1 Department of Medicine, University of Florida College of Medicine, and 2 Research Service, Malcom Randall Veterans Affairs Medical Center, Gainesville, Florida 32608-1197
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ABSTRACT |
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We investigated possible involvement of the actin cytoskeleton in the regulation of the L-arginine/nitric oxide (NO) pathway in pulmonary artery endothelial cells (PAEC). We exposed cultured PAEC to swinholide A (Swinh), which severs actin microfilaments, or jasplakinolide (Jasp), which stabilizes actin filaments and promotes actin polymerization, or both. After treatment, the state of the actin cytoskeleton, L-arginine uptake mediated by the cationic amino acid transporter-1 (CAT-1), Ca2+/calmodulin-dependent (endothelial) NO synthase (eNOS) activity and content, and NO production were examined. Jasp (50-100 nM, 2 h treatment) induced a reversible activation of L-[3H]arginine uptake by PAEC, whereas Swinh (10-50 nM) decreased L-[3H]arginine uptake. The two drugs could abrogate the effect of each other on L-[3H]arginine uptake. The effects of both drugs on L-[3H]arginine transport were not related to changes in expression of CAT-1 transporters. Swinh (50 nM, 2 h) and Jasp (100 nM, 2 h) did not change eNOS activities and contents in PAEC. Detection of NO in PAEC by the fluorescent probe 4,5-diaminofluorescein diacetate showed that Swinh (50 nM) decreased and Jasp (100 nM) increased NO production by PAEC. The stimulatory effect of Jasp on NO production was dependent on the availability of extracellular L-arginine. Our results indicate that the state of actin microfilaments in PAEC regulates L-arginine transport and that this regulation can affect NO production by PAEC.
L-arginine transporter; nitric oxide production; actin cytoskeleton; pulmonary endothelial cells; swinholide; jasplakinolide
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INTRODUCTION |
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PULMONARY ENDOTHELIAL CELLS are a rich source of nitric oxide (NO), a nitrogen-centered free radical with multiple and unique physiological bioregulatory activities (36). Pulmonary endothelial cells generate NO from L-arginine via the catalytic action of a NADPH-requiring, Ca2+/calmodulin-dependent (endothelial) NO synthase (eNOS) that is a peripheral membrane-associated protein (42). More recent studies have shown that eNOS, when appropriately acylated, targets to caveolae, specific cholesterol- and sphingolipid-rich microdomains of the plasma membrane (20, 47). The targeting of eNOS to caveolae in endothelial cells is promoted, in part, by direct interaction of the enzyme with the caveolar structural protein caveolin-1. There are data suggesting that the caveolar localization of eNOS in endothelial cells is critical for optimal NO production in stimulated and nonstimulated conditions (18, 30).
The activity of eNOS is not the only determinant controlling NO production by endothelial cells. Recent reports indicate that NO production, especially production stimulated by bradykinin (5), acetylcholine (1), shear stress (40), and endotoxin (44), is dependent on the availability and delivery of extracellular L-arginine. In endothelial cells, the delivery of extracellular L-arginine is mediated by the Na+-independent system y+ carrier, which is responsible for 70-95% of L-arginine uptake in endothelial cells (22, 56). Three system y+ cationic amino acid transporters (CAT-1, CAT-2B, CAT-3) have been cloned, and the characteristic features of these transporters have been described (see reviews in Refs. 12 and 17). In porcine pulmonary artery endothelial cells (PAEC), the vast majority of system y+-mediated Na+-independent L-arginine uptake is provided by the CAT-1 transporter (56). Recently, we reported that the constitutive CAT-1 transporter and eNOS can colocalize in the caveolae of porcine PAEC and can be coimmunoprecipitated, suggesting a protein-to-protein interaction between CAT-1 and eNOS in these cells (33). We suggested that formation of such a caveolar complex promotes optimal NO production through the directed delivery of substrate (i.e., L-arginine) to eNOS.
