© 1996 American Heart Association, Inc.
Articles |
Differential Regulation of L-Arginine Transport and Nitric Oxide Production by Vascular Smooth Muscle and Endothelium
From the Houston VA Medical Center and the Departments of Medicine (W.D., L.L., I.I., A.I.S.), Pharmacology (W.D.), and Molecular and Human Genetics (W.E.O.), Baylor College of Medicine, Houston, Tex.
Correspondence to Dr William Durante, Houston VA Medical Center, Bldg 109, Room 116, 2002 Holcombe Blvd, Houston, TX 77030.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Abstract Since NO production is dependent on the availability of L-arginine, we examined whether L-arginine transport and NO synthesis are coregulated by vascular smooth muscle cells and endothelial cells cultured from the same vessel wall source. L-Arginine transport by both bovine aortic smooth muscle cells (BASMCs) and endothelial cells (BAECs) was primarily Na+ independent (
70%) and was mediated by both a high- and
low-affinity transport system. Treatment of BASMCs with tumor
necrosis factor-
(TNF-) or interleukin-1ß (IL-1ß) resulted in
a significant increase in L-arginine transport (20%)
and in the induction of NO release. Exposure of BASMCs to interferon
gamma (IFN-
) or lipopolysaccharide (LPS) also stimulated
NO release but did not affect L-arginine transport. In
contrast, incubation of BAECs with TNF- or LPS strikingly enhanced
L-arginine uptake (2.5-fold), whereas IL-1ß and IFN-
had no effect. Treatment of BAECs with any of the inflammatory
mediators did not stimulate NO production. These results
demonstrate that L-arginine uptake and NO synthesis by
these cells are differentially regulated. In BASMCs, the coinduction of
L-arginine transport and NO formation may function to
provide increased levels of substrate to the cell during activation of
the NO synthase enzyme. In contrast, the selective stimulation of
L-arginine uptake in BAECs indicates that
L-arginine transport is dissociated from NO generation in
these cells.
Key Words: nitric oxide synthase • amino acids • smooth muscle • endothelium
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A cationic, dibasic, semiessential amino acid, L-arginine mediates numerous biological functions.1 2 It is an intermediate in the urea cycle and is a necessary precursor for protein, creatine, and polyamine biosynthesis. In addition, L-arginine stimulates the release of several hormones and may play a role in host immunity. More recently, L-arginine has been identified as the exclusive precursor for the multifunctional molecule NO.3 4 This simple diatomic gas is synthesized from the terminal guanidino nitrogen atoms of L-arginine by the action of a group of enzymes known as NOSs.5 In vascular cells, this oxidative reaction is mediated by two distinct classes of NOS. A constitutively active Ca2+-calmodulin–dependent isoform of the enzyme predominates in endothelial cells, whereas a Ca2-insensitive isoform is induced by inflammatory cytokines in vascular smooth muscle.6 7 8 9 In the circulation, the release of NO by vascular cells plays an important role in regulating blood flow by inhibiting vascular tone and platelet adhesion and aggregation.10 11 12 In addition, the production of NO by the blood vessel wall may limit intimal hyperplasia following local vascular injury by inhibiting smooth muscle proliferation and migration.13 14 15
Cellular NO production is absolutely dependent on the availability of L-arginine. This amino acid can be obtained from exogenous sources via a plasma membrane transporter or from intracellular sites by protein degradation or by endogenous synthesis. Recent studies indicate that NO synthesis by vascular smooth muscle is strictly dependent on the presence of extracellular L-arginine. Both the cytokine-stimulated release of NO by vascular smooth muscle cells and the NO-induced vascular hyporeactivity following endotoxin administration can be prevented by eliminating L-arginine from the extracellular environment.8 16 In addition, the recent finding that an inhibitor of L-arginine uptake, L-lysine, blocks IL-ß-stimulated NO production by vascular smooth muscle cells suggests that L-arginine transport can become the rate-limiting step in NO synthesis by these cells.17 In contrast, NO synthesis by endothelial cells appears to be less dependent on extracellular L-arginine. The exogenous administration of L-arginine does not affect NO release by endothelial cells in culture or in situ.18 19 Furthermore, incubation of endothelial cells for up to 24 hours in L-arginine–deficient media only marginally reduces NO synthesis.5 Since NO generation by vascular smooth muscle and by endothelium has contrasting requirements for extracellular L-arginine, the present study directly compared L-arginine transport by these different cell types cultured from the same vascular source, specifically BASMCs and BAECs. In addition, the ability of different inflammatory cytokines to modulate L-arginine uptake and NO production by these cells was studied.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cell Culture
BASMCs and BAECs (third passage) were provided by Dr Timothy Scott-Burden (University of Texas, Houston). These cells were isolated from the same bovine thoracic aorta by enzymatic digestion using standard methods and characterized morphologically and immunocytochemically as described previously.20 21 Cells were cultured serially, and responses were compared at identical passages. BASMCs were grown in MEM, whereas BAECs were propagated in DMEM. In both instances, media contained 600 µmol/L L-arginine and were supplemented with 20 mmol/L HEPES-NaOH, 10% (vol/vol) heat-inactivated FCS, 100 U/mL penicillin, and 100 U/mL streptomycin. Subcultured cells were seeded onto 12-well plates and used in passages 5 to 20. Once cells reached confluence (
3 days), the culture media were replaced with
serum-free media containing 0.1% (wt/vol) fatty acid–free BSA
for 24 hours. In some experiments, cells were exposed to TNF- (1 to
100 ng/mL), IL-1ß (1 to 100 ng/mL), IFN- (1 to 100 U/mL), LPS (0.3
to 3.0 µg/mL), or their respective vehicles for an additional 24
hours before their use.
