Classical isoforms of PKC as regulators of CAT-1 transporter activity in pulmonary artery endothelial cells

Karina Y. Krotova, Sergey I. Zharikov, and Edward R. Block

Department of Medicine, University of Florida College of Medicine; and Research Service, Malcom Randall VA Medical Center, Gainesville, Florida 32610


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We examined which isoforms of protein kinase C (PKC) may be involved in the regulation of cationic amino acid transporter-1 (CAT-1) transport activity in cultured pulmonary artery endothelial cells (PAEC). An activator of classical and novel isoforms of PKC, phorbol 12-myristate-13-acetate (PMA; 100 nM), inhibited CAT-1-mediated L-arginine transport in PAEC after a 1-h treatment and activated L-arginine uptake after an 18-h treatment of cells. These changes in L-arginine transport were not related to the changes in the expression of the CAT-1 transporter. The inhibitory effect of PMA on L-arginine transport was accompanied by a translocation of PKCalpha (a classical PKC isoform) from the cytosol to the membrane fraction, whereas the activating effect of PMA on L-arginine transport was accompanied by full depletion of the expression of PKCalpha in PAEC. A selective activator of Ca2+-dependent classical isoforms of PKC, thymeleatoxin (Thy; 100 nM; 1-h and 18-h treatments), induced the same changes in L-arginine uptake and PKCalpha translocation and depletion as PMA. The effects of PMA and Thy on L-arginine transport in PAEC were attenuated by a selective inhibitor of classical PKC isoforms Go 6976 (1 µM). Phosphatidylinositol-3,4,5-triphosphate-dipalmitoyl (PIP; 5 µM), which activates novel PKC isoforms, did not affect L-arginine transport in PAEC after 1-h and 18-h treatment of cells. PIP (5 µM; 1 h) induced the translocation of PKCepsilon (a novel PKC isoform) from the cytosolic to the particulate fraction and did not affect the translocation of PKCalpha . These results demonstrate that classical isoforms of PKC are involved in the regulation of CAT-1 transport activity in PAEC. We suggest that translocation of PKCalpha to the plasma membrane induces phosphorylation of the CAT-1 transporter, which leads to inhibition of its transport activity in PAEC. In contrast, depletion of PKCalpha after long-term treatment with PMA or Thy promotes dephosphorylation of the CAT-1 transporter and activation of its activity.

cationic amino acid transporter; L-arginine transport


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

VASCULAR ENDOTHELIAL CELLS are a rich source of nitric oxide, a free radical with unique physiological bioregulatory activities (23). Endothelial cells generate nitric oxide from L-arginine via the catalytic action of endothelial nitric oxide synthase (2). Recent reports indicate that nitric oxide production by endothelial cells is dependent on the availability of extracellular L-arginine (12, 14, 16, 17, 28), suggesting that delivery of extracellular L-arginine may be a regulatory factor in nitric oxide production. The transport of L-arginine, as well as other cationic amino acids, is mediated by the system y+ carrier in different types of cells (9). Three related cationic amino acid transporters (CAT-1, CAT-2B, and CAT-3) with characteristics of system y+ have been cloned and identified (6, 20). The fourth related transporter, CAT-2A, exhibits 10-fold lower substrate affinity and higher transport capacity than the other three members of the family and was cloned from murine liver (7). In endothelial cells, system y+ is represented by the CAT-1 transporter, which is responsible for 70-95% of L-arginine uptake (38).

Recently, we reported that the constitutive CAT-1 transporter localizes in caveolae of porcine pulmonary artery endothelial cells (PAEC) and can form a functional unit with endothelial nitric oxide synthase at the caveolar membrane (21). Caveolae are specialized microdomains of the plasma membrane defined by the presence of structural proteins called caveolins and by high cholesterol and sphingolipid contents (3). Caveolin functions as a scaffolding protein within caveolar membranes and therefore interacts with signaling proteins including G protein-mediated signaling molecules, calcium-mediated signaling molecules, tyrosine kinases-mitogen-activated protein kinase pathway components, and lipid signaling molecules (11, 32, 33). Localization of the CAT-1 transporter in the immediate vicinity of transduction cascades could contribute to the regulation of CAT-1 transport activity in endothelial cells.

