxic metals
Protective Antioxidant Inhibits leukocyte
adhesion Protects cells against oxidant injury
6
O2- PHYSIOLOGICAL FUNCTIONS
1 to 2 of the total oxygen uptake is partially
reduced to generate either H2O2 or superoxide
O2-. Increased formation of superoxide has been
implicated in the pathophysiology of several
cardiovascular and neuro- degenerative diseases.
The exact mechanisms are not clear
yet. Evidences indicate that formation of
superoxide-derived oxidants may be involved -
from natural or SOD catalyzed disproportionation
? H2O2 - from reaction with NO ? ONOO-
(peroxynitrite).
7
IMPLICATION OF O2- IN NO-ENDOTHELIAL ACTIVITY
O2- is one of the most important
physiopathological modulators of stationary and
efficient NO in blood vessels. The main vascular
source of O2- is the enzymatic activity of the
membrane NAD(P)H oxidase endothelium, smooth
muscle and adventitia. eNOS is also able to
generate O2- under specific conditions (absence
of tetrahydrobiopterin, BH4 (an eNOS cofactor)
or L-arginine). The three NOS isoforms are able
to produce NO and O2- via different mechanisms
and under specific conditions.
8
GENERAL DESCRIPTION OF VASCULAR PATHWAYS OF NO,
O2- ONOO-
O2
O2
L-Arginine
2
extracellular
NO
Ca
NAD(P)H
membrane
R
oxidase
intracellular
NOS
NAD(P)H
NAD(P)
NOS
2
Ca
NO
catalase
CaM
Ca/CaM
SOD Cu,Zn

-
ONOO
9
DIRECT INDIRECT PHYSIOLOGICAL EFFECTS OF NO
DIRECT EFFECTS Reactions in which NO
interacts directly with a biological target NO
concentrations lt 1 mM (normal tissues)
INDIRECT EFFECTS Reactions in which
NO-derived intermediates RNOS are Involved NO
concentrations gt 1 mM (up-regulation)
nitrosation
NO O2- or O2 ? RNOS
oxidation
nitration
10
GENERAL DESCRIPTION OF VASCULAR PATHWAYS OF NO,
O2- ONOO-
L-ARGININE
NADPH2
O2
FADH2 NADH2
BH4
NOS COUPLED OXIDO- REDUCTASE
204
XANTHINE
NOS
XANTHINE OXIDASE
PEROXIDASE ACTIVITIES
PLASMA MEMBRANE NAD(P)H OXIDASE
MITOCHONDRIAL RESPIRATION
OXIDIZED BH4
URIC ACID
LIPOXYGENASE
NADP
NAD(P)
CITRULLINE
HpETEs
FAD NAD
O2-
NO
NO
H2O
H2O2
SUPEROXIDE DISMUTASE
CATALASE
SPONTANEOUS DISMUTATION
ONOO-
NO2-
GLUTATHIONE PEROXIDASE
GS-SG
2GSH
11
O2- / NO CHEMICAL REACTIVITY
2 x 106 M-1 s-1
Cyt c Fe(III)
Cyt c Fe(II)
NO2-
2 x 106 M-2 s-1
natural disproportionation 5 x 105 M-1 s-1 at pH
7.4
O2
0.5 O2 0.5 H2O2
NO
O2-
SOD-catalyzed disproportionation 2 x 109 M-1 s-1
2 x 1010 M-1 s-1
catalase
ONOO-
H
pKa 6.8
O2 H2O
H NO3-
ONOOH
0.2 s-1
NO2-
12
DIFFUSION AND INTERACTION OF NO WITH O2- two
point sources model
NO/O2 interaction nitrosative stress
O2- source
direct effects
direct effects
oxidative stress
NO source
ONOO-

• maximum nitrosative stress near the source of NO
• direct effects of NO as it diffuses from its
  source
• as NO approaches the O2- source it reacts to form
  ONOO-
• as ONOO- diffuses into areas of O2- or NO excess,
  it decomposes
• oxidative stress is limited to sites close to the
  O2- source

13
CHARACTERIZATION OF NO AND O2- IN BIOLOGICAL
SYSTEMS Analusis, 28 (2000) 443-527, F. Bedioui,
coordinator

• NO monitoring
• spin trapping EPR (Y. Henry A. Guissani),
• chemiluminescence (S. L. Archer coll.),
  electrochemical
• NO-microsensors (F. Bedioui, M. Pontié,J. M. A.
  Devynck)
• - O2- monitoring
• cytochrome c UV-Visible, EPR spin trapping,
  fluorescence
• chemiluminescence (J. P. Souchard, F. Nepveu J.
  F. Arnal,
• J.P. Illiou N. Villeneuve, J. Vasquez-Vivar
  coll.),
• Electrochemical detection (C. McNeil coll.).
• - NO and oxidative stress
• electrochemical approach (C. Amatore coll.)
• - ONOO- formation, reactions and detection (R.
  Radi coll.).

