The caveolar nitric oxide synthase/arginine regeneration system for NO production in endothelial cells
Larry P. Solomonson*,
Brenda R. Flam,
Laura C. Pendleton,
Bonnie L. Goodwin and
Duane C. Eichler
Department of Biochemistry and Molecular Biology, University of South
Florida College of Medicine, Tampa, FL 33612, USA
*
Author for correspondence (e-mail:
lsolomon{at}hsc.usf.edu)
Accepted 6 March 2003
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Summary
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The enzyme endothelial nitric oxide synthase (eNOS) catalyzes the
conversion of arginine, oxygen and NADPH to NO and citrulline. Previous
results suggest an efficient, compartmentalized system for recycling of
citrulline to arginine utilized for NO production. In support of this
hypothesis, the recycling enzymes, argininosuccinate synthase (AS) and
argininosuccinate lyase (AL), have been shown to colocalize with eNOS in
caveolae, a subcompartment of the plasma membrane. Under unstimulated
conditions, the degree of recycling is minimal. Upon stimulation of NO
production by bradykinin, however, recycling is co-stimulated to the extent
that more than 80% of the citrulline produced is recycled to arginine. These
results suggest an efficient caveolar recycling complex that supports the
receptor-mediated stimulation of endothelial NO production. To investigate the
molecular basis for the unique location and function of endothelial AS and AL,
endothelial AS mRNA was compared with liver AS mRNA. No differences were found
in the coding region of the mRNA species, but significant differences were
found in the 5'-untranslated region (5'-UTR). The results of these
studies suggest that sequence in the endothelial AS-encoding gene, represented
by position -92 nt to -43 nt from the translation start site in the extended
AS mRNA 5'-UTRs, plays an important role in differential and
tissue-specific expression. Overall, a strong evidential case has been
developed supporting the proposal that arginine availability, governed by a
caveolar-localized arginine regeneration system, plays a key role in
receptor-mediated endothelial NO production.
Key words: nitric oxide, eNOS, endothelial nitric oxide synthase, arginine, citrulline, arginine regeneration system, argininosuccinate synthase, argininosuccinate lyase, caveolae, nitric oxide production
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Introduction
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Endothelial nitric oxide synthase (eNOS), the enzyme that catalyzes the
production of NO from the amino acid arginine in endothelial cells, plays a
key role in vasoregulation as well as in other important physiological
processes such as angiogenesis. Impaired production of endothelial NO has been
associated with hypertension, heart failure, hypercholesterolemia,
atherosclerosis and diabetes (Govers and
Rabelink, 2001
; Vallance and
Chan, 2001
; Maxwell,
2002
). Circulating effectors, such as bradykinin, bind to
receptors on the luminal surface of endothelial cells, signaling the transient
release of NO to the adjacent smooth muscle layer and resulting in relaxation
of the vessel wall.
The signal for eNOS activation is a transient increase in intracellular
calcium, which activates the enzyme through binding of a calcium-calmodulin
complex (Ca-Cam). Endothelial NOS activation also occurs in response to shear
stress (Govers and Rabelink,
2001
; Maxwell,
2002
). Consistent with the important physiological roles of eNOS,
the enzyme appears to be subject to multiple modes of regulation, in addition
to primary regulation through reversible Ca-Cam binding and activation. These
include reversible phosphorylation and palmitoylation, substrate and cofactor
availability, dimerization of enzyme subunits, intracellular translocation and
protein-protein interactions (Govers and
Rabelink, 2001
). Several of these potential modes of regulation
appear to be interrelated. As a component of caveolae, a subcompartment of the
plasma membrane that serves to sequester proteins involved in cell signaling,
eNOS may transiently interact with several different caveolar components.
Previous work from several different laboratories has suggested that a diverse
group of proteins, including calmodulin, caveolin-1, bradykinin B2 receptor,
heat shock protein 90, argininosuccinate synthase (AS), argininosuccinate
lyase (AL), Raf-1, Akt, extracellular signal-related kinase, eNOS interacting
protein, eNOS traffic inducer and unidentified tyrosine-phosphorylated
proteins (Hellermann et al.,
2000
; Govers and Rabelink,
2001
; Maxwell,
2002
; Nedvetsky et al.,
2002
), may be transiently and functionally associated with
eNOS.