The possible regulation of endothelial CAT-1 transporter by caveolar kinases, and in particular by protein kinase C (PKC), has been poorly studied. Graf and coauthors (13) showed that phorbol myristate acetate (PMA), a diacylglycerol analog that directly activates PKC, inhibits CAT-1 transport activity in EA.hy926 endothelial cells without reducing CAT-1 mRNA or protein expression. In contrast, PMA-mediated PKC activation in human umbilical vein endothelial cells has been reported to cause an increase in CAT-1 transport activity (27). To date, 12 isoforms of PKC have been identified. All PKC isoforms consist of an NH2-terminal regulatory region and a COOH-terminal catalytic region. PKC isoforms are divided into three subgroups on the basis of the properties of the regulatory regions (24). Classical PKC isoforms (alpha , beta 1, beta 2, and gamma ) require calcium, diacylglycerol, and phosphatidylserine for activation. Novel PKC isoforms (delta , epsilon , theta , eta ) require diacylglycerol and phosphatidylserine. Atypical PKC isoforms (iota , lambda , zeta ) and PKCµ require phosphatidylserine only. Thus each isoform of PKC could be regulated by different stimuli and regulate differential downstream signaling molecules.

In this study, we examined which isoforms of PKC may be involved in the regulation of CAT-1 transport activity in cultured PAEC. Our data demonstrate that classical isoforms of PKC are involved in the regulation of the CAT-1 transporter without affecting its expression in PAEC and provide a rationale for explaining the differences observed by others studying the effects of PMA on CAT-1-mediated L-arginine transport.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture and reagents. All experiments were performed with PAEC. 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 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.

Phorbol-12-myristate-13-acetate (PMA), 4alpha -phorbol, Go 6976, thymeleatoxin (Thy), and phosphatidyl-3,4,5-triphosphate-dipalmitoyl (PIP) were obtained from Calbiochem. Antibodies to PKC isoforms were purchased from BioTransduction Laboratories. Peptide corresponding to the fourth extracellular domain of the human CAT-1 transporter (T-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 Cocalico. Rabbit polyclonal antibodies to this peptide were prepared, purified by using HiTrap Protein G column (Amersham Pharmacia Biotech), and characterized as previously reported by our laboratory (35). All other chemicals were purchased from Sigma Chemical.

Measurements of CAT-1-mediated Na+-independent L-arginine uptake by PAEC. To remove residual culture medium, extracellular Na+, and drugs, cells grown in 24-well plates were washed with 0.5 ml of 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, 37°C, LiCl-Dulbecco solution). Transport assays were initiated by the addition of the same buffer (0.5 ml) containing 50 µM unlabeled L-arginine plus L-[3H]arginine (10 µCi/ml), and 1 min later transport was stopped by washing the cells four times with 2 ml of ice-cold LiCl-Dulbecco solution. 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 (uptake in the presence of 10 mM unlabeled L-arginine instead of 50 µM unlabeled L-arginine). Aliquots of solubilized cells were also taken for protein content measurements by use of the Lowry method (18).

Separation of membrane and cytosolic fractions from PAEC. Control cells and cells treated with drugs in 100-mm culture dishes were washed twice with cold PBS and then scraped in an ice-cold buffer (0.7 ml per dish) of the following composition (in mM: 20 Tris · HCl, pH 7.4, 10 EDTA, 5 EGTA, 5 2-mercaptoethanol, 10 benzamidine, 1 mg/ml leupeptin, 50 µg/ml PMSF, 0.1 mg/ml ovalbumin, and 0.1 µg/ml aprotinin). After incubation for 5 min on ice, cells were sonicated and centrifuged at 20,000 g for 30 min. The supernatant was saved as the cytosolic fraction. The pellet (membrane fraction) was resuspended in 200 µl of the solubilization buffer (150 mM NaCl, 10 mM Tris · HCl, pH 7.4, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate Na, 1 mM EDTA, and 1 mM EGTA), incubated on ice for 30 min, and sonicated three times for 15 s. The solubilizate was centrifuged at 20,000 g for 30 min, and the supernatant was referred to as the solubilizate of the particulate fraction. Aliquots of the cytosolic fraction and the solubilizate of the particulate fractions were mixed with Laemmli sample buffer (1:2 vol/vol) and used for SDS-PAGE after Western blot analysis of PKC isoforms.

