CONTEMPO UPDATES
LINKING EVIDENCE AND EXPERIENCE
Nitric Oxide Deficiency as a Cause
of Clinical Hypertension
Promising New Drug Targets for Refractory Hypertension
Gail D. Thomas, PhD
Weiguo Zhang, MD, PhD
Ronald G. Victor, MD
T
HE NITRIC OXIDE PATHWAY CON-
tributes to the blood pressure–
lowering effects of many com-
monly used therapeutic agents and
emerging basic research on this path-
way is elucidating potential new anti-
hypertensive drug targets. Understand-
ing the regulation of the nitric oxide
pathway also provides a conceptual
framework for understanding the po-
tential role played by nitric oxide in a
number of disorders, including con-
gestive heart failure, atherosclerosis,
nitrate tolerance, muscular dystro-
phy, circulatory shock, and male sexual
dysfunction.
Nitric Oxide Pathway
Nitric oxide is produced by the enzyme
nitric oxide synthase (NOS) during the
oxidation of the amino acid substrate
L-arginine to L-citrulline.1 Three dis-
tinct NOS isoforms are found in mam-
malian cells: endothelial NOS (eNOS or
NOS III), neuronal NOS (nNOS or NOS
I), and inducible NOS (iNOS or NOS II).
Under normal physiological condi-
tions, iNOS is undetectable in macro-
phages and other tissues, and thus
appears to play little if any role in the nor-
mally functioning cardiovascular sys-
tem. However, induction of iNOS expres-
sion by cytokines or inflammatory
mediators is thought to play a pivotal role
in causing hypotension during septic
shock. Isolated case reports suggest that
NOS inhibitors might be useful pressor
agents in treating septic shock,2 but this
has not been pursued systematically. In
contrast, both eNOS and nNOS are con-
stitutively expressed and are thought to
©2001 American Medical Association. All rights reserved.
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contribute to the normal regulation of
vasomotor tone and blood pressure
(FIGURE). In the vascular endothelium,
eNOS converts L-arginine to nitric oxide,
which diffuses to the adjacent vascular
smooth muscle where it activates a series
of G-kinases culminating in vasodila-
tion.3 This is the classic nitric oxide path-
way of endothelial-dependent vasodila-
tion.4 In addition, both nNOS and eNOS
are expressed in the central nervous sys-
tem and emerging evidence suggests that
neuronally derived nitric oxide is an
important component of the signal trans-
duction pathway that tonically restrains
sympathetic outflow from the brain-
stem, placing a brake on a-adrenergic
vasoconstriction.5 Thus, nitric oxide is
thought to reduce peripheral vasomo-
tor tone and blood pressure both by caus-
ing active vasodilation and decreasing
central sympathetic vasoconstrictor drive.
Interruption of either protective mecha-
nism could contribute to hypertension.
Decreased NOS Expression
as a Cause of Hypertension
Experimentally, the most efficient way
to reduce NOS expression is to develop
knockout mice by targeted disruption of
one of the 3 NOS genes. The eNOS
knockout mouse develops mild-to-
moderate hypertension. Basal blood pres-
sure levels are typically 20 mm Hg higher
than in control littermates.6 To deter-
mine if deficient eNOS expression is a
cause of clinical hypertension, numer-
ous investigators are searching for single
nucleotide polymorphisms or other
mutations in the NOS genes, but so far
there is no general consensus that any
of the NOS genes are linked to human
hypertension. 7 On the other hand,
increased NOS expression constitutes an
exciting potential new form of antihy-
(Reprinted) JAMA, April 25, 2001—Vol 285, No. 16
pertensive therapy. When normoten-
sive mice were engineered to overex-
press constitutively active eNOS, their
mean blood pressure levels were 18
mm Hg lower than normal.8 In sponta-
neously hypertensive rats, a single intra-
venous injection of human complemen-
tary DNA encoding eNOS almost
completely normalized blood pressure for
5 to 6 weeks.9 However, a number of tech-
nical problems with delivery systems
remain to be solved before cardiovascu-
lar gene therapy can be realized in clini-
cal medicine.
However, increased nitric oxide
production underlies some of the anti-
hypertensive effects of currently used
cardiovascular therapeutics. For ex-
ample, both exercise training and HMG-
CoA reductase inhibitors (statin drugs)
have been shown to increase eNOS
expression in the vessel wall10,11 and this
increase may account in part for the mod-
est blood pressure–lowering effects of
these interventions. Estrogen replace-
ment therapy increases expression of
both eNOS and nNOS in rats,12 and these
effects could underlie some of the car-
dioprotective effects of estrogen and ex-
plain why postmenopausal estrogens do
not cause hypertension. Some of the an-
tihypertensive action of angiotensin-
converting enzyme inhibitors is due to
the decreased breakdown of bradyki-
nin resulting in receptor-mediated acti-
Author Affiliations: Department of Internal Medi-
cine, Hypertension Division, and the Donald W. Rey-
nolds Cardiovascular Clinical Research Center, Uni-
versity of Texas Southwestern Medical Center, Dallas.
