Pertussis toxin activates L-arginine uptake in pulmonary endothelial cells through downregulation of PKC-{alpha} activity

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

Department of Medicine, University of Florida College of Medicine, Gainesville 32610; and Research Service, Malcom Randall Veterans Affairs Medical Center, Gainesville, Florida 32608

Submitted 17 July 2003 ; accepted in final form 12 December 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pertussis toxin (PTX) induces activation of L-arginine transport in pulmonary artery endothelial cells (PAEC). The effects of PTX on L-arginine transport appeared after 6 h of treatment and reached maximal values after treatment for 12 h. PTX-induced changes in L-arginine transport were not accompanied by changes in expression of cationic amino acid transporter (CAT)-1 protein, the main L-arginine transporter in PAEC. Unlike holotoxin, the {beta}-oligomer-binding subunit of PTX did not affect L-arginine transport in PAEC, suggesting that G{alpha}i ribosylation is an important step in the activation of L-arginine transport by PTX. An activator of adenylate cyclase, forskolin, and an activator of protein kinase A (PKA), Sp-cAMPS, did not affect L-arginine transport in PAEC. In addition, inhibitors of PKA or adenylate cyclase did not change the activating effect of PTX on L-arginine uptake. Long-term treatment with PTX (18 h) induced a 40% decrease in protein kinase C (PKC)-{alpha} but did not affect the activities of PKC-{epsilon} and PKC-{zeta} in PAEC. An activator of PKC-{alpha}, phorbol 12-myristate 13-acetate, abrogated the activation of L-arginine transport in PAEC treated with PTX. Incubation of PTX-treated PAEC with phorbol 12-myristate 13-acetate in combination with an inhibitor of PKC-{alpha} (Go 6976) restored the activating effects of PTX on L-arginine uptake, suggesting PTX-induced activation of L-arginine transport is mediated through downregulation of PKC-{alpha}. Measurements of nitric oxide (NO) production by PAEC revealed that long-term treatment with PTX induced twofold increases in the amount of NO in PAEC. PTX also increased L-[3H]citrulline production from extracellular L-[3H]arginine without affecting endothelial NO synthase activity. These results demonstrate that PTX increased NO production through activation of L-arginine transport in PAEC.

cationic amino acid transporter; regulation; G proteins; caveolae; protein kinase C-{alpha}


VASCULAR ENDOTHELIAL CELLS are a rich source of nitric oxide (NO), which has been shown to play an important role in diverse pathophysiological conditions (23). Endothelial cells generate NO from L-arginine via the catalytic action of endothelial nitric oxide synthase (eNOS; see Ref. 2). Although endothelial cells can synthesize some arginine, recent reports indicate that NO production by endothelial cells is dependent on the availability of extracellular L-arginine (12, 14, 28). Among several transport systems that provide delivery of extracellular L-arginine into mammalian cells (y+, bo,+, Bo,+, and y+L), system y+ is the main transport agency in endothelial cells (5, 11, 38). Encoded by cationic amino acid transporters (CAT-1, CAT-2A, CAT-2B, and CAT-3), system y+ is characterized by sodium independence and stimulation of transport by substrate on the opposite side of the membrane (trans-stimulation; see Ref. 7). In pulmonary artery endothelial cells (PAEC), system y+ is represented by the CAT-1 transporter, which is responsible for 70–95% of L-arginine uptake (38). Little is known about factors that regulate the CAT-1 transporter. It has been reported that protein kinase C (PKC) is involved in the regulation of the human (h) CAT-1 transporter (10). More recently, we reported that in PAEC CAT-1 transport activity is regulated by classical isoforms of PKC (18).

Recently, we found that long-term (several hours) treatment of PAEC with pertusssis toxin induced a significant activation of CAT-1-mediated L-arginine transport. Pertussis toxin (PTX) has been widely used as a tool for examination of G protein-regulated cellular signaling pathways. The PTX holotoxin consists of a {beta}-oligomer binding subunit and an S1 ADP-ribosyltransferase fragment that catalyzes ADP-ribosylation at a cysteine residue on the {alpha}-subunit of Gi of heterotrimeric G proteins, leading to inactivation of the inhibitory G{alpha} (16). It has been shown that, in addition to an effect on G protein-mediated pathways, PTX can have direct, i.e., in the absence of exogenous ligand/receptor stimulation, effects on cellular function. These effects include Ca2+ mobilization (31), cAMP synthesis, diacylglycerol generation (35), tyrosine phosphorylation (36), phosphatase activity (6), and cytoskeletal reorganization (9). In addition, it was demonstrated that PTX changes the activity of several kinases in endothelial cells as follows: p42/p44 MAPK (8), PKC (27), and p38 MAPK (9). It is interesting to note that, in endothelial cells, these kinases target to caveolae (see references in Ref. 33), specific cholesterol- and sphingolipid-rich microdomains of the endothelial plasma membrane, where the CAT-1 transporter is also localized (20). Colocalization of the CAT-1 transporter and caveolin-interacting signaling molecules creates a possibility that PTX activates L-arginine transport in endothelial cells through one of the signaling cascades.