Western blot analysis of contents of the CAT-1 transporter and PKC isoforms. Preparing the samples for the expression of PKC isoforms in cytosolic and particulate fractions by Western blot analysis has been described above. For the analysis of CAT-1 contents in PAEC, cells grown on 100-mm dishes were washed twice with ice-cold PBS without Ca2+ and 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 20,000 g for 30 min, and supernatants were taken for Western blot analysis. Samples (15-20 µg of protein) were separated by SDS-PAGE (7.5% acrylamide) and transferred to nitrocellulose. The expressed CAT-1 protein was blotted overnight (4°C) with anti-CAT-1 antibody at 1:1,000 dilution and horseradish peroxidase-conjugated goat anti-rabbit IgG (Cell Signaling) at 1:2,000 dilution (for 1 h). For detection, ECL Western blotting detection reagent (Amersham Pharmacia Biotech) was used. The expressed PKC isoforms were hybridized with mouse monoclonal anti-PKC antibodies. Alkaline phosphatase-conjugated goat anti-mouse secondary antibody (1:3,000 dilution; Bio-Rad) was used to visualize bound primary antibodies with chemiluminescence substrate (Immuno-Star; Bio-Rad). The density of the bands was quantitated by using a Fluor-S MultiImager system (Bio-Rad).

Statistics. 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 and the expression of PKC isoforms. 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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To study the effects of PKC activation on CAT-1-mediated transport in PAEC we treated cells with PMA or 4alpha -phorbol, an inactive analog of PMA. The effects of PMA on L-arginine uptake by PAEC were dependent on the duration of the cell treatment: a 1-h treatment with PMA inhibited L-arginine transport by ~30%, whereas long-term treatment (18 h) with PMA activated L-arginine uptake by PAEC by ~70%. 4alpha -Phorbol, did not affect L-arginine transport in PAEC after short-term or long-term treatment (Fig. 1, A and B). The changes in Na-independent L-arginine transport in PAEC induced by short-term and long-term PMA treatments were not related to the changes in CAT-1 expression evaluated by Western blot analysis in PAEC (Fig. 1, C and D).


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Fig. 1.   Effects of phorbol-12-myristate-13 acetate (PMA) on L-[3H]arginine transport and expression of the cationic amino acid transporter CAT-1 in pulmonary artery endothelial cells (PAEC). Cells were grown in 24-well plates until confluence. At 12-16 h before the treatment with drugs, cell medium (RPMI 1640) was changed to serum-free RPMI 1640. The drugs for treatments were dissolved in serum-free media. Cells were treated with DMSO (final concentration 0.1%), or 100 nM PMA (in 0.1% DMSO), or 100 nM 4alpha -phorbol (4 P, in 0.1% DMSO) for 1 h (A) or 18 h (B). After the treatment, cells were washed with warmed LiCl-Dulbecco solution and then incubated in LiCl-Dulbecco solution containing 50 µM L-[3H]arginine (10 µCi/ml) for 60 s at 37°C. The cells were then washed 4 times with 2 ml of ice-cold LiCl-Dulbecco and lysed, and radioactivity and protein contents of the lysates were determined. Data are expressed as a percentage of control (Cont) values derived from untreated cells (400-500 pmol/mg protein/min) (means ± SD mean, n = 4 experiments with 4 observations per experiment). *P < 0.05 vs. control. C: Western blot analysis of CAT-1 transporter expression in PAEC. At 12-16 h before the treatment with DMSO or 100 nM PMA, the medium was changed to serum-free medium. Cells were treated with 0.1% DMSO or 100 nM PMA in 0.1% DMSO for 1 h or 18 h. Lane 1, 0.1% DMSO; 1-h treatment. Lane 2, 100 nM PMA; 1-h treatment. Lane 3, 0.1% DMSO; 18-h treatment. Lane 4, 100 nM PMA; 18-h treatment. Representative immunoblot from 4 experiments is shown. D: CAT-1 contents (mean of the relative density units ± SE) from 4 experiments. No significant differences in CAT-1 protein level in the 4 groups were observed. Numbers on the horizontal axis correspond to the lanes in C.