L-Arginine Transport Assay
Transport of [3H]L-arginine by
vascular cells was determined essentially as described by Gazzola et
al.22 In all experiments, cells were washed twice with 2.0
mL of HEPES buffer (mmol/L: KCl 5, CaCl2 0.9,
MgCl2 1, D-glucose 5.6, and HEPES 25, pH 7.4)
containing either 140 mmol/L NaCl or choline chloride. Transport assays
were performed by incubating cells at 22°C with 0.5 mL HEPES buffer
containing [3H]L-arginine (2 µCi/mL).
Transport activity was terminated by aspirating the media and rapidly
washing the cells with ice-cold uptake buffer (five times with 1
mL). The cells were allowed to dry, and the cell-associated
radioactivity was extracted with 500 µL of 0.2% SDS in 0.2N NaOH and
then assayed by liquid scintillation spectrometry (Tri-Carb liquid
scintillation analyzer, model 1900 TR, Packard). Protein in the
NaOH extracts was measured by using the bicinchoninic acid method with
BSA as the standard.23 To correct for nonspecific uptake
or binding, cells were incubated in parallel wells with HEPES buffer
containing 10 mmol/L L-arginine, the fraction of the
radioactivity associated with the cells was determined, and this
fraction was then subtracted from each data point.
NO Measurement
The generation of NO by vascular cells was determined by
measuring the extracellular release of nitrite, the stable oxidation
product of NO,24 and by monitoring the intracellular
accumulation of L-citrulline.25 For nitrite
measurements, aliquots (400 µL) of culture medium were mixed with an
equal volume of Griess reagent [1% sulfanilamide/0.1%
N-(1-naphthyl)ethylenediamine dihydrochloride in 2%
phosphoric acid], and after color development, the optical densities
were determined at 540 nm (Ultraspec III, Pharmacia).26
Nitrite concentrations were determined relative to a standard curve by
using an aqueous solution of sodium nitrite, and background nitrite
values corresponding to serum-free medium were subtracted from
experimental values.
L-Citrulline formation was monitored by adding [14C]L-arginine (0.5 µCi) to vascular cells incubated in 500 µL of serum-free media. After a 6-hour incubation, the [14C]L-arginine–containing media were removed, and the cells were washed three times with 2 mL PBS. The reactions were stopped by adding 300 µL of ice-cold Tris buffer (pH 6.8) containing Triton X-100 (0.01%), and cells were scraped, vortexed, and centrifuged at 1000g for 1 minute. Aliquots (10 µL) of the supernatant were spotted onto thin-layer chromatography plates and developed in the solvent system chloroform/methanol/ammonium hydroxide/water (1:4:2:1 [vol/vol/vol/vol]). After drying, L-arginine products were detected by ninhydrin spray, and [14C]L-citrulline was identified by cochromatography with unlabeled L-citrulline, scraped, and quantified by scintillation counting. This particular solvent system was also used to determine the extent of L-arginine metabolism, since it adequately resolves L-arginine and its various metabolites.27
Intracellular L-Arginine Measurement
Vascular cells were scraped in ice-cold PBS and
centrifuged at 1000g for 5 minutes at 4°C. The
cell pellet was then lysed in distilled water with three cycles of
freeze/thaw, and the cell debris was removed by
centrifugation. L-Arginine measurements
were performed using ion exchange chromatography on a
Beckman model 6300 amino acid analyzer with ninhydrin
detection. All values were normalized to the protein content of the
supernatant solution.
Western Blotting
Vascular cells were scraped in ice-cold PBS,
centrifuged at 1000g for 5 minutes at 4°C, and
lysed in electrophoresis buffer (125 mmol/L Tris-HCl [pH 6.8], 12.5%
glycerol, 2.5% dithiothreitol, 2% SDS, and trace bromophenol blue).
Whole-cell lysates were boiled for 10 minutes, and SDS-PAGE was
performed on 7.5% gels with 25 µg of protein using the buffer system
of Laemmli.28 The separated blots were electrophoretically
transferred to nitrocellulose membranes, as described by Towbin et
al.29 Nitrocellulose blots were blocked for 1 hour in PBS
containing 0.1% Tween 20 and 3% nonfat milk and then incubated with
anti-murine iNOS IgG (1:2500 dilution) or anti-human ecNOS IgG
(1:1000 dilution) in Tween 20 (0.1%) containing PBS for 1 hour. The
membrane was then washed in PBS and incubated for 1 hour with either
anti-mouse (1:5000 dilution) or anti-rabbit (1:7500 dilution)
horseradish peroxidase–conjugated antibody. After further washing
with PBS, blots were then incubated in commercial ECL reagents
(Amersham Corp) and exposed to photographic film for 5 minutes.