Caveolae are specialized microdomains of the plasma membrane defined by their unique morphology and by the presence of one or more of a family of novel integral membrane proteins known as caveolins (2). Caveolae have been shown to be enriched in signaling molecules such as cell surface receptors, receptor tyrosine kinases, and G proteins (see reviews in Refs. 28 and 46). There are several reports demonstrating that plasmalemmal caveolae are closely related to cellular actin filaments (19, 29, 48). Recently, we reported that the CAT-1 L-arginine transporter interacts with the actin-binding protein fodrin and that proteolysis of fodrin can affect CAT-1 transport activity in PAEC (57). Therefore, it is tempting to speculate that the actin cytoskeleton may be involved in regulating caveolar function and, in particular, that changes in the organization of actin filaments may alter CAT-1-mediated L-arginine transport, the activity of eNOS, and/or NO production by endothelial cells. To test this, we exposed cultured porcine PAEC to agents with specific actin-binding activities and examined the state of the actin cytoskeleton (stress fibers), CAT-1-mediated L-arginine transport, eNOS activity, and NO production. Among the specific agents capable of affecting the actin cytoskeleton, we focused our study on two that easily penetrate cellular membranes and have opposite effects on actin microfilaments: swinholide A (Swinh) and jasplakinolide (Jasp) (50). Swinh is a microfilament-disrupting marine toxin that stabilizes actin dimers and severs actin filaments (7). Jasp binds to and stabilizes actin microfilaments and can promote actin polymerization (6, 8). Our results indicate that L-arginine transport in PAEC is regulated by the state of actin microfilaments and that changes in CAT-1 transport activity are predominantly responsible for changes in NO production by PAEC exposed to Swinh or Jasp.
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MATERIALS AND METHODS |
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Reagents.
We used commercial and noncommercial sources for Swinh and Jasp.
Commercial Jasp was obtained from Molecular Probes (Eugene, OR).
Commercial Swinh was obtained from Alexis Biochemicals. The commercially available agents induced the same changes in our experiments as the natural products isolated from marine sponges. Misakinolide A, latrunculin A and B, and halichondramide A were kindly
provided by Dr. Michael R. Bubb. All agents were stored in methanol at
20°C and were diluted into medium immediately before use. The final
methanol concentration in medium did not exceed 1%. All agents readily
enter living mammalian cells when added to culture medium
(50). L-[3H]arginine was
obtained from Amersham (Arlington Heights, IL). Mouse anti-eNOS
polyclonal antibody was obtained from Transduction Laboratories
(Lexington, KY). Fluorescence probe 4,5-diaminofluorescein diacetate
(DAF-2DA) used for NO detection was purchased from Calbiochem (San
Diego, CA). Peptide corresponding to the fourth extracellular domain of
the human CAT-1 transporter
(Y-F-G-V-S-A-A-L-T-L-M-M-P-Y-F-C-L-D-K-D-T-P-L-P-D-A-F-K-H-V-G-W-G) was synthesized by the Protein Core Facility of the Interdisciplinary Center for Biotechnology Research at the University of Florida. Rabbit
polyclonal antibodies to this peptide were prepared and characterized
as previously reported by us (54).
Tissue culture. Endothelial cells were isolated by collagenase treatment of the main pulmonary artery of 6- to 7-mo-old pigs and were cultured and characterized as previously reported (4). Third- to fifth-passage cells in monolayer culture were maintained in RPMI 1640 medium containing 4% fetal bovine serum and antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin, 10 µg/ml gentamicin, and 1.5 µg/ml Fungizone) and were used 2 or 3 days after confluence.
Analysis of the actin microfilament system of PAEC. For the fluorescence microscopy analysis of microfilaments, PAEC were grown to near confluence on Mat Tech chamber slides (Mat Tech, Ashland, MA). Swinh and Jasp were added to the culture medium at final concentrations of 50-100 nM, and the cells were examined after a 2-h incubation. For F-actin staining, treated and untreated PAEC were washed twice in phosphate-buffered saline (PBS), permeabilized with 0.05% Triton X-100 for 3 min at room temperature, and then fixed in 4% formaldehyde in PBS for 10 min at room temperature, washed three times with PBS, and blocked in 1% BSA and 50 mM L-lysine in PBS for 1 h at 4°C. Labeling of F-actin with fluorescein-phalloidin was done per the manufacturer's directions (Molecular Probes, Eugene, OR). Images were digitally recorded using a Zeiss Axiovert 100M microscope with a Zeiss LSM5 confocal attachment.