Because activation of PKC is accompanied by its translocation inside cells (10), we examined how the changes in L-arginine transport induced by PMA correlated with the redistribution of PKC isoforms and the CAT-1 transporter between cytosolic and particulate fractions in PAEC. For this, we studied the effects of PMA on L-arginine uptake and on expression of PKC isoforms and the CAT-1 transporter in parallel experiments. PKCalpha , PKCepsilon , and PKClambda /zeta were used as representatives of classical, novel, and atypical PKC isoforms, respectively. Cells were treated with 100 nM PMA for 10 min, 1 h, 4 h, and 18 h. A 10-min treatment of PAEC with PMA induced approximately a 20% decrease in L-arginine uptake, and the decrease was more profound (a 30% decrease) after 1 h of treatment (Fig. 2A). These treatments were not accompanied by a redistribution of the CAT-1 transporter between cytosolic and particulate fractions (Fig. 2, B and C). The same treatments induced almost a complete translocation of PKCalpha from the cytosolic to the particulate fraction (the ratio of cytosolic PKCalpha -to-particulate PKCalpha changed from 20:1 in control to 1:15 after a 10-min treatment with PMA; Fig. 3B). In control cells, the majority (80%) of PKCepsilon was expressed in the particulate fraction of PAEC, and PMA induced very little translocation of this isoform from the cytosolic to the particulate fraction after 10-min and 1-h incubations with PMA (the fraction of the particulate PKCepsilon in PAEC increased from 80% in control to 90 and 95% after treatments with PMA for 10 min and 1 h, respectively) (Fig. 3B). PKClambda /zeta localized mainly in the cytosolic fraction in control PAEC (the ratio of cytosolic PKClambda /zeta to particulate PKClambda /zeta is equal to ~4:1), and there were no significant changes in the distribution or expression of PKClambda /zeta after treatment with PMA (Fig. 3, A and B). After a 4-h treatment, PMA induced an ~60% increase in L-arginine uptake by PAEC, which approached a maximum (70%) after 18 h of treatment with PMA (Fig. 2A). The activation of L-arginine transport after the long-term treatments (4 and 18 h) with PMA was not accompanied by a redistribution of the CAT-1 transporter between cytosolic and particulate fractions (Fig. 2, B and C). At the same time, the long-term treatments with PMA induced a 65% depletion of particulate PKCalpha after the 4-h treatment and a 90% depletion after the 18-h treatment (Fig. 3, A and B). The treatment of PAEC with PMA for 4 h induced only a 16% depletion of PKCepsilon in the particulate fraction, which approached 79% after 18 h of treatment with PMA (Fig. 3, A and B). Thus the depletion of PKCepsilon was much slower than PKCalpha under the action of PMA, and PMA-induced activation of L-arginine transport in PAEC correlated more with the depletion of PKCalpha than PKCepsilon .


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Fig. 2.   Time dependence of PMA effects on L-arginine uptake and CAT-1 transporter distribution between cytosolic and particulate fractions in PAEC. Cells were grown in 24-well plates until confluence. At 12-16 h before treatment with PMA, cell medium (RPMI 1640) was changed to serum free. PMA (100 nM) was dissolved in serum-free RPMI 1640 containing 0.1% DMSO (final concentration). Cont cells were treated with RPMI 1640 containing 0.1% DMSO without PMA. Cells were treated with PMA for 10 min and 1, 4, and 18 h. A: L-arginine uptake. Treated cells were washed with warmed LiCl-Dulbecco and then incubated in 0.5 ml per well of LiCl-Dulbecco containing 50 µM L-[3H]arginine (10 µCi/ml) for 1 min at 37°C. The uptake was stopped by rapidly washing the cells with ice-cold LiCl-Dulbecco (4 times with 2 ml per well). To estimate the value of radioactivity binding to the surface of the cells, the same uptake experiments were performed in the presence of 10 mM of unlabeled L-arginine. The radioactivity accumulated by cells was quantitated, after solubilization of the cells in 0.2% SDS, by liquid scintillation spectrometry. Data represent means ± SD of 4 experiments with 8 replicates in each experiment. *P < 0.05 and **P < 0.01 vs. control. B: Western blot analysis of the CAT-1 distribution between the cytosolic (C) and the particulate (P) fractions. Representative immunoblot (from 3 experiments) is shown. C: densitometric analysis of the CAT-1 transporter contents in the C and P fractions. Each bar represents the mean of the relative density units ± SE from 3 experiments.