Materials
Fatty acid–free BSA, L-glutamine, LPS, SDS,
DMEM, ninhydrin spray, L-arginine, Triton X-100, Tween 20,
glycerol, dithiothreitol, L-citrulline, thin-layer
chromatography plates (silica gel-25), and the reagents
for nitrite determinations were purchased from Sigma Chemical Co; FCS,
penicillin, and streptomycin were from GIBCO; recombinant murine
TNF- and IFN- were from Genzyme; recombinant murine IL-1ß was
obtained from R & D Systems; MEM was from ICN Biomedicals; A23187 was
from Calbiochem-Novabiochem Corp; murine monoclonal antibody to mouse
macrophage iNOS and affinity-purified polyclonal rabbit
antibody to human ecNOS were obtained from Transduction Laboratories;
[3H]L-arginine (58 Ci/mmol) was from American
Radiolabeled Chemicals; and [14C]L-arginine
(300 mCi/mmol) was from Du Pont–New England Nuclear Research
Products.
Statistics
Results are expressed as mean±SEM. Statistical analysis
was performed with the use of Student's two-tailed t
test and an ANOVA when more than two treatments were compared. Values
of P<.05 were considered to be statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The time course of L-arginine uptake by both types of vascular cells is shown in Fig 1
. Specific transport of
50 µmol/L of [3H]L-arginine by BASMCs
increased over time and was linear for 10 minutes. In contrast,
uptake of L-arginine by BAECs was slower and showed
linearity for 15 minutes. The intracellular level of
L-arginine was 330 pmol/mg protein in BASMCs and
marginally decreased to 310 pmol/mg protein during the 30-minute time
course of L-arginine transport. In BAECs, basal
intracellular L-arginine concentration was 360
pmol/mg protein and remained at this level during the course of
L-arginine uptake. In addition, no significant
metabolism of L-arginine was detected by the
vascular cells. Even after 30 minutes of incubation, >95% of the
radioactivity in BASMCs and BAECs was present as
L-arginine. Thus, cellular efflux and
metabolism do not appear to significantly influence our
uptake studies.
|
) and BAECs (
). Transport was initiated by
the addition of 50 µmol/L [3H]L-arginine to
vascular cells incubated with choline-containing HEPES buffer, and
specific uptake was determined at indicated times. Results are
mean±SEM of four separate experiments, each performed in triplicate.
*Statistically greater (P<.05) increase in
L-arginine uptake by BASMCs compared with BAECs.
Substitution of choline in the uptake buffer with sodium at equimolar
concentrations significantly increased L-arginine transport
by both BASMCs and BAECs (Fig 2). However, 70% of
L-arginine uptake by vascular cells occurred through a
Na+-independent pathway. Given the predominance of
Na+-independent transport, all additional experiments
measured uptake over a time period of 10 minutes using
choline-containing buffer.
|
In subsequent kinetic studies, saturable uptake of radiolabeled
L-arginine (0.005 to 10 mmol/L) was determined. A
saturation plot of uptake velocity as a function of
L-arginine concentration by smooth muscle cells is shown in
Fig 3A. As evident from the Eadie-Hofstee plots, uptake
of L-arginine was biphasic, with two clearly separated
affinity states (Fig 3B). A high-affinity transporter having a
Michaelis constant (Km) of 25.0±0.5
µmol/L and a maximum transport velocity (Vmax) of
472±25 pmol/mg protein per minute was apparent. A second
low-affinity system was also demonstrated with a
Km of 754±68 µmol/L and a
Vmax of 1291±85 pmol/mg protein per minute. Kinetic
studies in endothelial cells also resolved
L-arginine transport into a high- and low-affinity
component (Fig 4). However, BAECs had a lower affinity
for L-arginine but a higher transport capacity than BASMCs.
In BAECs, the high-affinity transporter had a
Km of 93.8±6.0 µmol/L and a
Vmax of 846±66 pmol/mg protein per minute, whereas the
low-affinity system had a Km of
1076±51 µmol/L and a Vmax of 1704±107 pmol/mg protein
per minute. The differences in kinetics between BASMCs and BAECs were
not due to an overestimation in smooth muscle cell protein arising from
matrix secretion, since control experiments in which BASMCs were
selectively removed with EDTA (20 mmol/L) or lysed with ammonium
hydroxide (25 mmol/L) demonstrated that matrix protein contributed
<6% of total protein.