In this work, we studied the possible mechanisms by which long-term treatment of endothelial cells with PTX activates CAT-1-mediated L-arginine transport and how PTX-induced activation of L-arginine transport affects NO production by endothelial cells. We showed that long-term treatment of endothelial cells with PTX activated L-arginine transport through downregulation of PKC-{alpha} activity. The PTX-induced changes in L-arginine transport led to increased L-citrulline and NO production by endothelial cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents. Unless specified, reagents were obtained from Sigma Chemical (St. Louis, MO). PBS, Hanks' balanced salt solution (HBSS) without phenol red, DMEM media, RPMI 1640, FBS, antibiotics (penicillin-streptomycin, gentamicin), and an antimycotic (amphotericin B) were purchased from GIBCO. Forskolin, adenosine 3',5'-cyclic monophosphorothioate-Sp-isomer (Sp-cAMPS), adenosine 3',5'-cyclic monophosphorothioate-Rp-isomer (Rp-cAMPS), SQ-22536, Y-27632, PD-98059, SB-203580, phorbol 12-myristate, 13-acetate (PMA), wortmannin, Go 6976, nitro-L-arginine methyl ester (L-NAME), protease inhibitor cocktail (Set III), and phosphatase inhibitor cocktail (Set II) were obtained from Calbiochem. Antibodies to different PKC isoforms were purchased from BD Biosciences. L-[3H]arginine was obtained from Amersham. Pierce phosphocellulose units were obtained from Pierce. PTX and the {beta}-oligomer of PTX were purchased from List Biological Laboratories. Rabbit polyclonal CAT-1 antibodies were prepared, purified, and characterized as previously reported by our laboratory (37).

Cell culture. PAEC were obtained from the main pulmonary artery of 6- to 7-mo-old pigs and were cultured in RPMI 1640 medium supplemented with 10% (vol/vol) FBS, antibiotics (10 U/ml penicillin, 100 µg/ml streptomycin, and 20 µg/ml gentamicin), and an antimycotic Fungizone (2 µg/ml). Cells were maintained on 100-mm culture dishes at 37°C in a humidified atmosphere of 5% CO2-95% air and grew until confluence. Cells from primary dishes were detached with 0.05% trypsin, resuspended in fresh culture media, and passaged in 100-mm dishes or 24-well plates. Cells were used at passages 3–6.

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 measurements of protein content (19).

Western blot analysis of contents of the CAT-1 transporter. For analysis of CAT-1 contents in PAEC, cells grown on 100-mm dishes were washed two times with ice-cold PBS without Ca2+ and Mg2+ and were scraped in lysis buffer [10 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40 (NP-40), 0.4% deoxycholate, and 60 mM octylglucoside] containing protease inhibitor cocktail Set III (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 protein) were separated by SDS-PAGE (7.5% acrylamide) and transferred to nitrocellulose membranes. 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 density of the bands was quantitated using a Fluor-S MultiImager system (Bio-Rad).

PKC studies. Control PAEC and PAEC treated with PTX grown in 100-mm dishes were washed in PBS and scraped in lysis buffer [20 mM Tris·HCl, 150 mM NaCl, 0.1% Triton X-100, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM EGTA containing protease inhibitor cocktail Set III (Calbiochem), and phosphatase inhibitor cocktail Set II (Calbiochem)]. The lysates were centrifuged at 20,000 g for 10 min at 4°C. The supernatants (for each sample the same amount of protein, ~700–1,000 µg, was used) were incubated with 1 µg each of anti-PKC isoform ({alpha}, {epsilon}, {zeta}/{lambda}) antibodies or rabbit IgG (nonspecific activity) at 4°C overnight. Next protein G-agarose was added, and incubation was continued for 2 h. The agarose beads were washed two times with the lysis buffer containing 0.5 M NaCl and a third time with the appropriate kinase buffer. These washed beads were used as the immunoprecipitates from each PKC isoform antibody. Immunoprecipitates of each PKC isoform or rabbit IgG on beads were incubated with reaction mixtures containing the appropriate kinase buffer, 2 mM ATP, [32P]ATP, peptide substrate, and activator's solution (phosphatidylserine plus diacylglycerol/diolein). Only PKC-{alpha} kinase buffer contained CaCl2 because only classical isoforms of PKC need calcium for activation. Activator solution for PKC-{zeta}/{lambda} contained only phosphatidylserine because atypical isoforms of PKC need only phosphatidylserine for activation. The reactions were stopped with 75 mM phosphoric acid, and the reaction mixtures were spotted on phosphocellulose units and counted. Nonspecific activity was subtracted from the total counts per minute.