Corresponding Author and Reprints: Ronald G. Vic-
tor, MD, Department of Internal Medicine, Hyper-
tension Division, University of Texas Southwestern
Medical Center, 5323 Harry Hines Blvd, Room J4.134,
Dallas, TX 75390-8586 (e-mail: ronald.victor
@utsouthwestern.edu).
Contempo Updates Section Editors: Stephen J.
Lurie, MD, PhD, Senior Editor; Alice T. D. Hughes, MD,
Fishbein Fellow.
2055
 DRUG TARGETS FOR REFRACTORY HYPERTENSION
vation of eNOS and thus improved en-
dothelial-mediated vasodilation.1
In addition, excessive nitric oxide–
mediated vasodilation accounts for the
marked and sometimes fatal hypoten-
sive response to nitrates in patients receiv-
ing sildenafil, a phosphodiesterase inhibi-
tor. 13 Nitric oxide donors, such as
nitroglycerin, cause vasodilation by
increasing cyclic guanine monophos-
phate in the vessel wall and this increase
is augmented when breakdown of cyclic
guanine monophosphate is prevented by
phosphodiesterase inhibition.1,13
Endogenous NOS Inhibitors
as a Cause of Hypertension
The enzymatic activities of eNOS and
nNOS normally are not limited by sub-
strate availability because L-arginine is
both produced endogenously and is
plentiful in the diet. In normotensive
animals and humans, both NOS iso-
forms can be inhibited pharmacologi-
Figure. Modulation of the Nitric Oxide (NO) Pathway
Blood Vessel Wall
Exercise
Statins
Estrogen
L-Arginine
ENDOTHELIUM
NO
Nitroglycerin
ACEI Antioxidants
Angiotensin ΙΙ
Cytokines
• O2-
ONOO-
VASCULAR
SMOOTH MUSCLE
Factors that decrease NO availability are shown in red; factors that increase NO availability are shown in blue. Dashed lines indicate inhibitory effect; solid thin arrows
indicate positive effect. ACEI indicates angiotensin-converting enzyme inhibitors; BKase, bradykininase; ADMA, asymmetric dimethyl arginine; eNOS, endothelial NO
synthase; nNOS, neuronal NO synthase; ·02−, superoxide anion; ONOO, peroxynitrite; GTP, guanosine triphoshate; cGMP, cyclic guanosine monophosphate; GMP,
guanosine monophosphate; PKG, cyclic GMP-dependent protein kinase; PDE, phosphodiesterase.
2056 JAMA, April 25, 2001—Vol 285, No. 16 (Reprinted)
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cally by oral or intravenous adminis-
tration of methylated arginines, which
are competitive NOS inhibitors.6,7 These
pharmacologic agents cause marked
and dose-dependent elevations in blood
pressure level, suggesting that nitric ox-
ide production is one of the most im-
portant defense mechanisms against hy-
pertension.
The potential clinical relevance of these
experimental observations is that meth-
ylated arginines constitute putative en-
dogenous NOS inhibitors. Asymmetric
dimethyl arginine (ADMA) and sym-
metric dimethyl arginine are endoge-
nous substances first isolated from hu-
man plasma and urine. When infused
into the human brachial artery, ADMA
causes vasoconstriction whereas sym-
metric dimethyl arginine is inert.14 Be-
cause ADMA normally is cleared by the
kidney, the plasma concentration in-
creases 3- to 10-fold in humans and ani-
mals with renal failure.15 Thus, accumu-
ACEI
Bradykinin
BKase
Inactive
Peptides
eNOS ADMA
L-Citrulline
Guanylyl
Cyclase
Sildenafil
NO
GTP cGMP GMP
PDE
PKG
Vasodilation
©2001 American Medical Association. All rights reserved.