|
|
In the next series of experiments, we examined the capacity of
inflammatory cytokines to regulate L-arginine
transport in the two types of vascular cells. Treatment of BASMCs for
24 hours with TNF- (1 to 100 ng/mL) or IL-1ß (1 to 100 ng/mL)
stimulated L-arginine uptake by 20%. However, the
addition of IFN- (1 to 100 U/mL) or LPS (0.3 to 3.0 µg/mL) had no
effect on L-arginine transport (Fig 5A). In
contrast, treatment of BAECs with TNF- and LPS resulted in a marked
increase of up to 2.5-fold in L-arginine transport (Fig 5B). Incubation of BAECs with IL-1ß or IFN- had no effect on
L-arginine uptake. In both BASMCs and BAECs, inflammatory
cytokines selectively increased the Na+-independent
component of L-arginine transport. Treatment of vascular
cells with TNF- (100 ng/mL) in the presence of extracellular
Na+ did not further stimulate L-arginine
transport, indicating that cytokine treatment did not induce a
latent Na+-dependent transport system for
L-arginine (Fig 6).
|
|
Since TNF- elevated L-arginine transport in vascular
cells, kinetic studies were performed to determine the biochemical
mechanism(s) involved. Treatment of BAECs with TNF- (100 ng/mL) for
24 hours selectively increased the Vmax for
L-arginine uptake by both the high- and low-affinity
transport system, whereas the Km remained
unaltered (Table). In contrast, TNF- increased the
Vmax for only the high-affinity carrier in BASMCs
without affecting the Km of either
carrier system (Table).
|
Incubating BASMCs with TNF-, IL-1ß, IFN-, or LPS resulted in a
concentration-dependent increase in NO production, as
reflected by the extracellular accumulation of nitrite and the
intracellular formation of citrulline (Fig 7). In
contrast, treatment of BAECs with the same inflammatory mediators
failed to stimulate NO generation, as assessed by either nitrite
release or citrulline formation (Fig 8). Western blot
analysis revealed that TNF- (100 ng/mL) induced the
expression of iNOS protein in BASMCs but did not stimulate the
expression of iNOS in BAECs (Fig 9A). Alternatively,
immunoblotting indicated the presence of ecNOS in
BAECs, in both the presence and absence of TNF- (100 ng/mL),
but failed to detect ecNOS expression in BASMCs (Fig 9B). The selective
expression of ecNOS in BAECs was confirmed by the ability of the
calcium ionophore A23187 (10 µmol/L) to stimulate
[3H]L-citrulline formation from
[3H]L-arginine in BAECs (from 13±3 to 42±5
pmol/mg protein, P<.05) while having no effect on
[3H]L-citrulline production in BASMCs
(from 17±6 to 19±5 pmol/mg protein). The ionophore A23187 had
no effect on L-arginine transport by either cell type (data
not shown).
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The present study demonstrates that L-arginine transport by vascular cells is mediated by at least three distinct transport systems and that inflammatory mediators differentially regulate L-arginine uptake and NO production by these cells. In smooth muscle cells, TNF-
and IL-1ß coordinately
stimulate L-arginine transport and NO synthesis,
whereas in endothelial cells, TNF- and LPS
selectively enhance L-arginine transport without
stimulating NO release.
Plasma amino acid transport is a dynamic tissue-specific process
that involves discrete membrane-bound transport
proteins.30 Transport can be broadly classified into
Na+-dependent and Na+-independent systems. In
the present study, we demonstrate that L-arginine
transport in vascular cells is primarily Na+ independent
(70%) and that inflammatory cytokines selectively stimulate
Na+-independent uptake. Furthermore, our finding of both a
high- and low-affinity Na+-independent
L-arginine transport system in bovine vascular cells
supports previous studies that examined L-arginine uptake
in vascular cells derived from different animal species or from
different blood vessels17 31 32 33 and complements recent
reports that have identified both high- and low-affinity
Na+-independent cationic transport proteins in other cell
types.34 35
At physiological plasma concentrations (50 to 100 µmol/L), the high-affinity transporter would mediate most of the L-arginine transport by vascular cells.36 Interestingly, our kinetic studies revealed differences in L-arginine uptake between the two types of vascular cells derived from the same vessel wall preparation. Endothelial cells possess a lower affinity for L-arginine but a higher transport capacity than smooth muscle cells. This is reflected by a twofold to fourfold higher Km and a nearly twofold greater Vmax for both the high- and low-affinity transport systems. The physiological significance of this transport heterogeneity is not known but may reflect differences in substrate utilization and/or endogenous L-arginine biosynthetic activity between the two cell types.
Treatment of vascular cells with inflammatory mediators revealed
further differences in L-arginine transport and
metabolism between vascular smooth muscle and
endothelium. Incubation of smooth muscle cells with
TNF- or IL-1ß stimulates L-arginine transport in
conjunction with NOS activity, as determined by nitrite release and
citrulline formation. In contrast, treatment of
endothelial cells with TNF- or LPS results in a
striking increase in L-arginine uptake that is not linked
to an increase in NO production. These findings suggest that
L-arginine transport might be coupled to different
metabolic pathways in the two cells. Treatment of vascular
smooth muscle cells with inflammatory cytokines results in the
expression of iNOS,6 7 8 which is a high-output isoform
of the enzyme, the activity of which is strictly dependent on the
presence of extracellular L-arginine.37 This
isoform of NOS, however, is not present in all
endothelial cells. Although microvascular
endothelium expresses iNOS, recent reports indicate
that inflammatory mediators do not induce iNOS expression in
endothelial cells derived from large vessels but rather
regulate the activity and expression of the low-output ecNOS, which
is less dependent on extracellular
L-arginine.38 39 40 41 Our present findings
showing that inflammatory mediators fail to induce NO
production or iNOS protein in BAECs further support the absence
of iNOS expression in macrovascular endothelial cells.