Detection of NO produced by PAEC using a fluorescent indicator for NO DAF-FM diacetate. Control PAEC and PAEC treated with PTX were first washed in HBSS and then incubated with 5 µM 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) diacetate (Molecular Probes) for 15 min at room temperature in darkness. After the incubation, PAEC were washed to remove excess probe. Fresh HBSS was added to cells, and cells were incubated for an additional 10 min to allow complete deesterification of the intracellular diacetate. After this procedure, direct visualization of NO production with the fluorescent indicator was performed using a laser-scanning confocal microscope with excitation and emission maxima at 495 and 515 nm, respectively. Intensity of fluorescence was quantitated using LSM 510 (version 3.0 SP3) software for the Carl Zeiss Laser Scanning Microscope. Simultaneous visualization of cell morphology by differential interference contrast microscopy allowed us to select areas with confluent cells.

Detection of the amount of L-[3H]citrulline produced from extracellular L-[3H]arginine. Control PAEC and PAEC treated with PTX were washed with LiCl-Dulbecco solution (see the composition above) and then incubated in LiCl-Dulbecco solution containing 50 µM L-[3H]arginine for 15 min. After washing cells with ice-cold LiCl-Dulbecco solution, we measured the total amount of labeled amino acids (L-[3H]arginine plus L-[3H]citrulline formed from L-[3H]arginine) within PAEC. This value is an index of the total radioactivity taken up by PAEC for the period of incubation or, in other words, is an index of the activity of L-arginine transport. After separation of intracellular L-[3H]arginine and L-[3H]citrulline by ion-exchange chromatography, we detected the amount of L-[3H]citrulline inside PAEC and calculated the percentage of L-[3H]arginine converted to L-[3H]citrulline during the 15-min incubation, a measure of eNOS activity, in the same PAEC.

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 carried out during this investigation. Data are expressed as means ± SE. Comparisons between values were made using an unpaired, two-tailed Student's t-test and, for some experiments, one-way ANOVA, followed by the Tukey's test for multiple comparisons. A value of P < 0.05 was considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Long-term treatment of PAEC with PTX causes activation of L-arginine uptake. Long-term (24-h) treatment of PAEC with PTX induced activation of L-arginine transport into cells. The activation was dependent on the concentration of PTX in the culture media and was evident at concentrations as low as 100 ng/ml (Fig. 1A). The maximal stimulation (~2-fold) of L-arginine uptake was observed at a PTX concentration of 500 ng/ml. The effect of PTX on L-arginine transport was also dependent on the time of the treatment and appeared only after >3 h of treatment (Fig. 1B). At a concentration of 500 ng/ml, the maximal activation of L-arginine uptake by PAEC was observed after treatment for 12 h (Fig. 1B). PTX-induced changes in L-arginine transport were not accompanied by changes in the expression of CAT-1 protein (Fig. 1C). Cycloheximide did not change the effects of PTX on L-arginine uptake (Fig. 1D), suggesting that the synthesis of new proteins is not involved in the activating effects of PTX.



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Fig. 1. Effects of pertussis toxin (PTX) on L-arginine uptake and expression of the cationic amino acid transporter (CAT)-1 in pulmonary artery endothelial cell (PAEC) monolayers. A: confluent PAEC in 24-well plates were treated with concentrations of PTX in culture media ranging from 0 (control) to 1 µg/ml for 24 h. After treatment, the medium was discarded, and cells in each well were washed one time with 0.5 ml of LiCl-Dulbecco solution. Immediately after washing, transport of L-[3H]arginine was assayed as described in MATERIALS AND METHODS. Plot represents means ± SE of 4 experiments with 4 replicates/experiment. Beginning with a concentration of 100 ng/ml, PTX induced a significant increase in L-arginine uptake by PAEC with P < 0.01. B: cells were treated with 500 ng/ml PTX for different periods of time. Results are means of 3 experiments with 4 replicates/experiment. C: representative (of 3 experiments) Western blot image of CAT-1 expression in cultured PAEC. Cells grown in 100-mm dishes were treated with 500 ng/ml PTX for different periods of time. After treatments, cells were lysed with SDS lysis buffer, scraped off dishes, heated for 5 min, and microcentrifuged. Proteins in the supernatant were separated by 7.5% SDS-PAGE and immunoblotted with CAT-1 antibodies. Scanning densitometry analysis of bands from all 3 experiments did not reveal significant differences in CAT-1 protein levels in different groups. D: cultured cells were treated with 1 µM cycloheximide (Cycl) 1 h before the treatment with PTX (500 ng/ml; 18 h). Bars are means of 3 experiments with 4 replicates/experiment. *P < 0.05 vs. control.