14
THE COMMONLY USED NO-ELECTROCHEMICAL SENSOR
T. Malinski and Z. Taha, Nature, 358 (1992) 676
F. Lantoine, S. Trévin, F. Bedioui and J.
Devynck, J. Electroanal. Chem., 392 (1995) 85
15
AMPEROMETRIC MEASUREMENTS
MEASUREMENT CONDITIONS
Phosphate buffer (PBS 0.1 M)
NaCl145 mM
CaCl
1 mM

2
glucose5 mM
pH 7.2
Aerobic medium
MEASUREMENT TECHNIQUE
Differential Normal Pulse
Amperometry (DNPA) at 0.67 V/sce
M
F. Lantoine, S. Trévin, F. Bedioui and J.
Devynck, J. Electroanal. Chem., 392 (1995) 85
16
CALIBRATION OF THE NO-ELECTROCHEMICAL SENSOR
MEASUREMENT CONDITIONS
Phosphate buffer (PBS 0.1 M)
NaCl145 mM
CaCl
1 mM
2
glucose5 mM
pH 7.2
Aerobic medium
MEASUREMENT TECHNIQUE
Differential Normal Pulse
Amperometry (DNPA) at 0.67 V/sce
DETECTION LIMIT
1 nanomolar
F. Lantoine, S. Trévin, F. Bedioui and J.
Devynck, J. Electroanal. Chem., 392 (1995) 85
17
ELECTROCHEMICAL CHARACTERIZATION OF O2-
Electrode electrochemically pretreated carbon
microfiber
Xanthine H2O 2O2 uric acid 2H
2O2- XOD
1. Phosphate buffer solution PBS (pH
7.4) 2. PBS xanthine (240 mM) 3. PBS Xanthine
(240 mM) XOD (0.2 mM)
MEASUREMENT TECHNIQUE
Differential Pulse
Voltamperometry(DPV)
C. Privat, S. Trévin, F. Bedioui and J. Devynck,
J. Electroanal. Chem., 436 (1997) 261
18
ELECTROCHEMICAL CHARACTERIZATION OF ENZYMATIC
PRODUCTION OF O2-
Differential normal pulse amperogram obtained at
0.12V, at a bare carbon microfiber electrode in
PBS (pH 7.4). injection 1 xanthine
(500 µM) injections 2-5 xanthine oxidase
(0.1 to 0.9 µM) injection 6 superoxide
dismutase (0.064 µM) injection 7 superoxide
dismutase (total concentration 0.192 µM)
injection 8 superoxide dismutase (total
concentration 0.256 µM)
C. Privat, S. Trévin, F. Bedioui and J. Devynck,
J. Electroanal. Chem., 436 (1997) 261
19
ELECTROCHEMICAL CHARACTERIZATION OF ENZYMATICALLY
PRODUCED O2- AND ITS REACTIVITY WITH NO
S
O
D
DI (nA)I
N
O
6
4