lation of this endogenous NOS inhibitor
has been hypothesized (but not yet
proven) to contribute to hypertension in
patients with chronic renal failure. In ad-
dition, plasma ADMA concentrations
also are increased in animals and pa-
tients with atherosclerosis, possibly be-
cause of a local defect in the enzyme that
hydrolyzes ADMA in endothelial cells.16
Thus, excessive accumulation of ADMA
provides a potential explanation for the
frequent coexistence of hypertension and
atherosclerosis. In rats, oral L-arginine
has been shown to abrogate salt-
dependent hypertension and renal pa-
renchymal hypertension, 2 forms of hy-
pertension accompanied by increased
plasma ADMA concentrations.17 In the
clinical setting, long-standing salt-
sensitive hypertension and renal paren-
chymal hypertension often cannot be
optimally controlled with current anti-
hypertensive regimens. Large random-
ized clinical trials are needed to de-
Brainstem Vasomotor Center
L-Arginine
nNOS
NO
eNOS
L-Citrulline
ADMA
Decreased
Sympathetic
Outflow
SYMPATHETIC
BLOOD
NERVE
PATHWAY
VESSEL
Decreased Activation of
α-Adrenergic Receptors
Decreased
Vasoconstriction

termine if L-arginine constitutes an ef-
fective add-on antihypertensive therapy
in such refractory cases. Although total
body nitric oxide production is re-
duced in patients with chronic renal fail-
ure,18 the only small prospective study
to examine dietary supplementation with
L-arginine found no effect on blood pres-
sure or renal function in patients with
moderate renal insufficiency.19 The nega-
tive results may be explained by the nor-
mal plasma L-arginine concentrations in
the cohort and that blood pressure was
already well controlled with traditional
antihypertensive agents.
Superoxide Production
as a Cause of Nitric
Oxide-Deficient Hypertension
Nitric oxide–deficient hypertension may
also be caused by decreased nitric ox-
ide bioavailability due to oxidative inac-
tivation of nitric oxide by superoxide an-
ion and other reactive oxygen species.
While numerous enzyme systems pro-
duce reactive oxygen species, the best
studied are (1) xanthine oxidoreduc-
tase, (2) NOS, and (3) nicotinamide-
adenine dinucleotide phosphate
(NAD[P]H) oxidases, the latter being the
major source of reactive oxygen species
in the vessel wall. The activity of NAD
(P)H oxidases is increased by physical
stress (eg, increased blood pressure), cy-
tokines, and angiotensin II.20 There is in-
creasing experimental evidence that gen-
eration of superoxide anion by NAD
(P)H oxidases is a major mechanism by
which minimal elevations in plasma re-
nin and angiotensin II levels lead to sus-
tained elevations in blood pressure.20 By
the same token, reduced generation of
superoxide and restoration of endothe-
lial-dependent vasodilation appears to be
an important mechanism mediating the
antihypertensive and cardioprotective ef-
fects of angiotensin-converting enzyme
inhibitors and angiotensin-receptor
blockers.21,22
Superoxide dismutases are enzymes
produced in blood vessels that nor-
mally inactivate superoxide. Superox-
ide dismutase, when delivered in mem-
brane-permeable liposomes, normalized
blood pressure in rats made hyperten-
©2001 American Medical Association. All rights reserved.
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DRUG TARGETS FOR REFRACTORY HYPERTENSION
sive with infusion of angiotensin II.21 Fur-
thermore, administration of either su-
peroxide dismutase or tempol (4-
hydroxy-tempo, a superoxide dismutase
mimetic) normalized blood pressure in
spontaneously hypertensive rats but had
no effect on blood pressure in normal
rats.22 Nonetheless, it remains to be seen
whether antioxidants constitute an ef-
fective form of clinical antihypertensive
therapy. A recent study of 39 patients
with mild hypertension found that 1
month of monotherapy with 500 mg/d
of vitamin C decreased systolic blood
pressure by 11 mm Hg and diastolic
blood pressure by 6 mm Hg; these de-
creases were significantly larger than with
placebo.23 However, several other small
clinical studies showed negligible ef-
fects of vitamin C on blood pressure.24
The major limitation in bringing this
promising area of basic science to clini-
cal practice is the need to develop far
more effective antioxidants for human
use. Compared with tempol or liposome-
encapsulated superoxide dismutase used
in animal experiments, vitamin C is a
weak antioxidant. Increased antioxi-
dant production with improved endo-
thelial-dependent vasodilation is likely
to be a potential mechanism underly-
ing the mild antihypertensive effect of
regular aerobic exercise.25
Perspective
Experimental animal studies have pro-
vided a new conceptual framework for
understanding how nitric oxide defi-
ciency can cause hypertension and how
specific components of the nitric ox-
ide pathway may constitute novel an-
tihypertensive drug targets. The chal-
lenge now is to translate this research
from the bench to the clinic.
Funding/Support: This work was supported by the
Donald W. Reynolds Foundation and National Heart,
Lung, and Blood Institute grants HL64784 (Dr
Thomas), HL44010 (Drs Victor and Zhang), and
HL06296 (Drs Thomas and Victor).
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