Measurement of CAT-1-mediated Na+-independent L-arginine transport by PAEC. Transport of radiolabeled L-arginine by PAEC was measured as reported by our group (57, 58). In brief, to remove residual culture medium and extracellular Na+, cells grown in 24-well plates were washed with a buffer of the following composition (in mM): 140 LiCl, 5 KCl, 2 Na2HPO4, 1.2 MgSO4, 2.5 CaCl2, 11 glucose, and 10 HEPES-Tris (pH 7.4, LiCl-Dulbecco solution). Transport assays were initiated by the addition of the same buffer containing 50 µM unlabeled L-arginine plus L-[3H]arginine (5 µCi/ml), and 30 s later, transport was stopped by washing the cells four times with ice-cold buffer. After solubilization of the cells in 0.2% SDS, aliquots were added to scintillation fluid, and radioactivity was quantitated by liquid scintillation spectrometry. All measurements of CAT-1-mediated Na+-independent L-arginine uptake were corrected by subtracting the nonspecific component of uptake.
Determination of eNOS activity in PAEC. eNOS activity in intact cells was determined by monitoring the conversion of L-[3H]arginine into L-[3H]citrulline using methods previously described (16, 43). Control cells and cells incubated for 2 h with 50 nM Swinh, 100 nM Jasp, or both drugs together were washed once in 1 ml of warmed LiCl-Dulbecco solution and then incubated in 0.5 ml of LiCl-Dulbecco solution containing L-[3H]arginine (5 µCi/ml) for 15 min. After the 15-min incubation, PAEC were washed three times with 2 ml of ice-cold LiCl-Dulbecco solution containing 5 mM EDTA and were then lysed in 1 ml of 10 mM HCl containing 0.1% SDS. Two aliquots of lysates (50 µl each) were removed for measurements of protein content and radioactivity incorporated during the incubation with labeled L-arginine. To the remaining sample (0.9 ml), 0.1 ml of 0.2 M sodium acetate buffer, pH 13.0, containing 10 mM L-citrulline was added, and the sample was applied to a column of Dowex AG50WX 8 (H+ form) added as a 1:1 slurry in H2O. The effluents were collected in counting vials and, subsequent to elution of residual L-[3H]citrulline with 1 ml of H2O, 10 ml of scintillation fluid were added for determination of eluted radioactivity. eNOS activity is expressed as percent conversion of incorporated L-[3H]arginine into L-[3H]citrulline. Protein determinations were made by the method of Lowry et al. (31).
Determination of eNOS activity in the total membrane fraction from PAEC. Control and treated PAEC were scraped and homogenized in buffer A (50 mM Tris · HCl, pH 7.4, containing 0.1 mM each EDTA and EGTA, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml leupeptin). The homogenates were centrifuged at 100,000 g for 60 min at 4°C, and the total membrane fraction pellet was resuspended in buffer B (buffer A plus 2.5 mM CaCl2) and used for eNOS activity determinations by monitoring the formation of L-[3H]citrulline from L-[3H]arginine (38). Membranes (100-120 µg of protein) were incubated (total volume 0.4 ml) in buffer A with the addition of 1 mM NADPH, 100 nM calmodulin, 10 µM tetrahydrobiopterin, and 5 µM combined L-arginine and purified L-[3H]arginine for 30 min at 37°C. The measurement of L-[3H]citrulline formation was performed as described by us (38).
Western blot analysis of eNOS and CAT-1 contents in cell lysates.
