Research Service, Malcom Randall Department of Veterans Affairs Medical Center, and Department of Medicine, University of Florida, Gainesville, Florida 32608-1197
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ABSTRACT |
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In this study, we
investigated the possible interaction between the cationic amino acid
transporter (CAT)-1 arginine transporter and ankyrin or
fodrin. Because ankyrin and fodrin are substrates for
calpain and because hypoxia increases calpain expression and activity
in pulmonary artery endothelial cells (PAEC), we also studied the
effect of hypoxia on ankyrin, fodrin, and CAT-1 contents in PAEC.
Exposure to long-term hypoxia (24 h) inhibited L-arginine uptake by PAEC, and this inhibition was prevented by calpain inhibitor 1. The effects of hypoxia and calpain inhibitor 1 were not associated with changes in CAT-1 transporter content in PAEC plasma membranes. However, hypoxia stimulated the hydrolysis of ankyrin and fodrin in
PAEC, and this could be prevented by calpain inhibitor 1. Incubation of
solubilized plasma membrane proteins with anti-fodrin antibodies resulted in a 70% depletion of CAT-1 immunoreactivity and in a 60%
decrease in L-arginine transport activity in reconstituted proteoliposomes (3,291 ± 117 vs. 8,101 ± 481 pmol · mg
protein1 · 3 min
1 in
control). Incubation with anti-ankyrin antibodies had
no effect on CAT-1 content or L-arginine transport in
reconstituted proteoliposomes. These results demonstrate that CAT-1
arginine transporters in PAEC are associated with fodrin, but not with
ankyrin, and that long-term hypoxia decreases L-arginine
transport by a calpain-mediated mechanism that may involve fodrin proteolysis.
ankyrin; calpain; hypoxia; pulmonary artery endothelial cells; cationic amino acid transporter-1
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INTRODUCTION |
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NITRIC OXIDE (NO) released from endothelial cells is an
important mediator that can function as a vasodilator agent (21). NO is
enzymatically generated from L-arginine via a
Ca2+/calmodulin-dependent NO
synthase [endothelial NO synthase (eNOS) or type III NO
synthase] that is membrane associated (24). The main
transport agency that is responsible for 60-95% of
carrier-mediated arginine delivery into lung vascular endothelial cells
is the y+ transport system (14,
29). The system y+ transporter is
encoded by a family of genes collectively referred to as cationic amino
acid transporter (CAT) genes (8, 18). Recently, we reported the
existence of a complex between CAT-1 and eNOS within plasmalemmal
caveolae of porcine pulmonary artery endothelial cells (PAEC; see Ref.
19). We have also reported that exposure to hypoxia decreased
CAT-1-mediated L-arginine uptake by cultured PAEC (4, 30).
Short-term exposures (i.e., 4 h) to hypoxia decreased
L-arginine uptake by inducing membrane depolarization,
whereas the mechanism responsible for decreased L-arginine
transport by long-term exposures to hypoxia (i.e., 12-24 h) seemed
to involve additional regulatory pathways (30).
More recently, we have shown that hypoxia upregulates the catalytic activity and mRNA expression of calpain, a Ca2+-regulated neutral cysteine protease, in porcine PAEC (28). Among the major substrates for calpain are membrane-related proteins, including the actin-binding cytoskeletal proteins fodrin (a nonerythroid analog of spectrin) and ankyrin (9). Studies investigating the interactions between membrane proteins and cytoskeletal proteins have demonstrated that ankyrin and fodrin exhibit high-affinity binding sites for integral membrane proteins and are involved in the regulation of membrane protein function (1, 2, 20), including membrane transport functions (6, 10, 15, 22, 23, 25, 26).
Based on these observations, we investigated the possible interaction between the CAT-1 L-arginine transporter and the cytoskeletal proteins ankyrin and fodrin in PAEC. Taking into account that hypoxia upregulates calpain activity in PAEC and that ankyrin and fodrin are calpain substrates, we also studied changes in ankyrin, fodrin, and CAT-1 contents after exposure to hypoxia. Our results demonstrate that CAT-1 L-arginine transporters are associated with fodrin but not with ankyrin in plasma membranes of PAEC and suggest that inhibition of CAT-1-mediated L-arginine transport in PAEC after long-term exposure to hypoxia is related to the calpain-mediated hydrolysis of fodrin.