 

PTX-induced activation of L-arginine uptake involves G{alpha} ADP ribosylation. PTX catalyzes ADP-ribosylation at the cysteine residue of the inhibitory G{alpha}, making this subunit of G protein incapable of exchanging GDP for GTP. To confirm the involvement of G{alpha}i ADP ribosylation in PTX-induced activation of L-arginine uptake by PAEC, we studied the effects of the purified {beta}-oligomer binding subunit of PTX, which is responsible for cellular binding and toxin entry into cells but which is devoid of ADP ribosyltransferase activity. Unlike the holotoxin, the {beta}-oligomer-binding subunit did not affect L-arginine transport in PAEC (Fig. 2A), suggesting that G{alpha}i ribosylation is an important step in the activation of L-arginine transport by PTX. Ribosylation of the inhibitory G{alpha} under the action of PTX causes the block of an inhibitory influence of G{alpha}i, thus inducing permanent activation of adenylyl cyclase. Another toxin that is also able to induce permanent activation of adenylyl cyclase by activating the G{alpha} subunit of the stimulatory G protein is cholera toxin (ChTx). Unlike PTX, which stimulates cAMP production because of its ability to uncouple G protein-mediated inhibition of adenylyl cyclase, ChTx induces the same effect by a direct stimulation of G{alpha}s. To compare the effects of PTX and ChTx on L-arginine transport in PAEC, we treated PAEC with different concentrations of ChTx for 12 or 24 h and studied L-arginine transport and the expression of the CAT-1 transporter. Like PTX, ChTx activated L-[3H]arginine uptake by PAEC, and this effect was dependent on ChTx concentration and time of treatment (Fig. 2B). The treatment with 5 µg/ml ChTx for 24 h induced about a twofold increase in L-arginine transport in PAEC. These changes, as in the case with PTX, were not related to changes in the expression of the CAT-1 transporter in PAEC (Fig. 2C).



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Fig. 2. Effects of cholera toxin (ChTx) and purified {beta}-oligomer of PTX on L-arginine (L-Arg) uptake in PAEC. A: confluent PAEC in 24-well plates were treated with the PTX {beta}-oligomer binding subunit ({beta}-OM; 500 ng/ml) or the PTX holotoxin (500 ng/ml) for 18 h. After treatments, L-[3H]arginine transport in PAEC was assayed as described in MATERIALS AND METHODS. Bars represent means ± SE of 2 experiments with 8 replicates/experiment. *P < 0.01 vs. control or {beta}-OM. B: confluent PAEC in 24-well plates were treated with various concentrations of ChTx (0.5–10 µg/ml) for 12 or 24 h. After treatments, the medium with toxin was discarded, cells in each well were washed one time with 0.5 ml of LiCl-Dulbecco solution, and immediately after washing L-[3H]arginine transport in PAEC was assayed as described in MATERIALS AND METHODS. Plots represent means ± SE of 3 experiments with 4 replicates/experiment. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control. C: representative (of 3 experiments) Western blot image of CAT-1 expression in cultured PAEC. Cells grown in 100-mm dishes were treated with 5 µg/ml ChTx for different periods of time. After treatments, cells were lysed with SDS lysis buffer, scraped off dishes, heated for 5 min, and microcentrifuged. Proteins in the supernatant were separated by 7.5% SDS-PAGE and immunoblotted with CAT-1 antibodies. Scanning densitometry analysis of bands from all 3 experiments did not reveal significant differences in CAT-1 protein level in different groups.

 

Effects of activators and inhibitors of adenylate cyclase or PKA on L-arginine transport and PTX-induced changes in L-arginine uptake by PAEC. Based on the observation that PTX and ChTx had similar effects on L-arginine transport in PAEC, it was reasonable to suggest that these two toxins affected CAT-1 transport activity through activation of adenylyl cyclase followed by activation of cAMP-dependent protein kinase A (PKA). To check this hypothesis, we measured L-arginine transport into PAEC treated with an activator of adenylate cyclase (forskolin) or an activator of PKA (Sp-cAMPS). Figure 3A shows that neither forskolin (5–20 µM) nor a cell-permeable cAMP analog that directly activates PKA, Sp-cAMPS (5–40 µM), affected L-arginine transport in PAEC. In addition, neither a specific inhibitor of PKA, Rp-cAMPS (10 µM; Fig. 3B), nor a cell-permeable inhibitor of adenylate cyclase, SQ-22536 (10 µM; Fig. 3C), changed the activating effects of PTX on L-arginine uptake. These results suggest that activation of CAT-1 transport activity under the action of PTX for 24 h is not related to activation of adenylate cyclase or PKA.