PAEC were washed twice with ice-cold PBS without Ca2+ or
Mg2+ and scraped in lysis buffer (10 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 0.4% deoxycholate,
and 60 mM octylglucoside) containing protease inhibitor cocktail
(Calbiochem). The cell extracts were centrifuged at 100,000 g for 30 min, and the supernatants were kept at 80°C
until use. Samples (15-20 µg of protein) were separated by
SDS-PAGE (7.5% acrylamide) and transferred to nitrocellulose. The
nitrocellulose membranes were incubated in blocking solution (10 mM
Tris, 5% nonfat dry milk, and 100 mM NaCl, pH 7.5) for at least 1 h and then hybridized overnight at 4°C with a 1:1,000 dilution of
polyclonal anti-eNOS antibody or a 1:800 dilution of a polyclonal
anti-CAT-1 antibody. Alkaline phosphatase-conjugated secondary
antibodies (1:3,000 dilution; Bio-Rad) were used to visualize bound
primary antibodies with chemiluminescence substrate (Immuno-Star;
Bio-Rad). The density of the bands was quantitated using a Fluor-S
MultiImager system (Bio-Rad).
Analysis of NO production by PAEC. NO production was measured in real time using DAF-2DA, a specific fluorescence probe for NO detection (26). DAF-2DA is a membrane-permeable dye that is hydrolyzed inside the cells by cytosolic esterases releasing DAF-2. In the presence of NO, DAF-2 converts into a fluorescent product, DAF-2 triazole, which can be detected (37). For NO detection, PAEC were grown on 96-well, clear-bottom plates. Before the experiment, cells were washed once and incubated in 0.1 ml of Krebs-Henseleit solution [in mM: 132 NaCl, 3.9 KCl, 2.8 CaCl2, 1.5 KH2PO4, 1.2 MgCl2, 10 HEPES (pH 7.4), and 5.5 glucose] containing 20 µM DAF-2DA for 1 h at 37°C. After the incubation, cells were washed and incubated in 0.1 ml of Krebs-Henseleit solution containing 50 µM L-arginine and various concentrations of Swinh or Jasp. All measurements of specific DAF-2DA fluorescence were corrected by subtracting the nonspecific fluorescence in wells without addition of DAF-2DA and in wells without cells. Fluorescence was measured using an FL600 microplate fluorescence reader (Bio-TEK Instrument, Winooski, VT) every 5 min (excitation wavelength = 488 nm and emission wavelength = 510 nm).
Data analysis. In each experiment, treated and control PAEC were matched for cell line, age, number of passages, and number of days postconfluence to avoid variations in tissue culture factors that can influence the measurements of L-[3H]arginine uptake, eNOS activity, and NO production. Data are expressed as means ± SD. Comparisons between values were made using an unpaired, two-tailed Student's t-test. A value of P < 0.05 was considered statistically significant.
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RESULTS |
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Effects of Swinh and Jasp on L-arginine uptake and organization of the actin cytoskeleton in PAEC. To investigate whether changes in organization of the actin cytoskeleton affect L-arginine transport, cultured PAEC were exposed to several marine natural products that have been reported to target the actin cytoskeleton and to change its organization (50). Special attention was paid to Swinh and Jasp because of their opposing effects on microfilament structure.
PAEC were exposed to Swinh or Jasp for 0.5-12 h (Fig. 1A). Exposure to 10 nM Swinh had no significant effect on L-[3H]arginine uptake, whereas exposure to 20 nM and 50 nM Swinh resulted in significant dose-dependent and sustained decreases in L-[3H]arginine uptake. In contrast, exposure of PAEC to the microfilament-stabilizing agent Jasp (50 nM and 100 nM) resulted in significant increases in L-[3H]arginine uptake, which were maximal after 2 h of incubation. No evidence of cell toxicity, assessed by cell number, cell protein content, and lactate dehydrogenase release, was observed with the concentrations of Swinh or Jasp used in our experiments. The decrease in L-arginine transport observed in cells incubated with Swinh was also observed with other marine agents known to disrupt the actin microfilament system, including misakinolide A, which sequesters unpolymerized actin and caps the barbed (growing) ends, latrunculin A and B, which sequester actin monomers, and halichondramide, which severs microfilaments (Table 1).
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Effects of Swinh and Jasp on eNOS activity and content in PAEC.