Thus, the differential expression of NOS isoforms in vascular cells,
combined with their disparate requirements for extracellular
L-arginine, may explain the different responses of vascular
cells to inflammatory cytokines.
In smooth muscle cells, the coinduction of L-arginine
uptake and iNOS expression by TNF- and IL-1ß may provide a
mechanism by which increased levels of substrate are provided to smooth
muscle cells during activation of the iNOS enzyme. However, the
capacity of TNF- and IL-1ß to stimulate L-arginine
transport in BASMCs is limited (20%) and may thus further function
as a rate-limiting step in restricting the availability of
substrate, thereby preventing the release of cytotoxic levels of NO by
these cells. In addition, as we previously demonstrated in rat aortic
smooth muscle cells,17 not all inducers of iNOS can
regulate L-arginine transport in BASMCs. Although TNF-
and IL-1ß stimulate both L-arginine uptake and NO
production, IFN- and LPS selectively stimulate NO generation
without affecting L-arginine transport. In contrast, an
earlier study showed that LPS could coinduce L-arginine
uptake and iNOS expression in a murine macrophage cell
line.42 These results suggest that the coexpression of
L-arginine transport and iNOS activity is both stimulus and
cell specific.
Unlike smooth muscle cells, the production of NO by vascular endothelium is not associated with an increase in L-arginine transport. Treatment of endothelial cells with the calcium ionophore A23187 stimulates NO synthesis but fails to upregulate L-arginine uptake. Endothelial cells express the low-output ecNOS isoform; therefore, substrate demand is much lower than it is for vascular smooth muscle cells after the activation of NOS. Furthermore, since intracellular stores of L-arginine in endothelial cells range from 100 to 200 µmol/L, they should provide sufficient substrate for ecNOS.27 43 Interestingly, a recent report by Arnal et al44 demonstrates that the intracellular concentration of L-arginine in endothelial cells can be varied over 100-fold without changing NO production. These results suggest that NO synthesis by the vascular endothelium is independent of both extracellular and intracellular levels of L-arginine. Surprisingly, however, the administration of L-arginine in vivo has been demonstrated to restore endothelium-derived NO production in various pathological disorders,45 46 47 suggesting that under pathophysiological conditions L-arginine availability may be rate limiting. Alternatively, elevating levels of L-arginine in these disease states may have secondary effects on other systems that can regulate endothelial NO synthesis.44
In both vascular smooth muscle cells and endothelium,
inflammatory cytokines increase L-arginine
transport by increasing the transport capacity. Treatment of vascular
cells with TNF- selectively elevates the Vmax for
L-arginine transport without affecting the affinity for
L-arginine. Thus, the TNF-–mediated increase in
L-arginine transport activity in vascular cells most likely
results from the de novo synthesis of additional transport proteins.
This is consistent with our early finding in rat aortic smooth
muscle cells, which demonstrates that cytokine-induced
increases in L-arginine uptake are prevented by
cycloheximide.17
In conclusion, the present study demonstrates that multiple pathways for L-arginine uptake are present in vascular cells and that L-arginine transport and NO formation are differentially controlled in these cells. In smooth muscle cells, the coinduction of L-arginine uptake and NO release by certain cytokines may provide a mechanism by which increased levels of substrate are provided to the cell during activation of the iNOS enzyme. In contrast, the selective stimulation of L-arginine transport in endothelial cells indicates that L-arginine uptake is uncoupled from NO generation in these cells.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This study was supported in part by National Heart, Lung, and Blood Institute grant HL-36045, the Veterans Affairs Merit Review Board, and a Grant-in-Aid from the American Heart Association, Texas Affiliate, Inc. The authors thank Dr Michael H. Kroll for helpful discussions and suggestions and Kelly J. Peyton and Janet K. Hrbolich for expert technical assistance.
Received August 7, 1995; accepted March 21, 1996.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Barbul B. Arginine: biochemistry, physiology, and therapeutic implications. J Parenter Nutr. 1986;10:227-238.
2. Visek W. Arginine needs, physiological state and usual diets. J Nutr. 1986;116:36-46.
3. Granger DL, Hibbs JB Jr, Perfect JR, Durack DT. Specific amino acid (L-arginine) requirement for the microbiostatic activity of murine macrophages. J Clin Invest. 1988;81:1129-1136.
4.
Iyengar R, Steuhr DJ, Marletta MA.