A potential limiting factor for endothelial NO production is the
availability of the substrate, arginine. Intracellular levels of arginine have
been estimated to range from 100 µmol l-1 to 800 µmol
l-1, which is well above the Km value of 5
µmol l-1 for eNOS (Harrison,
1997
). Endothelial NO production can, nonetheless, be stimulated
by exogenous arginine (Vallance and Chan,
2001
). This phenomenon, termed the 'arginine paradox', suggests
the existence of a separate pool of arginine directed to endothelial NO
synthesis. As illustrated in Fig.
1, arginine has a number of metabolic roles in addition to NO
production, including production of major metabolites such as urea,
polyamines, creatine, ornithine and methylarginine derivatives. The observed
stimulation of endothelial NO production by exogenous arginine suggests that
the arginine directed to NO production may be segregated from bulk cellular
arginine utilized for these other metabolic roles.

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Fig. 1. Metabolic roles and fates of arginine. In addition to incorporation into
protein, arginine serves as a metabolic precursor for several important
metabolites, as indicated by the arrows. Also indicated is the two-step
conversion of citrulline to arginine.
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One possible site of control is at the level of arginine uptake. McDonald
et al. (1997
) showed that the
CAT1 transporter, responsible for 60-80% of total carrier-mediated arginine
transport into endothelial cells, colocalizes with eNOS in caveolae. They
proposed that the arginine utilized by eNOS might, at least in part, be
maintained by the CAT1 transporter. Another important mechanism for
controlling the availability of arginine directed to NO production may be the
regeneration of arginine from the other product of the eNOS-catalyzed
reaction, citrulline. Hecker et al.
(1990
) initially demonstrated
that citrulline, produced in the conversion of arginine to NO, can be recycled
to arginine. A possible link between NO production and arginine regeneration
from citrulline was subsequently established for other cell types
(Nussler et al., 1994
;
Shuttleworth et al., 1995
).
This regeneration is catalyzed by the enzymes AS and AL, both of which also
play an essential role in the urea cycle in liver. The potential importance of
this regeneration system for endothelial NO production was supported by a
report of two infants with a deficiency of AL who were shown to be
hypertensive (Fakler et al., 1995). Upon infusion of arginine, the blood
pressure of these infants decreased to near normal levels, suggesting a
critical role for arginine regeneration in the regulation of systemic blood
pressure. More recent evidence from DNA microarray analysis suggests an
important role for the arginine regeneration system by clearly demonstrating
significant and coordinate upregulation of AS-encoding gene expression in
response to shear stress stimulation of endothelial NO production
(McCormick et al., 2001
). It
was concluded that available arginine is a prerequisite for NO production and
that in the absence of synthesis of additional eNOS, shear stress-induced
increases in NO synthesis depend on an increase in synthesis of arginine from
citrulline through increased AS expression. Although supplemental arginine can
be beneficial in some cases (Wu and
Meininger, 2002
), in other cases it may lead to adverse effects
owing to the multiple metabolic roles of arginine
(Chen et al., 2003
;
Loscalzo, 2003
).
Recent work further supports the hypothesis that the arginine regeneration
system, comprised of a caveolar complex that includes eNOS, AS and AL, plays
an important, and most likely essential, role in the receptor-mediated
production of NO by vascular endothelial cells.
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Effects of exogenous arginine and citrulline on endothelial NO
production
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Endothelial NOS is localized in plasmalemmal caveolae. The localization of
eNOS in this signaling subcompartment of the plasma membrane may have
important implications with regard to the regulation and catalytic efficiency
of eNOS (Everson and Smart,
2001
; Shaul,
2002
). We have recently found evidence for an efficient cycling of
citrulline to arginine, raising the possibility of a channeling complex of
eNOS and the enzymes of the citrulline-arginine cycle (AS and AL) localized in
caveolae. Our initial research effort that led to this finding was designed to
test the hypothesis that an intracellular pathway exists for the generation of
methylarginines to regulate NO production in nitric oxide-producing tissues.
The goal of this initial work was to determine the physiological significance
of intracellular methylarginines as regulators of NOS activity. To examine the
levels of endogenous methylarginines, we developed methods that allowed for
the rapid and quantitative analysis (by HPLC) of arginine, citrulline and the
methylarginines from endothelial cell extracts. There was no apparent change
in levels of methylarginines following stimulation of endothelial cells with
either bradykinin or the calcium ionophore A23187. In an attempt to raise
intracellular methylarginine levels, and further test our hypothesis, we added
citrulline, which we expected to inhibit dimethylarginine
dimethylaminohydrolase, the enzyme that converts
NG-methylarginine or
NG,NG-dimethylarginine to citrulline
and monomethylamine or dimethylamine, respectively. The objective was to
determine whether inhibition of the degradation of methylarginines would
increase their intracellular concentrations and thereby inhibit NO production.
