Department of Medicine, University of Florida College of Medicine; and Research Service, Malcom Randall VA Medical Center, Gainesville, Florida 32610
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ABSTRACT |
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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 PKC (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 PKC
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 PKC
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 PKC
(a
novel PKC isoform) from the cytosolic to the particulate fraction and
did not affect the translocation of PKC
. 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 PKC
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 PKC
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
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INTRODUCTION |
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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 (,
1,
2, and
)
require calcium, diacylglycerol, and phosphatidylserine for activation.
Novel PKC isoforms (
,
,
,
) require diacylglycerol and
phosphatidylserine. Atypical PKC isoforms (
,
,
) 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.
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MATERIALS AND METHODS |
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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), 4Measurements 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.
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RESULTS |
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To study the effects of PKC activation on CAT-1-mediated transport
in PAEC we treated cells with PMA or 4-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%. 4
-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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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. PKC,
PKC
, and PKC
/
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 PKC
from the cytosolic to the particulate fraction (the ratio of cytosolic PKC
-to-particulate PKC
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 PKC
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 PKC
in PAEC
increased from 80% in control to 90 and 95% after treatments with PMA
for 10 min and 1 h, respectively) (Fig. 3B). PKC
/
localized mainly in the cytosolic fraction in control PAEC (the ratio
of cytosolic PKC
/
to particulate PKC
/
is equal to ~4:1),
and there were no significant changes in the distribution or expression
of PKC
/
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 PKC
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 PKC
in the particulate fraction, which approached 79% after
18 h of treatment with PMA (Fig. 3, A and
B). Thus the depletion of PKC
was much slower than PKC
under the action of PMA, and PMA-induced activation of
L-arginine transport in PAEC correlated more with the
depletion of PKC
than PKC
.
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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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Western blot analysis revealed that Thy (100 nM, 1 h) induced
translocation of PKC from the cytosolic to the particulate fraction,
changing the cytosolic PKC
-to-particulate PKC
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 PKC
(Fig. 6, A and
B). In contrast to PMA, PIP (5 µM, 1 h) did not
affect the translocation of PKC
(Fig. 6, A and
B). However, PIP (5 µM, 1 h) decreased expression of
PKC
in the cytosolic fraction and decreased the ratio of cytosolic
PKC
to particulate PKC
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
PKC
/
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 PKC
in
PAEC (compare Fig. 3, A and B and Fig. 6,
C and D).
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DISCUSSION |
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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
PKC (a classical isoform of PKC), PKC
(a novel isoform of PKC),
and PKC
/
(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). 4
-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 PKC
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 PKC
(Fig. 3). At the same time, there were no
changes in expression or translocation of PKC
/
. These results suggest that the expression of PKC
in the membrane fraction and CAT-1-mediated L-arginine transport in PAEC are
interrelated: the more PKC
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 PKC (Fig.
3, A and B), we evaluated the contribution of
PKC
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 PKC
from the cytosolic to the particulate fraction
and, unlike PMA, did not affect the translocation of PKC
(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 PKC
but not of PKC
(Fig. 6, A and B). Long-term
treatment with PMA and Thy induced almost complete depletion of PKC
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 PKC targets to plasma membrane caveolae
(22), where the CAT-1 protein is localized
(21). We suggest that translocation of PKC
to plasmalemmal caveolae induces phosphorylation of the CAT-1 transporter, which leads to inhibition of its transport activity in PAEC. In contrast, depletion of PKC
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 PKC
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.
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ACKNOWLEDGEMENTS |
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We thank Humberto Herrera for assistance with the tissue culture.
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FOOTNOTES |
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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.
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