n
M
XOD 0.5 mM
4
M

m
N
O
2

M
m
XA 400 mM


NO-sensor at 0.7 V
O2-- sensor at 0.1 V
C. Privat, S. Trévin, F. Bedioui and J. Devynck,
J. Electroanal. Chem., 436 (1997) 261
20
ELECTROCHEMICAL CHARACTERIZATION OF O2- IN
ALKALINE SOLUTION
?I (pA)
Differential normal pulse amperogram obtained at
0.12V, at a bare carbon microfiber electrode in
alkaline aqueous solution (pH 12.3) upon
successive additions of KO2
Calibration curve
21
ELECTROCHEMICAL CHARACTERIZATION OF O2- IN
ALKALINE SOLUTION
C. Privat, F. Bedioui, J M. A. Devynck and
coll, Free Rad. Biol. Med, 27 (1999) 554
spectrophotometry at a constant pH (8.4) of the
SOD-inhibitable reduction of ferricytochrome c
into ferrocytochrome c by O2.- solutions prepared
at different pH from the same amount of KO2
Evaluation of the pH-dependence of O2.-
measurements performed through differential
normal pulse amperometry at 0.1 V/SCE using an
electrochemically pretreated carbon microfiber
Variation of the amplitude of the oxidation
current of a 270 nM O2- solution as a function
of pH
22
APPLICATIONS OF NO AND O2- ELECTROCHEMICAL
SENSORS IN BIOLOGICAL SYSTEMS
1- Direct electrochemical monitoring of NO
Direct measurement of NO production in platelets
relationship with Ca2 flux Inhibition by
cholesterol oxides of NO release from human
endothelial Cells 2- Direct monitoring of NO
and influence of O2- and H2O2 Electrochemical
detection of NO production in human neutrophils
Agonist-evoked NO release from human endothelial
cells role of O2- 3- Direct electrochemical
monitoring of O2- Electrochemical determination
of O2- from a single neutrophil In situ
electrochemical release from interleukin-1-b-stimu
lated human vascular cells 4- Direct dual
electrochemical monitoring of NO and O2-
23
DIRECT MEASUREMENT OF NITRIC OXIDE PRODUCTION IN
PLATELETS RELATIONSHIP WITH CYTOSOLIC Ca2
CONCENTRATION F. Lantoine A. Brunet, F. Bedioui,
J. Devynck M. A. Devynck Biochem. Biophys.
Res. Commun., 215 (1995) 842

• EVIDENCES
• platelets are not only targets for NO produced by
  neutrophils,
• endothelial or smooth cells, but they are
  themselves also capable of its production.
• cNOS and iNOS isoforms have been identified in
  human platelets.
• It has been proposed that the agonist-induced
  rise in cytolosic Ca2 could activate the cNOS
• AIM
• Investigate the role of Ca2 rise in NO
  production in platelets stimulated by agonist
  triggering different transduction patways
• thrombin (elevates the cytosolic Ca2 from
  internal stores)
• ADP (opens receptor-operated Ca2 channel)
• Collagen (directly mobilizes Ca2 but only at
  high concentrations)