To our surprise, stimulation of NO production by bradykinin was increased by
the addition of citrulline, rather than decreased, and there was no apparent
change in methylarginine levels. To further examine the molecular basis for
the stimulation of NO production by citrulline, we compared the effect of
exogenous citrulline with the effect of exogenous arginine on NO production
and levels of intracellular arginine following bradykinin activation.
Surprisingly, added arginine did not cause as great an increase in endothelial
NO production as did added citrulline. In addition, there was a much larger
increase in intracellular arginine in response to exogenous arginine compared
with exogenous citrulline. Added citrulline caused only a modest increase in
intracellular arginine, while added arginine caused a substantial increase.
Thus, there appeared to be no correlation between total intracellular arginine
levels and endothelial NO production. To the best of our knowledge, this
represents the first attempt to correlate NO production with the levels of
intracellular arginine. Furthermore, the effects of arginine and citrulline on
NO production appeared to be synergistic, since a combination of arginine and
citrulline stimulated endothelial NO production more than did either arginine
or citrulline alone (Flam et al.,
2001
). Since arginine has a number of potential metabolic fates,
while citrulline has only one known metabolic fate
(Fig. 1), the efficiency of NO
production could be enhanced if a separate pool of arginine is maintained by
endothelial cells. Recycling the product of the NOS-catalyzed reaction,
citrulline, back to arginine via the enzymes of the arginine
regeneration system, AS and AL, would maintain this separate pool. The pool of
arginine used for NO synthesis would be essentially isolated from the bulk of
intracellular arginine through the efficient operation of an arginine
regeneration system. The apparent efficiency of the process suggests a
channeling of intermediates and a compartmentalized complex of eNOS and
enzymes of the arginine regeneration system. These results further support a
model in which eNOS is localized together with this arginine regenerating
system, and regulatory components, to ensure optimal efficiency of NO
production and regulation without affecting other arginine-dependent cellular
processes.
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Caveolar localization of arginine regeneration enzymes with eNOS
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Endothelial NOS is targeted by acylation to caveolae, where it interacts
with caveolin-1 (Everson and Smart,
2001
; Shaul,
2002
). In liver cells, the arginine-generating enzymes AS and AL
are associated with the outer mitochondrial membrane, reflecting the
functional role of these enzymes in the production of urea
(Cohen and Kuda, 1996
). To test
the model for a colocalization of AS and AL with eNOS, we used two different
fractionation protocols for the purification of caveolae
(Smart et al., 1995
;
Song et al., 1996
). Both
protocols generated a caveolar membrane fraction that was highly enriched in
caveolin-1, eNOS, AS and AL (Flam et al.,
2001
). These results support the proposal that a separate pool of
arginine, directed to NO synthesis, is effectively separated from the bulk of
intracellular arginine through the functional localization of arginine
regeneration enzymes and eNOS with plasmalemmal caveolae. A possible
consequence of this functional association would be the channeling of
intermediates through AS, AL and eNOS such that intermediates of the complex
would not equilibrate with bulk intracellular arginine.
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Degree of recycling
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Cellular activity of eNOS has been estimated by measuring the rate of
conversion of [3H]arginine to [3H]citrulline
(Hardy and May, 2002
). If
recycling of citrulline to arginine is tightly coupled to NO production, this
measurement would underestimate the cellular activity of eNOS. Estimating
cellular activity of eNOS by measuring rate of production of NO (as the
degradation product nitrite), on the other hand, should give a better estimate
of cellular activity of eNOS. To test this hypothesis, and to estimate the
degree of recycling of citrulline to arginine, we simultaneously measured the
apparent rate of arginine-to-citrulline conversion and the rate of production
of NO under both unstimulated and stimulated (addition of bradykinin)
conditions. The ratio of these activities was close to one under unstimulated
conditions. An increase in the ratio of NO produced to citrulline produced was
approximately eight upon exposure of endothelial cells to agonist (B. R. Flam,
D. C. Eichler and L. P. Solomonson, unpublished), indicating that recycling
and NO production were costimulated. These preliminary results suggest an
efficient caveolar complex for the regeneration of arginine directed to
receptor-mediated production of NO in endothelial cells and an efficiency of
greater than 80% for the recycling of citrulline to arginine under conditions
of maximum stimulation of NO production. Although recycling of citrulline to
arginine has been assumed to be important for conservation and efficient
utilization of arginine, the degree of recycling relative to NO production has
not, to the best of our knowledge, been quantified. Our results suggest that
this recycling, especially under stimulated conditions, may play a more
important role in endothelial NO production than previously recognized.