The measurements of L-[3H]citrulline
formation from L-[3H]arginine by intact PAEC
showed that Swinh (50 nM, 2-h treatment) decreased and Jasp (100 nM,
2-h treatment) increased the absolute amount of
L-[3H]citrulline produced by PAEC compared
with control cells. However, the calculation of the fraction of
L-[3H]arginine taken up by cells that was
converted to L-[3H]citrulline showed that the
velocity of L-[3H]citrulline formation, which
is a measure of eNOS activity, was comparable in control cells and
cells treated with one or both of the cytoskeleton-active agents (Fig.
3A). The measurements of eNOS
activity in the total membrane fractions isolated from PAEC also did
not show significant differences in eNOS activity between control
PAEC (10.0 ± 0.6 pmol L-citrulline · mg
protein1 · min
1) and PAEC incubated
for 2 h with 50 nM Swinh (8.4 ± 0.7 pmol L-citrulline · mg
protein
1 · min
1) or 100 nM Jasp
(8.3 ± 1.0 pmol L-citrulline · mg
protein
1 · min
1). Finally, Western
blot analysis of eNOS contents in cell lysates demonstrated that Swinh
(50 nM, 2-h treatment) and Jasp (100 nM, 2-h treatment) did not change
the amount of eNOS per microgram of cell protein (Fig. 3B).
Taken together, these data demonstrate that a 2-h treatment with Swinh
(50 nM) or Jasp (100 nM) did not significantly change eNOS expression
or eNOS activity in PAEC.
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Effects of Swinh and Jasp on NO production by PAEC.
Both Swinh and Jasp changed NO production by PAEC. As shown in Fig.
4A, Swinh (50-200 nM)
induced dose-dependent decreases in NO production in cultured PAEC
compared with control PAEC. The total amounts of NO produced by the
cultured PAEC after a 5-h incubation, as detected by the fluorescent
indicator DAF-2DA, were 40 and 370% less after treatment with Swinh in
concentrations of 50 and 200 nM, respectively, than in control cells
for the same period of time. Jasp resulted in a dose-dependent increase in NO production by cultured PAEC (Fig. 4A). PAEC treated
with 100 nM Jasp for 5 h produced about three times more NO than
untreated cells.
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DISCUSSION |
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Endothelial cells contain a well-developed actin microfilament cytoskeleton that has been shown to affect endothelial cell structure and function (21). Several reports suggest that structural changes in the endothelial cytoskeleton provide a transduction pathway between shear stress and the synthesis of NO (23, 25). Participation of the actin cytoskeleton in the regulation of NO production by PAEC may be mediated through effects on the availability of L-arginine, on eNOS activity, or on both. The current study was designed to determine whether changes in the actin cytoskeleton affect L-arginine uptake or eNOS activity or both, and if so, whether these effects would influence NO production. To change actin microfilament structure in PAEC, we chose two marine sponge toxins, Swinh and Jasp, possessing opposite effects on the microfilament structure (6-8). The advantages of these toxins are that they are readily cell permeable, they can be used for modification of the cytoskeleton without any additional cell treatments (50), and they can be used in endothelial cells in dosages that modulate the cytoskeleton (Fig. 2) but do not cause cytotoxicity.
Our results demonstrate that changes in actin microfilament structure affect CAT-1-mediated Na+-independent L-arginine transport activity in porcine PAEC. Stabilization of actin microfilaments by Jasp increased L-arginine uptake by PAEC, whereas disruption of microfilaments by Swinh and various other agents inhibited L-arginine transport. The effects of Swinh and Jasp were reversible, and both agents could abolish the effects of each other. These results indicate that the changes in L-arginine uptake induced by the action of Swinh and Jasp are directly connected to modifications of the actin cytoskeleton in PAEC. Because the changes in L-arginine uptake were not accompanied by changes in CAT-1 transporter expression in the PAEC, they are likely due to actin microfilament-induced changes in existing transporters or their environment.