Macrophage synthesis of nitrite, nitrate, and N-nitrosamines:
precursors and role of the respiratory burst. Proc Natl
Acad Sci U S A. 1987;84:6369-6373.
5. Palmer RMJ, Ashton AS, Moncada S. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature. 1988;333:664-666. [Medline] [Order article via Infotrieve]
6. Beasley DJ, Schwartz JH, Brenner BM. Interleukin-1 induces prolonged L-arginine-dependent cyclic guanosine monophosphate and nitrite production in rat vascular smooth muscle cells. J Clin Invest. 1991;87:602-608.
7. Busse R, Mulsch A. Induction of nitric oxide synthase by cytokines in vascular smooth muscle cells. FEBS Lett. 1990;275:87-90. [Medline] [Order article via Infotrieve]
8.
Durante W, Schini VB, Scott-Burden T, Junquero DC,
Kroll MH, Vanhoutte PM, Schafer AI. Inhibition of platelet
activation by an L-arginine derived substance released by
cultured vascular smooth muscle cells treated with
IL-1ß. Am J Physiol. 1991;261:H2024-H2030.
9.
Radomski MW, Palmer RMJ, Moncada S.
Glucocorticoids inhibit the expression of an inducible, but not
constitutive, nitric oxide synthase in vascular
endothelial cells. Proc Natl Acad Sci
U S A. 1990;87:10043-10047.
10. Gruetter CA, Barry BK, McNamara DB, Gruetter DY, Kadowitz PJ, Ignarro LJ. Relaxation of bovine coronary artery and activation of coronary arterial guanylate cyclase by nitric oxide, nitroprusside, and a carcinogenic nitrosoamine. J Cyclic Nucleotide Res. 1979;5:211-224. [Medline] [Order article via Infotrieve]
11.
Mellion BT, Ignarro LJ, Ohlstein EH, Pontecorvo EG,
Hyman AL, Kadowitz PJ. Evidence for the inhibitory
role of guanosine 3',5'-monophosphate in ADP-induced human
platelet
aggregation in the presence of nitric oxide and related
vasodilators. Blood. 1981;57:946-955.
12. Radomski MW, Palmer RMJ, Moncada S. Endogenous nitric oxide inhibits human platelet adhesion to vascular endothelium. Lancet. 1987;2:1057-1058. [Medline] [Order article via Infotrieve]
13. Garg UC, Hassid A. Nitric oxide-generating vasodilators and 8-bromo-cyclic GMP inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest. 1989;83:1774-1777.
14.
Scott-Burden T, Schini VB, Elizondo E, Junquero DC,
Vanhoutte PM. Platelet-derived growth factor suppresses
and fibroblast growth factor enhances cytokine-induced
production of nitric oxide by cultured smooth muscle cells:
effects on cell proliferation. Circ Res. 1992;71:1088-1100.
15.
Von Der Leyen HE, Gibbons GH, Morishita R, Lewis NP,
Zhang L, Nakajima M, Kaneda Y, Cooke JP, Dzau VJ. Gene therapy
inhibiting neointimal vascular lesion: in vitro transfer of
endothelial nitric oxide synthase gene.
Proc Natl Acad Sci U S A. 1995;92:1137-1141.
16. Schott CA, Gray GA, Stoclet JC. Dependence of endotoxin-induced vascular hyporeactivity on extracellular L-arginine. Br J Pharmacol. 1993;108:38-43. [Medline] [Order article via Infotrieve]
17.
Durante W, Liao L, Schafer AI. Differential
regulation of L-arginine transport and inducible NOS in
cultured vascular smooth muscle cells. Am J Physiol. 1995;268:H1158-H1164.
18. Mitchell JA, Hecker H, Anggard EA, Vane JR. Cultured endothelial cells maintain their L-arginine level despite the continuous release of EDRF. Eur J Pharmacol. 182:573-576.
19. Thomas G, Hecker M, Ramwell PW. Vascular activity of polycations and amino acids: L-arginine does not specifically elicit endothelium-dependent relaxation. Biochem Biophys Res Commun. 1989;158:177-180. [Medline] [Order article via Infotrieve]
20.
Johns RA, Peach MJ, Linden J, Tichotsky A.
NG-Monomethyl
L-arginine inhibits endothelium-derived
relaxing factor–stimulated cyclic GMP accumulation in cocultures
of endothelial and vascular smooth muscle cells by an
action specific to the endothelial cell.
Circ Res. 1990;67:979-985.
21.
Scott-Burden T, Resnik TJ, Baur U, Burgin M, Buhler
FR. Epidermal growth factor responsiveness in smooth muscle
cells from hypertensive and normotensive rats.
Hypertension. 1989;13:295-304.