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MATERIALS AND METHODS |
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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 (5). Third- to fifth-passage cells in monolayer culture in 24-well cluster trays were used for measurements of L-arginine transport, whereas cells in monolayer culture in 100-mm petri dishes were used for isolation of cellular plasma membranes. Cells were maintained in RPMI 1640 containing 4% FCS and antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin, 10 µg/ml gentamicin, and 1.5 µg/ml Fungizone) and were studied 3-5 days after confluence.
Exposure to hypoxia. Cells in 24-well cluster trays or 100-mm dishes were exposed at 37°C to normoxia (95% room air-5% CO2; 140-150 mmHg O2) or to hypoxia (0% O2-5% CO2-balance nitrogen; 7-12 mmHg O2) at 1 atm using sealed modular exposure chambers, as previously reported (4).
Measurements of CAT-1-mediated L-arginine transport by PAEC. Measurement of CAT-1-mediated transport of radiolabeled L-arginine (L-[3H]arginine; Amersham, Arlington Heights, IL) by normoxic and hypoxic cells was performed as previously described by Zharikov et al. (30). In brief, to remove residual culture medium and extracellular Na, cells were washed with a solution of the following composition (in mM): 140 LiCl, 5.9 KCl, 1.2 MgSO4, 1.0 CaCl2, 5.6 glucose, and 10 HEPES-Tris (pH 7.4; buffer A). Transport assays were initiated by the addition of the same buffer containing 50 µM unlabeled L-arginine plus L-[3H]arginine (5 µCi/ml; 60 Ci/mmol), and 30 s later, transport was stopped by washing the cells four times with ice-cold buffer A. To estimate the nonspecific uptake of L-arginine, the same experiments were carried out in parallel with buffer A containing 10 mM unlabeled L-arginine. After solubilization of the cells in 0.2% SDS, aliquots were added to scintillation fluid, and radioactivity was quantitated by liquid scintillation spectrometry. CAT-1-mediated L-arginine uptake was determined by subtracting the nonspecific component of uptake from total Na-independent uptake measured in buffer A.
Preparation of plasma membrane vesicles. The procedure for isolating plasma membrane vesicles yields a preparation that is free of mitochondria and microsomes and that has 15-fold enrichment of Na-K-ATPase (3); the procedure has been described by us in detail (29). Briefly, monolayer cultures were scraped into precooled Hanks' balanced salt solution containing 0.2 mM dithiothreitol, and the cell suspension was centrifuged at 800 g for 15 min. The cell pellet was resuspended at a ratio of 1 vol of pellet to 9 vol of buffer B [0.25 M sucrose, 1 mM MgSO4, and 10 mM HEPES-Tris (pH 7.4) containing 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 µg/ml leupeptin, and 2 µg/ml pepstatin A], and the suspended cells were disrupted by nitrogen cavitation using a precooled minicell disruptor. The broken cell suspension was centrifuged at 2,000 g for 5 min to remove nuclei, large aggregates, and unbroken cells. The postnuclear supernatant was centrifuged for 30 min at 85,000 g. The resulting pellet was resuspended in buffer B and layered over a discontinuous (15, 30, and 45%) sucrose gradient. The gradient was centrifuged at 100,000 g for 1 h. The bands at the 15 and 30% sucrose interfaces were collected, diluted with cold 10 mM HEPES-Tris, and centrifuged at 85,000 g for 30 min. The final pellet of plasma membrane vesicles was resuspended in 140 mM potassium phosphate buffer (pH 6.8) containing 1 mM MgSO4. Before solubilization of the plasma membrane vesicles (see below), system y+ transport activity was measured according to the procedure described earlier (29).