There are a number of observations that the activity of membrane
proteins can be regulated by the actin cytoskeleton (34). Dependence on the state of actin microfilaments has been reported for
Na+ channel (11) and
Na+-K+-ATPase (10) activities in
epithelial cells, for Ca2+ transport in fibroblasts
(55), for the activity of the cystic fibrosis
transmembrane conductance regulator (CFTR) in mouse adenocarcinoma cells and human epithelial cells (41), and for
Cl secretion in intestinal cells (32). The
precise mechanisms responsible for cytoskeletal regulation of the
activity of integral membrane proteins have not been worked out to
date. Several investigators have suggested that a regulatory role for
actin microfilaments may be mediated through changes in the secondary
structure of membrane proteins as a result of direct binding to the
cytoskeleton (10, 41) because distinct domains of some
membrane proteins, e.g., CFTR and
Na+-K+-ATPase, may represent relevant
actin-binding domains. Microfilament regulation of membrane proteins
may also be mediated by various actin-binding proteins. Recently, we
reported that the CAT-1 L-arginine transporter interacts
with the actin-binding protein fodrin in PAEC and that degradation of
fodrin affects L-arginine transport activity in these cells
(57). Therefore, it is possible that Swinh and Jasp
modulate the stability of a "CAT-1-fodrin-actin microfilament"
complex that affects transporter activity directly or affects the
localization, and in turn function, of the CAT-1 transporter in
specific membrane domains. Because the fodrin-based membrane skeleton
is a submembranous structure that binds a subset of membrane proteins
(3), the cytoskeleton changes induced by Jasp and Swinh
can enhance or weaken, respectively, the interaction of CAT-1 with
proteins that serve to regulate the L-arginine transporter system.
In contrast to L-arginine transport, neither eNOS activity nor eNOS protein content was altered by incubation with Swinh or Jasp for 2 h. Unlike the CAT-1 transporter, which is an integral membrane protein penetrating the plasma membrane, eNOS is a peripheral membrane protein that is targeted to specific cellular domains, including Golgi and plasmalemmal caveolae. Targeting of eNOS to plasmalemmal caveolae is dependent on the myristoylation (47) and palmitoylation (20) of eNOS. Sessa and colleagues (45) reported that eNOS association with Golgi membranes is also acylation dependent. Inhibition of acylation and mutation of the palmitoylation sites attenuate Golgi and caveolae targeting of eNOS (20, 45) and markedly reduce the stimulated production of NO (30). The biological function of Golgi-localized eNOS and the relationship between caveolar and Golgi pools of eNOS are not fully understood.
Several studies indicate that translocation of eNOS between the Golgi and plasmalemmal caveolae is a cytoskeleton-dependent process. For example, it has been shown that bradykinin-induced translocation of eNOS in endothelial cells is accompanied by rearrangement of F-actin (35, 53) and by translocation of a dimeric actin-cross-linking protein, filamin (52, 53). Moreover, blocking filamin phosphorylation prevents bradykinin-induced F-actin rearrangement and filamin and eNOS translocation in these cells (53). However, Venema and colleagues (51) reported that bradykinin-induced translocation of eNOS from Triton X-100-soluble membranes to the cytoskeleton produced no detectable change in eNOS activity of cell lysates. Furthermore, time-dependent changes in the eNOS activity of the Triton X-100-soluble (presumably Golgi-localized membranes) and Triton X-100-insoluble (presumably caveolar) fractions were proportional to the changes in the amount of the enzyme detected on immunoblots. The authors concluded that association of eNOS with cytoskeletal elements did not function as a mechanism for regulation of enzyme activity. This conclusion is in good agreement with our results showing that 2-h incubations with Swinh and Jasp do not alter eNOS activity or expression in cultured PAEC. However, our results do not preclude the possibility that longer incubations with these agents may result in alterations in eNOS activity or expression.