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Fig. 3. Effects of activators and inhibitors of adenylate cyclase or cAMP-dependent protein kinase (PKA) on L-arginine transport and PTX-induced changes in L-arginine uptake by PAEC. A: PAEC grown on 24-well plates were treated with an activator of adenylate cyclase, forskolin (5 or 20 µM), or an activator of PKA, Sp-cAMPS (5 or 40 µM), for 24 h. After treatments, L-[3H]arginine uptake by PAEC was measured as described in MATERIALS AND METHODS. Bars represent means ± SE of 3 experiments with 4 replicates/experiment. No significant changes in L-arginine uptake were observed compared with control cells. B: PAEC grown on 24-well plates were treated with PTX (500 ng/ml) for 24 h in the absence or presence of a specific inhibitor of PKA, Rp-cAMPS (10 µM). After treatment, L-[3H]arginine transport in PAEC was assayed. Bars represent means ± SE of 3 experiments with 4 replicates/experiment. *P < 0.01 vs. control cells. C: PAEC grown on 24-well plates were treated with PTX (500 ng/ml) for 24 h in the absence or presence of a cell-permeable inhibitor of adenylate cyclase, SQ-22536 (10 µM). After treatment, activity of L-[3H]arginine transport was assayed as described in MATERIALS AND METHODS. Bars represent means ± SE of 2 experiments made with 4 replicates/experiment. *P < 0.01 vs. control.

 

Inhibitors of the Rho kinase, MAPK, mitogen/extracellular signal-regulated kinase, and phosphatidylinositol 3-kinase pathways do not modify the PTX-induced increase in L-arginine uptake by PAEC. PTX is able to activate different transduction cascades in endothelial cells (8, 9, 27). To test which signaling pathways may be involved in the activation of L-arginine transport in PAEC by PTX, we studied the effects of different kinase inhibitors on PTX-activated L-arginine transport. A cell-permeable and selective inhibitor of Rho-associated protein kinase, Y-27632 (10 µM), did not affect L-arginine uptake by PAEC (neither after 1 h of treatment nor 24 h of treatment) and did not change the activation of L-arginine uptake induced by PTX (Fig. 4, A and B). A selective inhibitor of mitogen/extracellular signal-regulated kinase, PD-98059 (30 µM; Fig. 4C), a highly specific inhibitor of p38 MAPK, SB-203580 (1 µM; Fig. 4D), and an inhibitor of phosphatidylinositol 3-kinase, wortmannin (100 nM; Fig. 4E), did not affect L-arginine transport in PAEC and did not modify the PTX-induced activation of L-arginine transport in these cells, suggesting that these transduction cascades are not involved in the effects of long-term treatment with PTX on L-arginine transport.



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Fig. 4. Effects of inhibitors of some signaling pathways on PTX-induced activation of L-arginine uptake by PAEC. A: confluent PAEC were treated with PTX (500 ng/ml), with a highly potent, cell-permeable and selective inhibitor of Rho-associated protein kinases, Y-27632 (10 µM), or with PTX and Y-27632 together for 18 h. After the treatments, L-[3H]arginine uptake by PAEC was assayed as described in MATERIALS AND METHODS. Bars represent means ± SE of 3 experiments with 4 replicates/experiment. *P < 0.05 vs. control, nontreated PAEC. Long-term treatment with Y-27632 did not affect L-arginine transport in PAEC and did not change PTX-induced activation of L-arginine transport. B: confluent PAEC were incubated in serum-free RPMI 1640 media (Cont) or in media containing PTX (500 ng/ml) for 18 h. After the treatment, the media were discarded, and cells were treated for an additional 1 h with Y-27632 (10 µM). At the end of the second incubation, L-[3H]arginine was assayed as described in MATERIALS AND METHODS. Bars represent means ± SE of 3 experiments made with 4 replicates/experiment. *P < 0.05 vs. control, nontreated PAEC. C: confluent PAEC were treated with PTX (500 ng/ml), with a selective inhibitor of mitogen/extracellular signal-regulated kinase (MEK), PD-98059 (30 µM), or with PTX and PD-98059 together for 18 h. After the treatments, L-[3H]arginine uptake by PAEC was assayed as described in MATERIALS AND METHODS. Bars represent means ± SE of 2 experiments with 4 replicates/experiment. *P < 0.05 vs. control, nontreated PAEC. D: confluent PAEC were treated with PTX (500 ng/ml), with a highly specific inhibitor of p38 MAPK, SB-203580 (1 µM), or with PTX and SB-203580 together for 18 h. After the treatments, L-[3H]arginine uptake by PAEC was assayed as described in MATERIALS AND METHODS. Bars represent means ± SE of 3 experiments with 4 replicates/experiment. *P < 0.01 vs. control, nontreated PAEC. E: confluent cells grown on 24-well plates were incubated in serum-free RPMI 1640 without (1) or with (2) PTX (500 ng/ml) at 37°C for 18 h. After these treatments, the media were discarded and changed for fresh serum-free RPMI 1640 (4) or RPMI 1640 containing a selective inhibitor of phosphatidylinositol 3-kinase, wortmannin (100 nM) (3). PAEC were then incubated for an additional 1 h at 37°C. L-[3H]arginine uptake by PAEC was assayed as described in MATERIALS AND METHODS. Bars represent means ± SE of 2 experiments made with 4 replicates/experiment. *P < 0.01 vs. control, nontreated PAEC.