Solubilization of plasma membrane proteins. Plasma membrane proteins were solubilized by the method described by Tamarappoo et al. (27) with several minor modifications. Plasma membrane vesicles were mixed with an equal volume of solubilization buffer containing 2.5% sodium cholate, 4 M urea, 1 mM EDTA, 100 mM KCl, 1 mM PMSF, 2 µg/ml leupeptin, and 2 µg/ml pepstatin A (pH 7.4, 10 mM HEPES-Tris). The mixture was incubated for 30 min on ice and then centrifuged at 100,000 g for 1 h. The solubilized proteins in the supernatant were precipitated with a final concentration of 20% polyethylene glycol (PEG-8000) by the addition of 2 vol of 30% PEG-8000 and incubation for 20 min. The mixture was centrifuged at 100,000 g for 1 h, and the resulting pellet was washed three times in 150 mM KCl, 1 mM MgSO4, and 10 mM HEPES-Tris (pH 7.4) to remove residual PEG-8000. The washed pellets were resuspended in 20% glycerol, 2 mM EDTA, 2 mM dithiothreitol, 0.2% sodium cholate, 0.25% asolectin, and 10 mM HEPES-Tris (pH 7.4; STAB buffer) and were used in immunodepletion experiments immediately or after overnight storage at 4°C.
Immunodepletion of system y+ transport activity with anti-fodrin and anti-ankyrin antibodies. A 1-ml aliquot of anti-mouse IgG covalently linked to agarose beads (Sigma) was incubated for 1 h with 10 µg of polyclonal anti-ankyrin (Cortex Biochem, San Leandro, CA) or anti-fodrin (gift from Dr. Michael Kilberg, University of Florida) antibodies on ice and then centrifuged. The agarose beads were washed one time with STAB buffer and were mixed with solubilized plasma membrane proteins in STAB buffer. After incubation for 1 h on ice, the beads and solubilized plasma membrane proteins were centrifuged, and the resulting supernatants were carefully separated from the beads. The beads and an aliquot of the supernatants were used for immunoblot analysis (see below), and the rest of the proteins in the supernatants were reconstituted into proteoliposomes for the measurement of CAT-1-mediated L-arginine transport activity.
Reconstitution procedure. Reconstitution of proteins into liposomes was performed following the protocol described by Fafournoux et al. (11). A stock solution of asolectin (a mixture of soybean phospholipids; Sigma) was prepared by suspending the dry phospholipids in 140 mM potassium phosphate buffer (pH 6.8) containing 1 mM MgSO4 (PPB buffer) under an atmosphere of nitrogen followed by sonication. The liposomal suspension was centrifuged at 1,500 g for 2 min to remove aggregates of undissolved phospholipids. Reconstitution of amino acid transport activity was performed by mixing 0.5 mg of solubilized plasma membrane proteins with 10 mg of asolectin liposomes in a total volume of 1 ml. The mixture was frozen in liquid nitrogen, thawed at room temperature, diluted with at least 10 vol of PPB buffer, and then sonicated for 20 s. The proteoliposomes were pelleted by centrifugation at 100,000 g for 2 h and then resuspended in PPB buffer for use in transport assays. L-Arginine transport assay. Transport assays in plasma membrane vesicles and in proteoliposomes were performed as previously described by us (29). Briefly, plasma membrane vesicles or proteoliposomes loaded with PPB buffer (30 µl) were added to 270 µl of external solution containing 140 mM NaSCN, 1 mM MgSO4, 10 mM HEPES-Tris (pH 7.4), and 50 µM L-[3H]arginine. After incubation for 3 min at 37°C, the reactions were terminated by the addition of 5 ml of ice-cold 140 mM NaCl (stop solution) followed by filtration through glass-fiber Whatman GF/C filters presoaked in 0.3% polyethylenimine to decrease the nonspecific absorption of L-[3H]arginine on the filter. The filters were washed four times with 5 ml of stop solution, dried, and counted using liquid scintillation spectrometry. Zero-time blank values (membrane vesicles or proteoliposomes added after stop solution) were subtracted from all experimental values. Western blots. Whole cell extracts or plasma membrane fractions from normoxic and hypoxic PAEC, as well as beads and supernatants from the immunodepletion experiments (see above), were subjected to immunoblot analysis. For immunoblotting, samples (15-20 µg of protein) were denatured with Laemmli buffer, heated to 95°C for 5 min, and electrophoresed on 7.5% polyacrylamide