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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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., equal4 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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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.

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 alpha -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.

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 protein-1 · 3 min-1) compared with control proteoliposomes incubated with nonimmune IgG (8,101 ± 481 pmol · mg protein-1 · 3 min-1). 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


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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., equal4 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.


    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.


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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5.   Block, E. R., J. M. Patel, and D. Edwards. Mechanism of hypoxic injury of pulmonary artery endothelial cell plasma membranes. Am. J. Physiol. Cell Physiol. 257: C223-C231, 1989[Abstract/Free Full Text].

6.   Bourguignon, L. Y. W., and H. Jin. Identification of the ankyrin-binding domain of the mouse T-lymphoma cell inositol 1,4,5-trisphosphate (IP3) receptor and its role in the regulation of IP3-mediated internal Ca2+ release. J. Biol. Chem. 270: 7257-7260, 1995[Abstract/Free Full Text].

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