 

Activating effects of PTX on L-arginine transport in PAEC are mediated through classical isoforms of PKC. Patterson and co-authors (27) showed that PTX activates PKC in endothelial cells with the maximal effect after 30 min treatment and a lower effect after 2 h treatment. The activating effect of PTX (30 min treatment) was similar to the effect of the phorbol ether PMA (100 nM, 15 min treatment). We could find no data in the literature about the effects of long-term treatment with PTX, which we used in our experiments, on the activity of PKC. It is well established, however, that long-term stimulation of PKC with, for example, PMA leads to almost complete depletion of PKC in endothelial cells (18). Therefore, we tested the effects of long-term treatment with PTX on the expression and activity of different isoforms of PKC in PAEC. We observed that PTX (500 ng/ml, 18 h treatment) did not change the expression of PKC isoforms defined by Western blot analysis (Fig. 5B). At the same time, PTX induced about a 40% decrease in PKC-{alpha} activity and did not affect the activities PKC-{epsilon} and PKC-{zeta}/{lambda} (Fig. 5A). These results suggested that the changes in PKC-{alpha} activity may be involved in the changes of L-arginine transport induced by long-term treatment with PTX. Therefore, we assayed L-arginine uptake in PTX-treated PAEC after additional treatment with PMA (100 nM, 60 min), an activator of classical and novel isoforms of PKC, with a specific inhibitor of PKC-{alpha}, Go 6976 (1 µM), or with PMA and Go 6976 together. We observed that PMA eliminated the activating effects of PTX on L-arginine transport in PAEC (Fig. 6), whereas an inhibitor of PKC-{alpha} attenuated the blocking effects of PMA on PTX-induced activation of L-arginine uptake. Go 6976 alone did not affect PTX-induced changes in L-arginine transport in PAEC. These data indicate that PTX-induced activation of L-arginine transport in PAEC is mediated, at least partly, through downregulation of PKC-{alpha}.



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Fig. 5. Effect of PTX on the activity and expression of PKC isoforms in PAEC. A: confluent cells were treated with 500 ng/ml PTX for 18 h. After treatment, activities of protein kinase C (PKC)-{alpha} (as a representative of classical isoforms of PKC), PKC-{epsilon} (as a representative of novel isoforms of PKC), and PKC-{zeta}/{lambda} (as a representative of atypical isoforms of PKC) were determined as described in MATERIALS AND METHODS. Bars represent the mean activity determined in 3 experiments made in triplicates. *P < 0.01 vs. respective control cells. B: control cells and cells treated with PTX (500 ng/ml; 18 h) in 100-mm culture dishes were washed two times with ice-cold PBS without Ca2+ and Mg2+ and were 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 (Set III; Calbiochem). Samples (15 µg protein) were separated by SDS-PAGE and transferred to nitrocellulose membranes. 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). Representative immunoblots (from 3 experiments) are presented. The density analysis of the bands did not reveal significant differences in the expression of PKC isoforms in control (1) and PTX-treated (2) PAEC.

 


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Fig. 6. Effect of an activator and an inhibitor of PKC-{alpha} on L-arginine transport in PAEC treated with PTX. Confluent PAEC were treated with PTX (500 ng/ml) for 18 h. After this treatment, the media were discarded, and PAEC were treated additionally with dimethyl sulfoxide (DMSO, 0.01%) or PMA (100 nM) in 0.01% DMSO, an inhibitor of PKC-{alpha}, Go 6976 (1 µM), in 0.01% DMSO, or PMA plus Go 6976 in 0.01% DMSO for 1 h. Next, L-[3H]arginine uptake by PAEC was assayed. Data are expressed as a percentage of control (Cont) values derived from untreated cells [572 ± 55 (SE) pmol·mg protein-1·min-1; n = 3 experiments with 4 observations/experiment]. P < 0.05 vs. control, nontreated PAEC (*), PAEC treated with PTX for 18 h (**), or PMA (***).