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Molecular basis for functional role and location of endothelial
AS
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In liver tissue, AS plays an essential role in urea synthesis and appears
to be associated with the outer mitochondrial membrane
(Cohen and Kuda, 1996
). By
contrast, endothelial AS appears to be the rate-limiting enzyme in the
recycling of citrulline to arginine used for NO synthesis and is localized in
caveolae (Flam et al., 2001
).
Immunoblotting experiments suggested small differences in subunit molecular
masses and isoelectric points of endothelial AS compared with liver AS (B. R.
Flam, D. C. Eichler and L. P. Solomonson, unpublished). We speculated that
these differences could be due to a splice variant, but analysis of the coding
sequence of AS mRNA indicated no differences between the mRNA from endothelial
cells and liver (Pendleton et al.,
2002
). Because upstream and downstream untranslated regions (UTRs)
of mRNA can influence regulation of gene expression, we carried out both
5'-RACE (rapid amplification of cDNA ends) and 3'-RACE analysis to
investigate possible differences in the UTRs. We found AS mRNA species with
three different length 5'-UTRs in endothelial cells
(Fig. 2). Only one of these
products, the shortest 5'-UTR of 43 nt, was quantitatively expressed in
liver. No significant variation was found in the 3'-UTR. The
5'-RACE analysis identified endothelial AS mRNA species with extended
5'-UTRs of 66 nt and 92 nt, in addition to a major 43 nt 5'-UTR AS
mRNA (Fig. 2). Compositional
analysis revealed that all three AS mRNA 5'-UTRs were enriched in G+C
content (approximately 76%) and were likely to form complex and stable
secondary structures. An upstream open reading frame (uORF) that was
out-of-frame with the AS mRNA AUG start codon was detected in the 66 nt and 92
nt 5'-UTRs. RNase protection analysis (RPA) and real-time reverse
transcriptase-PCR (RT-PCR) verified and quantified the differential expression
of the extended 5'-UTR species relative to the major 43 nt 5'-UTR
AS mRNA. Estimates from RPA of the amount of the 92 nt and 66 nt species,
relative to the 43 nt species, were approximately 15% and 13%,
respectively.
Features of mRNA UTRs, specifically uORFs, are regarded as important
determinants of translational efficiency and may have important biological
implications for the regulation of translation. We therefore designed
experiments to determine to what extent the various 5'-UTRs of AS mRNA
influenced translation. Translational efficiencies for the 66 nt and 92 nt AS
5'-UTR constructs were 70% and 25%, respectively, of the translational
efficiency for the 43 nt 5'-UTR AS mRNA. Sequential deletions, starting
with the 5'-terminus of the 92 nt 5'-UTR construct, resulted in a
corresponding increase in translational efficiency, but the most pronounced
effect resulted from mutation of the uORF, which restored translational
efficiency to that observed with the 43 nt species. When the different AS mRNA
5'-UTRs, cloned in front of a luciferase reporter gene, were transfected
into endothelial cells, the pattern of luciferase expression was nearly
identical to that observed for the different 5'-UTR AS mRNAs in
endothelial cells. These results suggest that a complex
transcriptional/translational infrastructure exists to coordinate AS
expression and NO production (Pendleton et
al., 2002
).
 |
Model for coupling of arginine regeneration to endothelial NO
production
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A model depicting our view of the coupling of arginine regeneration to
endothelial NO production through the compartmentalized complex of AS, AL and
eNOS is shown in Fig. 3. This
coupling may be largely 'disengaged' under unstimulated conditions but is
'engaged' and tightly coupled in response to agonists such as bradykinin. The
molecular determinants and mechanisms involved in this coupling are not fully
understood at this time. Based on our studies, and evidence from other labs,
we believe the coupling of arginine regeneration to endothelial NO production
is important for the overall regulation of endothelial NO production and may
be essential for agonist-stimulated endothelium-dependent vasorelaxation.

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Fig. 3. Model for the coupling of endothelial NO production to the regeneration of
the substrate, arginine, from the product, citrulline. Shown is the CAT1
transporter involved in arginine transport and the complex of
argininosuccinate synthase and argininosuccinate lyase with endothelial nitric
oxide synthase.
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Acknowledgments
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The work described here was supported by the American Heart Association
National Grant 9750222N, American Heart Association Florida Affiliate Grant
9950864V and the Mary and Walter Traskiewicz Memorial Fund.
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