22. Gazzola GC, Dall'Asta V, Franchi-Gazzola R, White MF. The cluster tray method for rapid measurement of solute fluxes in adherent cultured cells. Anal Biochem. 1981;115:368-374. [Medline] [Order article via Infotrieve]
23. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985;150:76-85. [Medline] [Order article via Infotrieve]
24. Marletta MA, Yoon PS, Iyengar R, Leaf CD, Wishnok JS. Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate. Biochemistry. 1988;27:8706-8711. [Medline] [Order article via Infotrieve]
25. Durante W, Cheng K, Sunahara RK, Schafer AI. Ethanol potentiates interleukin-1ß stimulated inducible NOS expression in cultured vascular smooth muscle cells. Biochem J. 1995;308:231-236.
26.
Greene B, Pacitti AJ, Souba WW. Characterization
of L-arginine transport by pulmonary artery
endothelial cells. Am J Physiol. 1993;264:L351-L356.
27.
Sessa WC, Hecker M, Mitchell JA, Vane JR. The
metabolism of L-arginine and its significance
for the biosynthesis of endothelium-derived
relaxing factor: L-glutamine inhibits the generation of
L-arginine by cultured endothelial
cells. Proc Natl Acad Sci U S A. 1990;87:8607-8611.
28. Laemmli UK. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature. 1970;227:680-685. [Medline] [Order article via Infotrieve]
29.
Towbin H, Staehelin T, Gordon J.
Electrophoretic transfer of proteins from polyacrylamide gels
to nitrocellulose sheets: procedure and some applications.
Proc Natl Acad Sci U S A. 1979;76:4350-4354.
30.
Christensen HN. Role of amino acid transport and
countertransport in nutrition and metabolism.
Physiol Rev. 1990;70:43-77.
31. Green LC, Wagner DA, Glogowski G, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrite, nitrate, and [15N] in biological fluids. Anal Biochem. 1982;125:131-138. [Medline] [Order article via Infotrieve]
32. Lind DS, Copeland EM III, Souba WW. Endotoxin stimulates arginine transport in pulmonary artery endothelial cells. Surgery. 1993;114:199-205. [Medline] [Order article via Infotrieve]
33. Schmidt K, Klatt P, Mayer B. Characterization of endothelial cell amino acid transport systems involved in the actions of nitric oxide synthase inhibitors. Mol Pharmacol. 1993;44:615-621. [Abstract]
34.
Closs EI, Albritton LM, Kim JW, Cunningham JM.
Identification of a low affinity, high capacity transporter of cationic
amino acids in mouse liver. J Biol Chem. 1993;268:7538-7544.
35. Kim JW, Closs EI, Albritton LM, Cunningham JM. Transport of cationic amino acids by the mouse ecotropic retrovirus receptor. Nature. 1991;352:725-728. [Medline] [Order article via Infotrieve]
36. Altman P. Blood nitrogenous substances. In: Dittmer DS, ed. Blood and Other Body Fluids. Washington, DC: Federation of American Societies for Experimental Biology; 1961:47-88.
37. Hibbs JB, Vavrin Z Jr, Taintor RR. L-Arginine is required for expression of the activated macrophage effector mechanism causing selective metabolic inhibition of target cells. J Immunol. 1987;138:550-565. [Abstract]
38.
Kilbourn RG, Belloni P.
Endothelial cell production of nitrogen oxides
in response to interferon-K in combination with tumor necrosis factor,
interleukin-1, or endotoxin. J Natl Cancer
Inst. 1990;82:772-776.
39.
Lamas ST, Michel T, Brenner BM, Marsden PA.
Nitric oxide synthesis in endothelial cells: evidence
for a pathway inducible by TNF-. Am J Physiol. 1991;261:C634-C641.
40. Gross SS, Jaffe EA, Levi R, Kilbourn RG. Cytokine-activated endothelial cells express an isotype of nitric oxide synthase which is tetrahydrobiopterin-dependent, calmodulin-independent, and inhibited by arginine analogs with a rank-order of potency characteristic of activated macrophages. Biochem Biophys Res Commun. 1991;178:823-829. [Medline] [Order article via Infotrieve]
41. Rosenkranz-Weiss P, Sessa WC, Milstien S, Kaufman S, Watson CA, Prober JS. Regulation of nitric oxide synthesis by proinflammatory cytokines in human umbilical vein endothelial cells: elevations in tetrahydrobiopterin levels enhance endothelial nitric oxide synthase specific activity. J Clin Invest. 1994;93:2236-2243.
42. Bogle RG, Baydoun AR, Pearson JD, Moncada S, Mann GE. L-Arginine transport is increased in macrophages generating nitric oxide. Biochem J. 1992;284:15-18.
43.
Hecker M, Sessa WC, Harris HJ, Anggard EA, Vane
JR. The metabolism of L-arginine and its
significance for the biosynthesis of
endothelium-derived relaxing factor: cultured
endothelial cells recycle L-citrulline to
L-arginine. Proc Natl Acad Sci U S A. 1990;87:8612-8616.
44. Arnal JF, Munzel T, Venema RC, James NL, Bai C, Mitch WE, Harrison DG. Interactions between L-arginine and L-glutamine change endothelial NO production: an effect independent of NO synthase substrate availability. J Clin Invest. 1995;95:2565-2572.