 

PTX activates NO production by PAEC. Measurements of NO production by PAEC using a specific probe for NO, DAF-FM (15), revealed that long-term treatment with PTX (500 ng/ml) increases the amount of NO in PAEC (Fig. 7). Detection of fluorescence of DAF-FM showed that PTX induced about a twofold increase in NO production [Fig. 7, compare A2 (control) with A4 (the treatment with PTX for 18 h)]. PMA (100 nM, 1 h after the treatment with PTX; Fig. 7A6) attenuated the effects of PTX on NO production. To test whether PTX-induced changes in NO production are related to activation of L-arginine transport in PAEC, we measured formation of L-citrulline, a product of L-arginine conversion to NO under the action of eNOS, the rate of L-arginine delivery, and eNOS activity in the same PAEC. Figure 8A shows that the treatment with PTX activates the formation of L-[3H]citrulline during the incubation of cells with L-[3H]arginine. The formation of L-[3H]citrulline was eNOS-mediated because the eNOS inhibitor, L-NAME (100 µM), blocked the production of L-[3H]citrulline from extracellular L-[3H]arginine in control and PTX-treated PAEC (Fig. 8A). Increased production of L-[3H]citrulline under the action of PTX, theoretically, might result from an activation of eNOS or might be the result of activated delivery of extracellular L-[3H]arginine in PAEC. Figure 8B shows that PTX increases accumulation of L-[3H]arginine in PAEC. The accumulation of L-[3H]arginine in control PAEC was insensitive to the action of L-NAME (100 µM), and L-NAME did not change the activation of L-[3H]arginine accumulation induced by PTX (Fig. 8B). The measurements of the percentage of intracellular L-[3H]arginine converted to L-[3H]citrulline showed that PTX did not change eNOS activity in PAEC (Fig. 8C), although L-NAME (100 µM) significantly decreased eNOS activity in control PAEC and in PTX-treated PAEC (Fig. 8C). These results demonstrate that PTX-induced activation of NO production detected by a fluorescent probe is caused by the activation of L-arginine transport in PAEC.



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Fig. 7. Effects of PTX on nitric oxide (NO) production by PAEC. A: A1, A3, and A5 represent differential interference contrast photomicrographs of PAEC. A2, A4, and A6 represent fluorescence of DAF-FM in PAEC. PAEC were treated with PTX (500 ng/ml) for 18 h followed by treatment with DMSO (0.01%; A3 and A4) or with PMA (100 nM in DMSO; A5 and A6) for 1 h. A1 and A2 represent images of control, nontreated PAEC. DAF-FM fluorescence images were obtained 10 min after loading PAEC with 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) diacetate as described in MATERIALS AND METHODS. Images of nontreated and treated PAEC were obtained by laser scanning confocal microscopy using the same hardware and time course settings. Representative images from 4 experiments are shown. B: quatitative analysis of intensities of DAF-FM fluorescence in control nontreated PAEC and PAEC treated with PTX (500 ng/ml) for 18 h followed by 1 h treatment with DMSO (0.01%) or PMA (100 nM in DMSO). Bars represent mean intensities ± SE detected from several images (at least 4) in 4 independent experiments. *P < 0.01 vs. control, nontreated cells.

 


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Fig. 8. Effect of PTX on L-[3H]citrulline production from extracellular L-[3H]arginine. A: PAEC grown in 24-well plates were treated with PTX (500 ng/ml), with nitro-L-arginine methyl ester (L-NAME, 100 µM), or with PTX and L-NAME together for 18 h. After these treatments, PAEC were washed and incubated in LiCl-Dulbecco solution containing 50 µM L-[3H]arginine for 15 min at 37°C. The reaction was stopped by washing the wells 4 times with ice-cold LiCl-Dulbecco solution. To each well, 0.1 ml of 96% alcohol was added to extract radioactive amino acids from the cells. After drying, 1 ml of the 10 mM HEPES-EDTA-EGTA buffer (pH 5.5) was added to each well. For the separation of labeled L-citrulline from labeled L-arginine, columns filled with the resin AG-50W-X8 Na form (Bio-Rad) were used; 0.75 ml of a 1-ml sample were loaded to the columns, and the amount of L-[3H]citrulline in the column effluents was counted. The remaining 0.25 ml of the 1-ml samples were taken for counting the total radioactivity accumulated by PAEC during the 15-min incubation (L-[3H]arginine plus L-[3H]citrulline) and for protein measurements. Bars represent means ± SE of 4 experiments made in 4 replicates. *P < 0.01 vs. control, nontreated PAEC. **P < 0.05 vs. control. B: PAEC were treated in the same way as in A. Bars represent the mean values of L-[3H]arginine accumulated in PAEC for the period of incubation with labeled L-arginine. *P < 0.01 vs. control, nontreated PAEC. C: bars represent ratios of L-[3H]arginine converted to L-[3H]citrulline during the 15-min incubation, which can be considered as a measure of endothelial nitric oxide synthase (eNOS) activities at different treatments. **P < 0.05 vs. control, nontreated PAEC.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Despite a number of articles describing changes in L-arginine transport in different types of mammalian cells under various physiological and pathological states, there are few studies that have examined signaling pathways that regulate L-arginine transport. Several groups have discussed the involvement of PKC in the regulation of L-arginine transporters (4, 10, 13, 18, 26, 29). The CAT-1 transporter contains three putative sites for phosphorylation by PKC localized in the fifth and sixth intracellular loops according to the model by Albritton and co-authors (1). Recently, it was shown that, in human endothelial EA.hy926 cells and in oocytes expressing the hCAT-1 transporter, the activation of PKC downregulates the transport activity of hCAT-1 (10). More recently, using porcine PAEC, we have reported that it is the activation of the classical isoforms of PKC that downregulate the CAT-1 transporter, whereas phorbol ether-induced depletion of the classical isoforms of PKC in porcine PAEC upregulated CAT-1 transport activity (18).