24
RESULTS NO-electrochemical sensor calibration
in the platelet suspension
Differential normal pulse amperogram obtained at
0.75 V, at a nafion and nickel porphyrin coated
carbon microfiber electrode in a platelet
suspension containg 5x107platelets/mL in
Ca2-free PBS 5 mM glucose 0.5 mM Mg2 (pH
7.4) at 37 C. injections a 20 nM of NO each
injections b 40 nM of NO each
25
1- NO production 2- changes in the cytosolic
Ca2 concentrations in platelet suspension
(5x107 /mL), in the presence of 1mM extracellular
Ca2, treated by (a) 3 mg/mL collagen (b) 0.05
U/mL thrombin (c) 1 mM ADP (dashed line
presence of 30 nM Ca2)
RESULTS
1
2
1- Simultaneous NO and Ca2 determination clearly
demonstrate that the rise in Ca2 does not
parralel the stimulation of NO production. 2-
Both thrombin and ADP evoke Ca2 transients 3-
Collagen-induced NO production is increased by L-
arginine and decreased by L-NMMA (NOS
inhibitor) 4- Although cytosolic Ca2
participates in the regulation of NO synthesis,
it is not the messenger for cNOS activation
26
FURTHER FINDINGS
Based on these results, it has been proposed
that NO production in platelets is normally weak
but becomes functionally important when collagen
fibrils of the sub-endothelium are accessible (i.
e. when endothelial cells are absents). When
expressed by cell, NO production by platelets is
approximately 1000 times lower than that of
vascular endothelial cells. NO production by
collagen-stimulated platelets may partially
substitute for endothelial cells to exert a
vasorelaxing effect on smooth muscle cells and
participate in the local control of vascular
tone.
27
INHIBITION BY CHOLESTEROL OXIDES OF NO RELEASE
FROM HUMAN VASCULAR ENDOTHELIAL V. Deckert, A.
Brunet, F. Lantoine, G. Lizard, E. Millanvoye-van
Brussel, S. Monier, L. Lagrost, M. David-Dufilho,
P. Gambert M. A. Devynck Arterioscler Thromb
Vasc Biol., 18 (1998) 1054.
EVIDENCES Arteries from hypercholesterolemic
and atherosclerotic patients exhibt marked
attenuation of endothelium dependent
relaxation. Investigations revealed that
cholesterol derivatives oxidized in position 7,
ie, 7-ketocholesterol for example, unlike
unmodofied cholesterol, can reduce maximal
arterial relaxation. AIM Since the
endothelium-dependent relaxation of arteries is
mainly regulated by NO production, the current
study is related to the effect of cholesterol
oxides on NO released by cultured HUVECs
stimulated with histamine.
28
RESULTS time course of histamine-induced NO
production by HUVECs
concentration- and time- dependent effects of
7-ketocholesterol and cholesterol
on histamine-induced release of NO from HUVECs
HUVECs were preincubated (b) or not (a) with
7-ketochlesterol for 3 hours, then washed and
stimulated by 10 mM histamine
1- pretreatment of HUVECs with cholesterol did
not alter histamine-activated NO production. 2-
in contrast, pretreatment of HUVECs with
7-ketocholesterol decreased NO release. 3-
evidences showed that 7-ketocholesterol did not
reduce NO release by altering the Ca2-dependent
NOS activation steps
29
ELECTROCHEMICAL DETECTION OF NITRIC OXIDE
PRODUCTION IN HUMAN POLYMORPHONUCLEAR NEUTROPHIL
LEUKOCYTES G. Lärfars, F. Lantoine, M. A. Devynck
H. Gyllenhammer Scan. J. Clin. Lab. Invest., 59
(1999) 361.
EVIDENCES In inflamation and for protection
against bacterial infections the production of NO
by human polymorphonuclear leukocytes PMNs may
play an important role. AIM Study of NO
production from human PMNs by N-formyl-methionyl-l
eucyl-phenylalamin fMLR, a synthetic analogue of
naturally occurring bacterial chemotatic . The
influence of O2- and H2O2 was evaluated through
their accelerated disappearence due to SOD and
catalase.
30
RESULTS NO-electrochemical sensor calibration
in phosphate buffer PBS or Hanks balanced salt
solution HBSS without and with PMNs (1.75 x 106
/mL).
NO (nM)
NO (nM)
From differential normal pulse amperograms
obtained at 0.75 V, at a nafion and nickel
porphyrin coated carbon microfiber electrode
31
RESULTS evaluation of the importance of the
reaction between NO, O2- and H2O2
time course of NO production by PMNs (1.75 x 106
/mL) pre-incubated with L-arginine (1 mM) or
L-NMMA (1 mM) and stimulated or not with fMLR
(100 nM) in the presence of SOD (150 U/mL)
time course of NO production by PMNs (1.75 x 106
/mL) when fMLR (100 nM), SOD (150 U/mL) or
catalase (300 U/mL) were added in the presence of
L-arginine (1 mM)
32
ANALYSIS OF AGONIST-EVOKED NITRIC OXIDE RELEASE
FROM HUMAN ENDOTHELIAL CELLS ROLE OF SUPEROXIDE
ANION M. David-Dufilho, A. Brunet, C. Privat and
M. A. Devynck Clin. Exp. Pharm. Physiol., 28
(2001) in press
EVIDENCES In endothelial cells O2- is mainly
produced by NADPH oxidase and also by NOS when
the availability of its substrate, L-arginine, or
the cofactor BH4 is reduced. AIM Study of the
time-course of NO release from cultured HUVECs
through its electrochemical detection. The
effect of O2- production on NO release was
investigated through the effects of scavengers
and inhibitors of its synthesis.
33
RESULTS effects of O2- degradation and synthesis
on thrombin- induced NO production by HUVECs in
the presence of L-arginine
ONOO- production by dichlorofluorescein
oxidation and electrochemical monitoring of NO
reveal that agonist such as thrombin simultaneousl
y stimulates transient O2- production the
duration of NO release was increased only in the
presence of extracellular L-arginine and
exogenous SOD. It was decreased in presence
SOD-inhibitor
a control b SOD ? catalysis of O2-
disproportionation c DETC (diethylthiocarbamate
1mM) an exogenous SOD-inhibitor
34
ELECTROCHEMICAL DETERMINATION OF SUPEROXIDE
ANIONS GENERATED FROM A SINGLE NEUTROPHIL K.
Tanaka, F. Kobayashi, Y. SIsogai and T.
Iizuka Bioelectrochem. Bioenerg., 26 (1996) 413
EVIDENCES Neutrophils play an important role in
the defense mechanism by generating O2- when
stimulated.The stimulis often used in experiments
are either phagocytosable particles (bacteria,
virus, particules coated with an immunoglobulin
such as IgG) or soluble factors such as phorbol
myristate acetate PMA, and calcium ionophores.
When stimulated, neutrophils increase their
oxygen consumption and produce superoxide
anion. AIM Development of a technique for
bringing a single neutrophil cell into contact
with the surface of a microelectrode and detect
stimulated-O2- production.
35
RESULTS current time pofil obtined from a single
neutrophil with a carbon microelectrode in HBSS
Chronoamperometry of a carbon electrode (6 mm)
coated with IgG in HBSS, at 0.1 V/Ag-AgCl
neutrophil
pipette
Carbon fiber
a addition of few drops of 1x107 cells/mL
porcine neutrophils. b contact of a single cell
with the surface of the IgG modified electrode c
addition of a drop of 5 mg/mL SOD
Key-step bringing of the cell in contact with
the electrode
36
SUPEROXIDE RELEASE FROM INTERLEUKIN-1B-STIMULATED
HUMAN VASCULAR CELS IN SITU ELECTROCHEMICAL
MEASUREMENT C. Privat, O. Stepien, M.
David-Dufilho, A. Brunnet, F. Bedioui, P. Marche,
J. Devynck and M. A. Devynck Free Rad. Biol.
Med., 27 (1999) 554
EVIDENCES O2- has been proposed to be directly
or indirectly involved as a signaling messenger
in vascular dysfunction. Limited knowledge about
its in-situ production by vascular
cells. AIM Study of the responses of HUVECs
and internal mammary smooth muscle cells IMASMCs
to an inflammatory stimulus with the help of an
electrochemical sensor
37
RESULTS electrochemical and spectrophotometric
characterizations of O2- production by
stimulated cultured HUVECs
O2-(µM)
IL-1ß 0.5 U/ml
IL-1ß 0.5U/ml
6
Stimulated cells
70
4
0
Stimulated cells
O2- (nM)
2
SOD 100 U/ml
70
unstimulated
0
0
unstimulated
0
20
40
60
80
16 min
Uv-visible characterization (cytochrome c method)
Electrochemical characterization Differential
normal pulse amperometry at 0.1 V/sce of a
carbon micro fiber positioned at 10 mm above the
cultured cells
38
RESULTS electrochemical characterizations of O2-
production by stimulated cultured IMASMCs
I
L
-
1
ß

