Vol. 278, Issue 1, L111-L117, January 2000
Association of L-arginine transporters with
fodrin: implications for hypoxic inhibition of arginine
uptake
S. I.
Zharikov and
E. R.
Block
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 |
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
protein
1 · 3 min1 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 |
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 |
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.
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RESULTS |
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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Fig. 1.
Effect of calpain inhibitor 1 (N-acetyl-Leu-Leu-norleucinal) on
Na-independent
L-[3H]arginine
transport activity (system y+)
in pulmonary artery endothelial cells (PAEC). Cells grown in 24-well
cluster trays were exposed to normoxia (Nor; 95% room air-5%
CO2) or hypoxia (Hyp; 0%
O2-5%
CO2-95% nitrogen) for 24 h in the
presence and absence of 20 µM calpain inhibitor 1 (CI1). Immediately
after exposure, the medium was discarded, and cells in each well
were washed one time with 0.5 ml of LiCl-Dulbecco's solution. After
cells were washed, transport of L-arginine was measured as
described in MATERIALS AND METHODS. prot, Protein. Results
are means of 3 experiments (±SE) with 8 replicates per experiment.
* P < 0.001 vs. normoxia
without calpain inhibitor 1;
** P < 0.001 vs. hypoxia
without calpain inhibitor 1 and P > 0.05 vs. normoxia without calpain inhibitor 1.
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Analysis of L-arginine transporter (CAT-1)
contents in plasma membranes of normoxic and hypoxic
PAEC. To determine the content of CAT-1 transporters in
plasma membranes of normoxic and hypoxic PAEC, we performed Western
blot analysis using antibodies to CAT-1 on plasma membrane fraction
proteins harvested from cultured PAEC exposed to normoxia or hypoxia
for 24 h in the presence and absence of calpain inhibitor 1 (20 µM; Fig. 2). Neither hypoxia nor calpain inhibitor 1 changed the content of L-arginine
transporters in plasma membranes. These results provide evidence that
the hypoxia- and calpain inhibitor 1-induced changes in
L-arginine uptake mediated by system
y+ observed in Fig. 1 are not
associated with changes in the expression of CAT-1 transporters in the
PAEC plasma membranes.
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Fig. 2.
Western blot analysis of L-arginine transporter
[cationic amino acid transporter (CAT)-1] contents in
normoxic and hypoxic PAEC. Cells grown in 100-mm dishes were exposed to
normoxia or hypoxia for 24 h in the presence and absence of 20 µM
calpain inhibitor 1 (N-acetyl-Leu-Leu-norleucinal). Plasma
membrane fraction proteins were separated by 7.5% SDS-PAGE and
immunoblotted with CAT-1 antibodies as described in MATERIALS AND
METHODS. A: representative
immunoblot. Lane 1, normoxia without
calpain inhibitor 1; lane 2, normoxia
in the presence of calpain inhibitor 1; lane
3, hypoxia without inhibitor; lane
4, hypoxia in the presence of calpain inhibitor 1. No.
at left, molecular mass. B:
CAT-1 contents (mean of the relative density units ± SE) from 3 experiments. No significant differences in CAT-1 protein levels in the
4 groups were observed.
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Immunoblot analysis of ankyrin and fodrin contents in
plasma membrane fractions from PAEC. Because ankyrin
and fodrin are substrates for calpain in a variety of cells (9) and
participate in regulating the activity of many membrane proteins, we
examined the effects of hypoxia and calpain inhibition by calpain
inhibitor 1 on fodrin and ankyrin contents in PAEC. Plasma membrane
fraction proteins isolated from PAEC exposed to normoxia or hypoxia for 24 h in the absence and presence of calpain inhibitor 1 (20 µM) were
separated by 7.5% SDS-PAGE and then analyzed for ankyrin and fodrin
contents using antibodies to these cytoskeletal proteins (Fig.
3). Immunoblotting for ankyrin demonstrated
a decrease in ankyrin content and an increase in a 150-kDa
immunoreactive ankyrin fragment in hypoxic cells (Fig.
3A). Immunoblotting for fodrin revealed a decrease in fodrin content in the absence of any
immunoreactive degradation products in hypoxic cells (Fig.
3B). Calpain inhibitor 1 increased
ankyrin and fodrin contents in plasma membrane fractions of normoxic
and hypoxic PAEC. These results suggest that hypoxia stimulates
hydrolysis of ankyrin and fodrin and that calpain inhibitor 1 prevents
this hydrolysis in normoxic and hypoxic PAEC.
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Fig. 3.
Immunoblot analysis and quantification of ankyrin
(A) and fodrin
(B) levels in plasma membrane
fractions isolated from PAEC exposed to normoxia or hypoxia for 24 h in
the absence and presence of calpain inhibitor 1 (N-acetyl-Leu-Leu-norleucinal; 20 µM). Plasma membrane fractions were isolated from PAEC as described
in MATERIALS AND METHODS. Plasma membrane proteins were
separated by 7.5% SDS-PAGE and immunoblotted with anti-ankyrin
(A) or anti-fodrin
(B) antibodies. Representative
immunoblots are shown. Data show quantification of blots for intact
ankyrin (A) and  -fodrin
(B; mean of 3 experiments ± SE).