45. Cooke JP, Singer AH, Tsao P, Zera P, Rowan RA, Billingham ME. Antiatherogenic effects of L-arginine in the hypercholesterolemic rabbit. J Clin Invest. 1992;90:1168-1172.
46. Pieper GM, Peltier BA. Amelioration by L-arginine of a dysfunctional arginine/nitric oxide pathway in diabetic endothelium. J Cardiovasc Pharmacol. 1995;25:397-403. [Medline] [Order article via Infotrieve]
47.
Hamon M, Vallet, Bauters C, Wernert N, McFadden
EP, Lablanche JM, Dupuis B, Bertrand ME. Long-term oral
administration of L-arginine reduces the intimal thickening
and enhances neoendothelium-dependent
acetylcholine-induced relaxation after arterial
injury. Circulation. 1994;90:1357-1362.
This article has been cited by other articles:
 |
|
 |
|
  X. Jiang, F. Yang, H. Tan, D. Liao, R. M. Bryan Jr, J. K. Randhawa, R. E. Rumbaut, W. Durante, A. I. Schafer, X. Yang, et al. Hyperhomocystinemia Impairs Endothelial Function and eNOS Activity via PKC Activation Arterioscler Thromb Vasc Biol, December 1, 2005; 25(12): 2515 - 2521. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Schwartz, I. F. Schwartz, E. Gnessin, Y. Wollman, T. Chernichovsky, M. Blum, and A. Iaina Differential regulation of glomerular arginine transporters (CAT-1 and CAT-2) in lipopolysaccharide-treated rats Am J Physiol Renal Physiol, April 1, 2003; 284(4): F788 - F795. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. E. Mann, D. L. Yudilevich, and L. Sobrevia Regulation of Amino Acid and Glucose Transporters in Endothelial and Smooth Muscle Cells Physiol Rev, January 1, 2003; 83(1): 183 - 252. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Sala, B. M. Rotoli, E. Colla, R. Visigalli, A. Parolari, O. Bussolati, G. C. Gazzola, and V. Dall'Asta Two-way arginine transport in human endothelial cells: TNF-alpha stimulation is restricted to system y+ Am J Physiol Cell Physiol, January 1, 2002; 282(1): C134 - C143. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. K. Kiemer and A. M. Vollmar Induction of L-Arginine Transport Is Inhibited by Atrial Natriuretic Peptide: A Peptide Hormone as a Novel Regulator of Inducible Nitric-Oxide Synthase Substrate Availability Mol. Pharmacol., September 1, 2001; 60(3): 421 - 426. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A.C. Mendes Ribeiro, T.M.C. Brunini, J.C. Ellory, and G.E. Mann Abnormalities in L-arginine transport and nitric oxide biosynthesis in chronic renal and heart failure Cardiovasc Res, March 1, 2001; 49(4): 697 - 712. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. DURANTE, L. LIAO, S. V. REYNA, K. J. PEYTON, and A. I. SCHAFER Physiological cyclic stretch directs L-arginine transport and metabolism to collagen synthesis in vascular smooth muscle FASEB J, September 1, 2000; 14(12): 1775 - 1783. [Abstract] [Full Text]
|
|
|
|
 |
|
 Y. Hattori, K. Kasai, and S. S. Gross Cationic amino acid transporter gene expression in cultured vascular smooth muscle cells and in rats Am J Physiol Heart Circ Physiol, June 1, 1999; 276(6): H2020 - H2028. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. M. Edwards, E. J. Stack, and W. Trizna Interaction of L-Arginine Analogs with L-Arginine Uptake in Rat Renal Brush Border Membrane Vesicles J. Pharmacol. Exp. Ther., June 1, 1998; 285(3): 1019 - 1022. [Abstract] [Full Text]
|
|
|
|
 |
|
 G. M. Pieper Review of Alterations in Endothelial Nitric Oxide Production in Diabetes : Protective Role of Arginine on Endothelial Dysfunction Hypertension, May 1, 1998; 31(5): 1047 - 1060. [Full Text] [PDF]
|
|
|
|
|
|
R. DEVES and C. A. R. BOYD Transporters for Cationic Amino Acids in Animal Cells: Discovery, Structure, and Function Physiol Rev, April 1, 1998; 78(2): 487 - 545. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Durante, L. Liao, K. J. Peyton, and A. I. Schafer Lysophosphatidylcholine Regulates Cationic Amino Acid Transport and Metabolism in Vascular Smooth Muscle Cells. ROLE IN POLYAMINE BIOSYNTHESIS J. Biol. Chem., November 28, 1997; 272(48): 30154 - 30159. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J. Hutchison, M. S. Reitz, K. Sudhir, R. E. Sievers, B.-Q. Zhu, Y.-P. Sun, T. M. Chou, P. C. Deedwania, K. Chatterjee, S. A. Glantz, et al. Chronic Dietary L-Arginine Prevents Endothelial Dysfunction Secondary to Environmental Tobacco Smoke in Normocholesterolemic Rabbits Hypertension, May 1, 1997; 29(5): 1186 - 1191. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||