1
0
0
U
/
m
L
9
0

n
M
2
0

m
i
n

u
n
s
t
i
m
u
l
a
t
e
d
Electrochemical characterization Differential
normal pulse amperometry at 0.1 V/sce of a carbon
micro fiber positioned at 10 mm above the
cultured cells
39
SUPEROXIDE GENERATIONFROM CONSTITUTIVE NITRIC
OXIDE SYNTHASE IN ASTROCYTES IN VITRO REGULATES
EXTRACELLULAR NITRIC OXIDE AVAILABILITY C. M.
Tolias, C. McNeil, J. Kazlauskaite and E. W.
Hillhouse Free Rad. Biol. Med., 26 (1999) 99
EVIDENCES NO is synthesized from cNOS in
neurones. Evidences of O2- production by cNOS in
neurones under specific conditions, such as
substrate deprivation has indicated an additional
level of complexity in the function of this
enzyme. AIM Study of the responses of primary
rat brain cell cultures to exposure to alkaline
medium which results in rapid cell death and
simultaneous release of both NO and O2-
40
RESULTS electrochemical characterization of NO
and O2- simultaneous production by cultured
primary rat brain cells.
cytochrome c coating layer
bulk solution
cytochrome c Fe (II)
Gold ( or Platinum) electrode at E15-25 mV vs
Ag/AgCl
O2
e-
O2-
O2-
cytochrome c Fe (III)
O2- electrochemical sensor
Traces of NO/O2- from primary rat brain cell
cultures demonstrating simultaneous release of
both free radicals after exposure to a highly
alkaline solution (NaOH 2 M). CPTIO
2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazolin
e-1-oxyl-3-oxide, an NO harvester. The
NO-electrochemical sensor is ISO-NO Mark II, from
WPI
41
ACKNOWLEDGEMENTS
Laboratoire d Électrochimie et Chimie
Analytique UMR 7575 CNRS-ENSCP Dr J. Devynck Dr
S. Trévin Dr F. Lantoine Dr C. Privat Laboratoire
de Pharmacologie Cardiovasculaire UMR 8604
CNRS-CHU Necker Dr M. A. Devynck Dr M. David
Dufilho A. Brunet Finances CNRS (Ultimatech),
MESR, Swedish Medical research council