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Fig. 3.   Effect of PMA on expression of PKC isoforms in C and P fractions isolated from PAEC. Cells were grown in 100-mm culture dishes until confluence. At 12-16 h before the treatment with PMA, cell medium (RPMI 1640) was changed to serum-free. PMA (100 nM) was dissolved in serum-free RPMI 1640 containing 0.1% DMSO (final concentration). Cont cells were treated with RPMI 1640 containing 0.1% DMSO without PMA. Cells were treated with PMA for 10 min and 1, 4, and 18 h. For Western blot analysis of PKC isoforms, C and P fractions were isolated from control and treated PAEC as described in MATERIALS AND METHODS. Western blot analysis of PKC isoforms was performed as described in MATERIALS AND METHODS. A: representative immunoblots (from 3 experiments) for distribution of PKCalpha , PKCepsilon , and PKClambda /zeta between the C and P fractions after treatment with PMA. B: densitometric analysis of PKCalpha , PKCepsilon , and PKClambda /zeta contents in the C and P fractions. Each bar represents the mean of the relative density units ± SE from 3 experiments. *P < 0.05 vs. the particulate fraction after a 10-min treatment with 100 nM of PMA; **P < 0.05 vs. the particulate fraction in control.

To determine which PKC isoforms are responsible for the changes in L-arginine transport in PAEC induced by PMA, we used more specific PKC activators, e.g., Thy, which selectively activates Ca2+-dependent classical isoforms of PKC and PIP, which activates Ca2+-insensitive isoforms of PKC. As shown in Fig. 4, Thy (100 nM) induced the same changes in L-arginine uptake by PAEC as PMA (100 nM): it decreased L-arginine uptake after a 1-h treatment and increased uptake after an 18-h treatment. Unlike PMA and Thy, PIP (5 µM) did not affect L-arginine transport after any duration of treatment. The effects of PMA and Thy on L-arginine transport in PAEC were attenuated by a selective inhibitor of Ca2+-dependent isoforms of PKC, Go 6976 (1 µM), which does not affect the kinase activity of the Ca2+-independent isoforms (Fig. 5).


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Fig. 4.   Effects of thymeleatoxin (Thy) and dipalmitoyl-phosphatidylinositol-3,4,5-triphosphate (PIP) on L-arginine transport in PAEC. Cells were grown in 24-well plates until confluence. At 12-16 h before the treatment with drugs, cell medium was changed to serum-free. The drugs for treatments were dissolved in serum-free media containing 0.1% DMSO. Control medium contained only 0.1% DMSO. Cells were treated with PMA or Thy (both 100 nM) or PIP (5 µM) for 1 h (A) or 18 h (B). After treatment, the uptake experiment was performed. Data represent means ± SD, n = 3 experiments with 4 observations per experiment. *P < 0.01 vs. control; **P < 0.001 vs. control.



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Fig. 5.   Go 6976 (Go) blocks the effects of PMA and Thy on L-[3H]arginine transport in PAEC. Cells were grown in 24-well plates until confluence. At 12-16 h before treatment with drugs, cell medium was changed to serum-free. Cells were treated with PMA or Thy (both 100 nM) in the absence or presence of a selective inhibitor of Ca2+-dependent PKC isozymes, Go 6976 (1 µM), for 1 h (A) or 18 h (B). Control medium contained only 0.1% DMSO. After treatment, cells were washed with warmed LiCl-Dulbecco, and then the L-[3H]arginine uptake experiment was performed as described. Data represent means ± SD; n = 4 experiments with 4 observations per experiment. *P < 0.001 vs. control; **P < 0.01 vs. PMA and Thy without Go 6976, respectively.

Western blot analysis revealed that Thy (100 nM, 1 h) induced translocation of PKCalpha from the cytosolic to the particulate fraction, changing the cytosolic PKCalpha -to-particulate PKCalpha ratio from 22:1 to 1:14, an effect similar to the action of PMA (Fig. 6, A and B). At the same time, Thy did not affect the translocation of PKCepsilon (Fig. 6, A and B). In contrast to PMA, PIP (5 µM, 1 h) did not affect the translocation of PKCalpha (Fig. 6, A and B). However, PIP (5 µM, 1 h) decreased expression of PKCepsilon in the cytosolic fraction and decreased the ratio of cytosolic PKCepsilon to particulate PKCepsilon from 1:2 in control to 1:5 (the same ratio after the treatment with PMA is 1:2.5) (Fig. 6, A and B). Neither Thy nor PIP affected the translocation of PKClambda /zeta after a 1-h treatment (Fig. 6, A and B). After long-term treatment, Thy (100 nM, 18 h), like PMA (100 nM, 18 h), induced almost full depletion of PKCalpha in PAEC (compare Fig. 3, A and B and Fig. 6, C and D).