Our results also show that Swinh- and Jasp-induced rearrangements in the actin cytoskeleton alter NO production by PAEC. The changes in NO production paralleled the changes in L-arginine transport in these cells, i.e., stabilization of actin microfilaments increased NO production and L-arginine uptake, whereas the disruption of microfilaments by Swinh decreased NO production and L-arginine transport in a concentration-dependent manner. All of these changes occurred without any change in eNOS activity in the PAEC. These results suggest that NO production by PAEC is dependent on L-arginine transport. The dependence of NO production by vascular endothelial cells on extracellular L-arginine has been observed in a number of in vitro and in vivo studies (1, 14, 15, 39). These observations, i.e., that extracellular L-arginine administration seems to drive NO production even when intracellular levels of L-arginine are available in excess, have been referred to as the "arginine paradox" (27). Recently, we reported (33) that CAT-1 L-arginine transporters interact with and colocalize with eNOS molecules, forming a functional "CAT-1-eNOS" complex in the caveolae of cultured PAEC that can provide directed delivery of extracellular L-arginine to eNOS for NO synthesis. The parallel changes in L-arginine transport and NO production during the toxin-induced rearrangements of the actin cytoskeleton observed in our experiments fit well with the concept of such a complex.
The existence of CAT-1-eNOS complexes in PAEC is also supported by our
data that removal of extracelluar L-arginine from the medium affected Jasp-stimulated NO production. Thus the stimulatory effect of Jasp on NO production was significantly dependent on the
presence of extracellular L-arginine (Fig. 4B).
However, Jasp could stimulate NO production after the removal of
extracellular L-arginine from the medium to achieve an
"L-arginine-free" medium. Recently, Closs et al.
(13) suggested the existence of two pools of intracellular
L-arginine in endothelial cells, one of which ("not
freely exchangeable with extracellular space") might be a
membrane-encircled compartment that can be used by eNOS for NO
synthesis. If such a pool exists in porcine PAEC, then the stimulatory
effect of Jasp on NO production in our cells in
L-arginine-free medium may be related to an effect of Jasp
on the accessibility of L-arginine from this intracellular
pool to eNOS. However, there might also be another explanation for the
stimulatory effect of Jasp on NO production after removal of
extracellular L-arginine from the medium. Accumulation of
L-arginine by cells depends on membrane potential (9,
24, 49, 58) and, at the steady state, there is an equilibrium
between the inward directed, transporter-mediated flux of
L-arginine driven by membrane potential and the outward directed efflux supported by an electrochemical L-arginine
gradient (56). At the normal membrane potential (about
60 mV), the estimated ratio of intracellular to extracellular
L-arginine in cultured PAEC should be 1:10. After removal
of extracellular L-arginine, at the same membrane
potential, a portion of intracellular L-arginine will be
released from cells and a new steady-state equilibrium will be
reestablished at new concentrations of intracellular and extracellular
L-arginine. Quantitation of the medium concentration of
L-arginine after changing an
L-arginine-containing medium to an
L-arginine-free medium by HPLC showed that, under the
conditions of our experiments, extracellular (i.e., released)
L-arginine concentration in the L-arginine-free
medium was 7 ± 2 µM. This released L-arginine can
be delivered by CAT-1 transporters to eNOS for NO production, and this
delivery can be affected by Jasp. This may account for why Jasp is able
to stimulate NO production in L-arginine-free medium.
In conclusion, our results indicate that the state of actin microfilaments in PAEC can regulate Na+-independent CAT-1-mediated L-arginine transport and that this regulation affects NO production in these cells in the absence of a change in eNOS activity or content. The dependence of NO production by PAEC on CAT-1 transporter activity adds further support to the concept of CAT-1-mediated directed delivery of L-arginine to eNOS.
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ACKNOWLEDGEMENTS |
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We thank Humberto Herrera for assistance with tissue culture, Dr. Laszlo Prokai for help in quantitation of extracellular L-arginine by HPLC, and Janet Wootten for editorial assistance.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-52136 (Method to Extend Research in Time Award) and by the Medical Research Service, Department of Veterans Affairs.
Address for reprint requests and other correspondence: E. R. Block, Research Service (151), Dept. of Veterans Affairs Medical Center, 1601 SW Archer Road, Gainesville, FL 32608-1197 (E-mail: edward.block{at}med.va.gov).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 16 August 2000; accepted in final form 16 October 2000.
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