gel in the presence of SDS. Separated proteins were electrotransferred to nitrocellulose membranes, incubated with 5% fat-free milk (Bio-Rad) for 2 h, and then probed with anti-CAT-1, anti-fodrin (both anti-CAT-1 and anti-fodrin antibodies were kindly provided by Dr. Michael Kilberg), or anti-ankyrin (Cortex Biochem, San Leandro, CA) antibodies. The membrane was incubated with primary antibodies overnight at 4°C and was washed with 50 ml of 0.1% Tween 20, 20 mM Tris · HCl, pH 7.5, and 150 mM NaCl (TTBS) three times for 10 min. Secondary goat anti-rabbit or goat anti-mouse IgG conjugated to alkaline phosphatase (Bio-Rad) was diluted in TTBS plus 2% nonfat milk and incubated with the membranes at room temperature for 1-2 h. Depending on the primary antibodies, the secondary antibodies were diluted from 1:2,000 to 1:30,000. After the membranes were washed with TTBS, enhanced chemiluminescence (Immun-Star; Bio-Rad) was used to visualize the reactive proteins followed by densitometric quantification using a Fluor-S MultiImager system (Bio-Rad). Statistical analysis. Data are expressed as means ± SE. Comparisons between values were made using an unpaired two-tailed Student's t-test. A P value of < 0.05 was considered statistically significant. ![]() |
RESULTS |
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Effect of calpain inhibition on
L-[3H]arginine
uptake by normoxic and hypoxic PAEC.
To confirm the participation of calpain in the regulation of
L-arginine transport in PAEC, we exposed cultured cells to
normoxia or hypoxia for 24 h in the presence or absence of calpain
inhibitor 1 (N-acetyl-Leu-Leu-norleucinal; Sigma).
As shown in Fig. 1, incubation of normoxic
cells in the presence of 20 µM calpain inhibitor 1 increased
L-arginine transport in PAEC
(P < 0.001) compared with that in
normoxic cells incubated in the absence of calpain inhibitor 1, suggesting that activation of calpain inhibits system
y+-mediated L-arginine
transport activity. Exposure of PAEC to hypoxia for 24 h caused a 50%
inhibition of L-arginine uptake
(P < 0.001). Exposure to hypoxia in
the presence of calpain inhibitor 1 decreased the magnitude of the
hypoxia-induced reduction in L-arginine transport and
returned L-arginine transport to the level observed in
normoxic cells, suggesting that calpain activation is responsible, at
least in part, for the reduction in the system
y+-mediated L-arginine
transport in hypoxic PAEC. Similar changes in CAT-1-mediated
L-arginine transport activity in normoxic and hypoxic PAEC
were observed with calpeptin (Calbiochem-Novabiochem, La Jolla, CA) and
with E-64d (Calbiochem-Novabiochem), two other calpain-specific
inhibitors (data not shown).
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DISCUSSION |
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We have previously reported that exposure to hypoxia inhibits
CAT-1-mediated L-arginine transport in porcine PAEC (4,
30). Short-term exposures to hypoxia, i.e., 4 h, inhibit
L-arginine transport by altering membrane potential, but
the mechanism responsible for inhibition of transport associated with
long-term exposures (i.e., 12-24 h) to hypoxia is not known. We
have also recently reported that exposure to hypoxia upregulates
calpain mRNA expression and activity (28). In the present study, we
show that inhibition of CAT-1-mediated L-arginine transport
by exposure to 24 h of hypoxia can be significantly
ameliorated by the presence of an inhibitor of calpain activity, i.e.,
calpain inhibitor 1, calpeptin, or E-64d, indicating that calpain plays
a role in mediating the inhibition of L-arginine transport
in PAEC exposed to long-term hypoxia. It should be noted that the
addition of calpain inhibitor 1 to hypoxic PAEC did not result in the
full recovery of L-arginine uptake to levels observed in
normoxic cells treated with calpain inhibitor 1 (Fig. 1). This suggests
that there may be an additional set of non-calpain-mediated factors
that contribute to the inhibition of L-arginine transport
in hypoxic PAEC. Calpain also appears to play a role in the regulation
of L-arginine transport under normoxic conditions, since
transport was significantly higher in normoxic PAEC incubated in the
presence of a calpain inhibitor than in normoxic cells incubated in the
absence of a calpain inhibitor.