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Fig. 6.   Western blot analysis of the effects of Thy and PIP on expression of PKC isoforms in PAEC. Before treatments, PAEC were kept in serum-free media for 12-16 h. A and B: cells were treated with 0.1% DMSO (Cont), PMA (100 nM), Thy (100 nM), or PIP (5 µM) for 1 h. After the treatments, C and P fractions were isolated as described in MATERIALS AND METHODS. Western blot analysis of PKC isoforms was performed as described in MATERIALS AND METHODS. A: representative immunoblots (from at least 3 experiments) for distribution of PKCalpha , PKCepsilon , and PKClambda /zeta between C and P fractions after treatment with PMA, Thy, or PIP. B: densitometric analysis of PKCalpha , PKCepsilon , and PKClambda /zeta contents in the C and P fractions. Each bar represents the mean of the relative density units ± SE from 4 experiments. *P < 0.01 vs. the control cytosolic fraction; **P < 0.01 vs. the control particulate fraction. C and D: cells were treated with 0.1% DMSO (Cont) or Thy (100 nM) for 1 or 18 h. After the treatments, C and P fractions were isolated as described in MATERIALS AND METHODS. Western blot analysis using PKCalpha antibody was performed as described in MATERIALS AND METHODS. C: representative blot from 3 experiments with the same results. D: densitometric analysis of PKCalpha contents in C and P fractions. Each bar represents the mean of the relative density units ± SE from 3 experiments. *P < 0.001 vs. the control cytosolic fraction; **P < 0.001 vs. the control particulate fraction; ***P < 0.001 vs. the particulate fraction after the treatment with Thy for 1 h.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The possible involvement of PKC in the regulation of the L-arginine transporters in different cell types has been discussed for the last several years (5, 13, 15, 27, 29, 37). There are two lines of evidence suggesting that PKC may participate in the regulation of CAT-1 activity. First, the CAT-1 protein contains three putative sites for phosphorylation by PKC localized in the fifth and sixth intracellular loops, according to the model by Albritton and coauthors (1). Second, it has been reported by two groups (19, 21) that the CAT-1 protein is localized in caveolae. It has also been reported that activated PKC is located in caveolae (22, 30), allowing for the possibility of CAT-1 and PKC interaction.

PKC is a family of at least 12 related kinases (24). In our investigation, we studied, in parallel with CAT-1-mediated L-arginine transport, expression and translocation of PKCalpha (a classical isoform of PKC), PKCepsilon (a novel isoform of PKC), and PKClambda /zeta (an atypical isoform of PKC) in PAEC. We showed that an activator of classical and novel isoforms of PKC, phorbol ether PMA, inhibited L-arginine transport in PAEC after a 1-h treatment and activated L-arginine uptake after an 18-h treatment of cells (Fig. 1). 4alpha -Phorbol, which is incapable of affecting PKC activity, did not change L-arginine transport after 1-h or 18-h treatments. More detailed studies of the time dependence of PMA actions on L-arginine transport showed that the inhibitory effect of PMA started after 10 min of treatment and changed to activation after 4-h treatment of cells (Fig. 2). The inhibitory effect of PMA on L-arginine transport in PAEC was accompanied by a fast translocation of PKCalpha from the cytosol to the membrane fraction, whereas the activating effect of PMA on L-arginine transport was accompanied by full depletion of the expression of PKCalpha (Fig. 3). At the same time, there were no changes in expression or translocation of PKClambda /zeta . These results suggest that the expression of PKCalpha in the membrane fraction and CAT-1-mediated L-arginine transport in PAEC are interrelated: the more PKCalpha expressed in the membrane fraction, the lower L-arginine transport.

