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Production of nitric oxide (NO·) is one of
the most important roles of endothelial cells. It is critical in
the regulation of blood pressure and vessel homeostasis. In many
types of cardiovascular diseases with oxidative stress such as hypercholesterolemia
atherosclerosis diabetes cigarette smoking or hypertension the activity
of endothelial nitric oxide synthase (eNOS) is diminished causing
decrease in bioavailability nitric oxide. It has been suggested
that the oxidation of tetrahydrobipterin (BH4) is a major cause
of eNOS dysfunction under conditions of oxidative stress. This project
will study the effects of oxidative stress on the production of
nitric oxide in endothelial cells as well as the recovery of eNOS
activity by supplementation of endothelial cells with BH4. BH4 serves
as a critical co-factor for the eNOS. A deficiency of BH4 results
in a condition known as eNOS uncoupling which is associated with
increase in superoxide and decrease in NO· production. Uncoupling
of eNOS in the endothelium may lead to oxidative stress and endothelial
dysfunction (ED) via at least 3 mechanisms. First the diminished
enzymatic production of NO· may decrease cellular antioxidant
activity. Second the enzyme begins to produce superoxide contributing
to oxidative stress. Finally it is likely that eNOS can become partially
uncoupled such that both superoxide and nitric oxide are produced
simultaneously. Under this circumstance eNOS may become a peroxynitrite
(ONOO¯) generator leading to a vicious cycle of oxidative stress.
BH4 has been shown to be a target for oxidation by ONOO¯. To
gain further insight into these interactions the effects of oxidative
stress (superoxide production by xanthine oxidase or treatment with
(ONOO¯) on bovine aortic endothelial cells (BAECs) and the
recovery of eNOS by supplementation with BH4 will be studied.
The endothelial nitric oxide synthase (eNOS) is an
enzyme that catalyses the conversion of L-arginine NADPH and oxygen
to nitric oxide. NO· is a gas that acts as a hormone produced
by eNOS with antitrombotic antiatherogenic and vasodilator actions.
The dysfunction of eNOS with a decrease in BH4 content in endothelial
cells produced superoxide instead of NO. The loss of these functions
is now known as endothelial dysfunction (ED). There are a number
of diseases associated with ED such as hypercholesteremia and atherosclerosis.
These are associated with impaired nitric oxide production by the
endothelium and increased production of superoxide which in turn
reacts with nitric oxide at a extremely rapid rate of 6.7x109mol/L-1
x s-1 forming the strong biological oxidant peroxynitrite (ONOO¯)
a potent oxidizing agent. ONOO¯ causes lipid peroxidation and
protein damage. One of the most recently described reactions of
ONOO¯ is an oxidation of tetrahydrobiopterin (BH4) which may
lead to uncoupling of eNOS in vivo. We hypothesis that oxidative
stress uncouples eNOS and causes a decrease in NO· production
while causing an increase in superoxide generation due to oxidation
of BH4 therefore supplementation of endothelial cells with BH4 will
recover the normal functions of eNOS.
Measurements of nitric oxide with Fe(DETC)2
NO· production in BAECs has been measured with
colloid solution of Fe(DETC)2. To prepare Fe(DETC)2 colloid 3.2
mM sodium-diethyldithiocarbamate and 1.6 mM FeSO4 were separately
dissolved in nitrogen bubbled.
Experiments were performed to determine if intracellular
BH4 was a target of oxidation by extracellular superoxide produced
by xanthine oxidase. Superoxide production by xanthine oxidase has
been verified by spin probe CPH following accumulation of CP-nitroxide
radical. It has previously been shown that O2·- and H2O2
react minimally with BH4. The activity of eNOS was determined by
measuring NO· production in BAECs using the NO-specific spin
probe colloid Fe(DETC)2. Colloid Fe(DETC)2 in a cell-free sample
did not yield an ESR signal. Non-treated control cells demonstrated
a strong ESR signal of NO·-Fe(DETC)2 demonstrating coupled
eNOS function (Fig 3). NO·-Fe(DETC)2 spectra of control cells
was set as 100% for the comparison with the following results. Treatment
of control cells with BH4 did not significantly changed NO•
production. Treatment of cells with the inhibitor of BH4 synthesis
DAHP for 24 hours decreased NO· production (Fig. 2 3). The
supplementation of DAHP treated BAECs with BH4 recoverd eNOS function
(Fig. 3).Treatment of cells with Xanthine and Xanthine Oxidase decreased
NO· production and BH4 treatment showed significant recovery
of eNOS functions. Cells that were treated with Xanthine Xanthine
Oxidase and superoxide dismutase (SOD) showed no change in nitric
oxide level. Treatment with BH4 resulted in significant increase
in NO• production implying the presence of uncoupled eNOS.
SOD did not show significant protection of endothelial cells from
xanthine oxidase. The reaction between NO• and O2·-
occurs 3 times faster than the reaction of SOD with O2·-
suggesting that the activity of SOD was not enough or there was
not enough of BH4 for de novo synthesized eNOS in the presence of
H2O2.
The experiments with the endothelial cells treated with Xanthine
Oxidase showed significant uncoupling of eNOS. BH4 supplementation
fully restores eNOS function which provides further evidence that
BH4 may be beneficial for treatment of endothelial dysfunction under
oxidative stress.
We would like to thank Dr. Samuel Dudley for his contributions
to the study. Funding Attributions This material is based upon work
supported by the Howard Hughes Medical Institute under Grant No.
52003727 and by the National Science Foundation HBCU-UP program.
There is a significant amount of evidence that suggest
oxidant stress cell impairment from too much oxygen changes many
functions of the endothelial cells a layer of cells that line the
inside of certain body cavities. In diseases such as arteriosclerosis
(hardening of the arteries) hypertension and cigarette smoking there
seems to be a decrease in nitric oxide (NO·) production and
an increase in the generation of superoxide (O-2 ) and other reactive
oxygen species (ROS) which causes damage to important tissues of
your nervous system joints internal organs and blood vessels. There
are many enzymatic systems capable of producing ROS for example
xanthine oxidase NADPH oxidase and uncoupled endothelial nitric
oxide synthase (eNOS). The endothelial nitric oxide synthase (eNOS)
contributes to the regulation of blood pressure by the production
of nitric oxide (NO·). Nitric Oxide (NO·) compound
produced from L arginine by eNOS. NO· a gas that acts as
an intracellular and intercellular messenger in a wide range of
processes in the vascular and nervous systems. NO· regulates
blood pressure and blood flow by passing through the cell layers
of the vessel causing the smooth muscles that encase it to relax
and the blood vessel to dilate. A lack of co factors for eNOS results
in changes in cellular signaling such that eNOS is not activated
properly. The improper activation of eNOS results in endothelial
dysfunction failure of hormonal functions of the cells lining blood
vessels so that the hormones no longer have control over widening
of arteries is lost. A decline in NO· absorbtion rate causes
an increase in the production of superoxide (O-2) a free radical
produced by several enzyme sytems. The reaction between NO·
and O-2 generates peroxynitrite (ONOO-) a potent oxidizing agent.
The reaction between NO· and O-2 is three times faster than
the reaction rate of O-2 with superoxide dismutase (SOD) which stabilizes
O-2 . Given this rate there will always be some NO· and O-2
reaction taking place but antioxidant defenses decrease the interaction
and help maintain a balance between NO· and O-2.
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