Lane 1, normoxia without calpain
inhibitor 1; lane 2, normoxia in the
presence of calpain inhibitor 1; lane
3, hypoxia without inhibitor; lane
4, hypoxia in the presence of calpain inhibitor 1. Nos.
at left, molecular mass in kDa.
* P < 0.01 vs. normoxia
without calpain inhibitor 1;
** P < 0.01 vs. hypoxia
without calpain inhibitor 1.
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Immunoprecipitation of solubilized CAT-1 protein with
anti-fodrin and anti-ankyrin antibodies. To demonstrate
the possible interaction between CAT-1 transporters and ankyrin or
fodrin, we evaluated whether immunoprecipitation of ankyrin and fodrin from solubilized plasma membrane proteins of normoxic PAEC resulted in
coprecipitation of CAT-1 and loss of CAT-1-mediated
L-arginine transport. To do this, solubilized plasma
membrane proteins were incubated with rabbit anti-fodrin or mouse
anti-ankyrin antibodies for 1 h at 4°C after which IgG covalently
linked to agarose beads was added. The mixtures were incubated for an
additional 1 h at 4°C and then centrifuged. After
centrifugation, the supernatants were carefully removed, and aliquots
of the proteins in the supernatants and those precipitated with the
beads were subjected to Western blot analysis with CAT-1 antibodies.
The proteins remaining in the supernatants were reconstituted into
proteoliposomes and assayed for CAT-1-mediated L-arginine
transport activity. Western blot analysis demonstrated that sodium
cholate solubilized nearly all of the CAT-1 transporters from PAEC
plasma membranes (compare Fig. 4,
A and
B, lanes
1 and 2). Incubation
of the solubilized plasma membrane proteins with anti-fodrin antibodies
resulted in nearly a 70% decrease in CAT-1 content in the supernatants (Fig. 4A, lane
3 vs. lane 4) and in
the appearance of CAT-1 immunoreactivity in the pellet of beads (Fig.
4A, lane
5 vs. lane 6),
suggesting that anti-fodrin antibodies are able to immunoprecipitate
CAT-1 transporters. In contrast, anti-ankyrin antibodies did not
precipitate solubilized CAT-1 transporters (Fig.
4B, lane
3 vs. lane 4 and lane 5 vs. lane
6). The specific interaction of CAT-1 transporters with fodrin, but not with ankyrin, was also confirmed in the
experiments using reconstituted proteoliposomes (Table
1). The proteoliposomes reconstituted after
immunodepletion with anti-fodrin antibodies revealed a 60% decrease in
CAT-1-mediated L-arginine transport activity (3,291 ± 117 pmol · mg
protein1 · 3 min1) compared with
control proteoliposomes incubated with nonimmune IgG (8,101 ± 481 pmol · mg
protein1 · 3 min1). CAT-1-mediated
L-arginine transport activity in reconstituted proteoliposomes after immunodepletion with anti-ankyrin antibodies did
not differ significantly from that in the control proteoliposomes (Table 1).
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Fig. 4.
Immunoprecipitation of solubilized CAT-1 protein with anti-fodrin
(A) or anti-ankyrin
(B) antibodies. PAEC plasma
membranes were solubilized in buffer containing 1.25% sodium cholate,
2 M urea, 1 mM EDTA, 100 mM KCl, 1 mM phenylmethylsulfonyl fluoride,
2 µg/ml leupeptin, and 2 µg/ml pepstatin A. Solubilized
proteins were separated from detergent-resistant parts of membranes by
centrifugation. Solubilized proteins were incubated with anti-fodrin
(A) or anti-ankyrin
(B) antibodies and then with IgG
covalently linked to agarose beads as described in MATERIALS AND
METHODS. After centrifugation, proteins in the supernatants
and those precipitated with the beads were subjected to Western blot
analysis. Representative immunoblots are shown. Data show relative
CAT-1 contents in different samples (means ± SE).
Lane 1, solubilized proteins;
lane 2, detergent-resistant pellet;
lane 3, control supernatant
(incubation with nonimmune IgG); lane
4, supernatant after incubation with anti-fodrin
(A) or anti-ankyrin
(B) antibodies;
lane 5, control beads (incubation with
nonimmune IgG); lane 6, beads
incubated in the presence of anti-fodrin
(A) or anti-ankyrin
(B) antibodies. No. at left,
molecular mass. * P < 0.01 vs.
control supernatant (lane 3);
** P < 0.01 vs. control beads
(lane 5).
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Table 1.
CAT-1-mediated L-arginine transport activity in
proteoliposomes reconstituted after immunodepletion of plasma membrane
proteins by anti-ankyrin or anti-fodrin antibodies
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DISCUSSION |
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 |
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 |
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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