Calpain is the name used to describe a family of Ca2+-regulated nonlysosomal neutral cysteine proteases with a variety of endogenous substrates (9). Although the effects of exposure to long-term hypoxia on L-arginine transport and the effects of calpain inhibitor 1 on L-arginine transport in normoxic PAEC could be explained by calpain-mediated proteolysis of CAT-1 protein, neither hypoxia nor calpain inhibitor 1 affected the plasma membrane content of CAT-1. These results suggest that an alternate mechanism exists to explain the effects of calpain on CAT-1-mediated L-arginine transport in PAEC.
A number of reports have documented important links between the cytoskeletal proteins ankyrin and fodrin and integral plasma membrane proteins (1, 2, 20). Recently, Handlogten et al. (15) reported an association between the hepatic system A amino acid transporter and ankyrin/fodrin complexes, the first such association between an organic solute transporter and ankyrin/fodrin complexes. Ankyrin and fodrin have also been reported to serve as in vivo and in vitro substrates for calpain (9). Our results demonstrate that exposure to hypoxia for 24 h results in a significant decrease in the plasma membrane contents of ankyrin and fodrin that can be prevented in the case of fodrin, or significantly abated in the case of ankyrin, by calpain inhibitor 1. These observations suggest that calpain may regulate L-arginine transport activity in PAEC through calpain-mediated fodrin or ankyrin hydrolysis.
If fodrin and/or ankyrin is one of the regulatory elements for CAT-1-mediated L-arginine transport in PAEC, we might expect a protein-protein interaction between ankyrin and/or fodrin and CAT-1 protein. To test this possibility, we conducted coimmunoprecipitation experiments with anti-ankyrin or anti-fodrin antibodies and solubilized PAEC plasma membrane proteins. Incubation with anti-fodrin antibodies resulted in immunoprecipitation of nearly 70% of the CAT-1 transporters in the solubilized plasma membranes, whereas incubation with anti-ankyrin antibodies did not precipitate CAT-1 protein from the plasma membrane. We also observed a 60% decrease in CAT-1-mediated L-arginine transport activity in reconstituted proteoliposomes after immunoprecipitation of solubilized plasma membrane proteins with anti-fodrin antibodies. Anti-ankyrin antibodies had no effect on CAT-1-mediated L-arginine transport in the reconstituted proteoliposomes. Taken together, these results support a protein-protein interaction between fodrin and CAT-1 protein in porcine PAEC plasma membranes and suggest that calpain-mediated proteolysis of fodrin is an important mechanism responsible for the reduction of CAT-1-mediated L-arginine transport in PAEC exposed to long-term hypoxia.
The functional significance of the linkage that we have identified between CAT-1 and fodrin in PAEC was not addressed in this study. However, CAT-1 is a caveolar protein in porcine PAEC (19), and direct interaction between caveolar proteins and fodrin has been documented for various membrane proteins (7, 12, 23). Because morphological and biochemical observations have shown that caveolae in endothelial cells tend to exist in parallel with actin filaments (16, 17), it has been suggested that fodrin may mediate the linkage between caveolar proteins and actin (12). Therefore, we propose, based on our present results, that long-term exposure to hypoxia results in calpain-mediated fodrin proteolysis that, in turn, disrupts the functional association between CAT-1 and actin microfilaments leading to inhibition of L-arginine transport in PAEC. The evidence presented by us is the first to suggest that L-arginine transport in mammalian cells is regulated by the actin cytoskeleton. Additional studies will be needed to confirm this thesis and to define further the regulatory influences of the actin cytoskeleton on L-arginine transport and NO production by PAEC.
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ACKNOWLEDGEMENTS |
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We thank Humberto Herrera for assistance with tissue culture, Dr. Michael Kilberg for providing anti-CAT-1 and anti-fodrin antibodies, and Janet Wootten for editorial assistance.
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: E. R. Block, Research Service (151), Malcom Randall Dept. of Veterans Affairs Medical Center, 1601 SW Archer Rd., Gainesville, FL 32608-1197 (E-mail: edward.block{at}med.va.gov).
Received 5 April 1999; accepted in final form 27 July 1999.
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