Because PMA can activate the classical and the novel isoforms of PKC and, in our experiments, induced a small translocation of PKCepsilon (Fig. 3, A and B), we evaluated the contribution of PKCepsilon in PMA-induced changes in L-arginine transport in PAEC. For this, we used more specific activators of different PKC isoforms and compared their effects on L-arginine transport and PKC distribution in PAEC with the effects of PMA. We used Thy, an activator of classical Ca2+-dependent isoforms of PKC (25, 31, 36), and PIP, an activator of novel Ca2+-independent isoforms of PKC (8, 34). Thy exerted the same influence on L-arginine uptake as PMA: it inhibited uptake after short-term treatment and activated uptake after long-term treatment (Fig. 4). After short-term treatment, Thy induced translocation of PKCalpha from the cytosolic to the particulate fraction and, unlike PMA, did not affect the translocation of PKCepsilon (Fig. 6, A and B). PIP did not affect L-arginine transport in PAEC after short-term as well as after long-term treatments (Fig. 4) and induced translocation of PKCepsilon but not of PKCalpha (Fig. 6, A and B). Long-term treatment with PMA and Thy induced almost complete depletion of PKCalpha in PAEC. In addition, a specific inhibitor of the classical isoforms of PKC, Go 6976, was able to reverse the effects of PMA and Thy on L-arginine transport in PAEC. Together, these data indicate that only classical isoforms of PKC participate in the regulation of CAT-1 transport activity in PAEC.

Because PMA did not change the expression of the CAT-1 transporter in PAEC (Fig. 1, C and D) and did not change the distribution of the CAT-1 transporter between cytosolic and particulate fractions (Fig. 2, B and C), it is probable that PMA induces changes in L-arginine transport in PAEC through posttranslational modification of the CAT-1 transporter. It is known that under activation PKCalpha targets to plasma membrane caveolae (22), where the CAT-1 protein is localized (21). We suggest that translocation of PKCalpha to plasmalemmal caveolae induces phosphorylation of the CAT-1 transporter, which leads to inhibition of its transport activity in PAEC. In contrast, depletion of PKCalpha after long-term treatment with PMA or Thy promotes dephosphorylation of the CAT-1 transporter and activation of transport activity. It is very likely that PKCalpha directly regulates CAT-1 transport activity rather than regulating through downstream signaling pathways that may involve ERK1/2 or p38 MAP kinase. Our preliminary results show that a specific inhibitor of MAP kinase kinase, PD-98059, and a specific inhibitor of p38 MAP kinase, SB-203580, do not change the effects of PMA on L-arginine uptake in PAEC (data not shown).

According to the literature, PMA exerts different effects on L-arginine transport in different types of cells. For example, in human hepatoma cells (5), in human endothelial EA.hy926 cells (13), and in oocytes expressing the CAT-1 transporter (13), PMA induced inhibition of L-arginine uptake, whereas in macrophages (15, 29), human umbilical vein endothelial cells (27), and Caco-2 intestinal epithelial cells (26), PMA activated L-arginine uptake. The differences in the effects of PMA reported by these groups can be explained by different duration of treatment with PMA or by different permeability or sensitivity of the different cell types to PMA or by different expression of various PKC isoforms in the different cell types. Because different cell types express and use different members of the CAT family for the delivery of L-arginine, it is also possible that the different members of the CAT family are regulated in different ways under activation of PKC.

In conclusion, our results demonstrate that classical isoforms of PKC regulate CAT-1 transport activity in PAEC and that this regulation is independent of the expression of the CAT-1 transporter.


    ACKNOWLEDGEMENTS

We thank Humberto Herrera for assistance with the tissue culture.


    FOOTNOTES

This work was supported in part by American Heart Association Florida/Puerto Rico Affiliate Grant 0235018B (to S. I. Zharikov), by American Lung Association Grant RG-005-N (to S. I. Zharikov), by an American Lung Association of Florida Grant (to S. I. Zharikov), by National Heart, Lung, and Blood Institute Grant HL-52136 (M.E.R.I.T.) (to E. R. Block), and by the Medical Research Service, Department of Veterans Affairs (to E. R. Block).

Address for reprint requests and other correspondence: E. R. Block, Dept. of Medicine, Box 100277, J. Hillis Miller Health Center, 1600 SW Archer Road, Gainesville, FL 32610 (E-mail: blocker{at}medicine.ufl.edu).

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.

First published January 31, 2003;10.1152/ajplung.00308.2002

Received 13 September 2002; accepted in final form 23 January 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 284(6):L1037-L1044