In an attempt to find other signaling pathways regulating the activity of the CAT-1 transporter, we used PTX. The advantage of using PTX is its ability to stimulate different transduction cascades involving different protein kinases. The PTX holotoxin consists of an S1 ADP-ribosyltransferase fragment, the targets of which include the {alpha}-subunit of Gi and Go of heterotrimeric G proteins, and a {beta}-oligomer containing five subunits (S2-S6), that is responsible for the binding of PTX to cell receptors and for the delivery of the S1 fragment in the cell. Most of the effects of PTX on cellular function are determined by its ability to ADP-ribosylate the G{alpha}i/o subunits (16). Such a ribosylation leads to inhibition of the G{alpha}i/o-dependent pathways and activation of the G{beta}{gamma}-dependent pathways. It has been shown in numerous cell systems, however, that binding of the {beta}-oligomer to the cell surface is sufficient to activate some cellular signaling cascades (8, 17, 24, 31, 34). In our experiments, the treatment of PAEC with the purified {beta}-oligomer of PTX (Fig. 2A) did not affect CAT-1 transport activity, suggesting that the activating effect of PTX holotoxin on L-arginine transport is mediated through ADP-ribosylation of G proteins.

ADP-ribosylation of G{alpha}i triggers numerous signaling pathways related to inhibition of G{alpha}i and activation of the G{beta}{gamma}-dimer. Inhibition of G{alpha}i leads to activation of adenylyl cyclase, increased production of cAMP, and, in turn, activation of PKA. A possible involvement of this pathway in PTX-induced activation of L-arginine uptake by PAEC has been seemingly supported by our data that ChTx, which activates PKA through the activation of G{alpha}s, stimulated L-arginine uptake by PAEC (Fig. 2B). However, neither an activator of adenylyl cyclase, forskolin, nor an activator of PKA, Sp-cAMPS, affected L-arginine transport in PAEC (Fig. 3). In addition, a specific inhibitor of PKA, Rp-cAMPS, did not attenuate PTX-induced activation of L-arginine uptake (Fig. 3). These data indicate that activation of L-arginine transport after long-term treatment with PTX or ChTx is not related to increased cAMP production or PKA activation.

Other signaling cascades triggered under the inhibition of G{alpha}i by PTX are 1) the Srk-Rho kinase pathway (25) and 2) the p38 MAPK cascade (9). An inhibitor of Rho kinase, Y-27632, and an inhibitor of p38 MAPK, SB-203580, did not affect L-arginine transport in control PAEC and did not affect the changes in L-arginine transport in PTX-treated cells (Fig. 4, A, B, and D), suggesting that the Src-Rho kinase and p38 MAPK pathways are not involved in the PTX-induced activation of L-arginine transport in PAEC. We realize that this conclusion should be made with caution because the effects of long-term treatment with toxins on signaling cascades in cells are much more poorly studied than the effects of short-term treatments (up to 2 h). It is known that persistent activation of G{alpha}s via ChTx leads to its complete downregulation (21, 32). Our experiments show that persistent treatment of PAEC with PTX (18 and 24 h) leads to a significant downregulation of G{alpha}i (data not shown), suggesting that transduction cascades that normally are under the inhibitory influence of G{alpha}i should be active after the long-term treatment with PTX.

The testing of possible involvement of signaling cascades related to G{beta}{gamma} in PTX-induced changes in L-arginine transport showed that only an activator of PKC, PMA, was able to block the effects of PTX on L-arginine uptake (Figs. 4 and 6). PMA activates classical and novel isoforms of PKC; therefore, it is difficult to conclude from the experiments with PMA which isoforms of PKC are responsible for ameliorating the effects of PTX on L-arginine transport. In our porcine PAEC, classical isoforms of PKC are represented by PKC-{alpha}. PKC-{beta} and PKC-{gamma} were not detectable on Western blots using antibodies from different commercially available sources directed against PKC-{beta} and PKC-{gamma} (data not shown). Incubation of PTX-treated cells simultaneously with PMA and a specific inhibitor of PKC-{alpha}, Go 6976, attenuated the blocking effects of PMA on PTX-induced changes in L-arginine transport (Fig. 6), suggesting that PTX affects L-arginine transport in PAEC, at least partly, through downregulation of classical isoform PKC-{alpha}. In addition, analysis of the activity of different isoforms of PKC in PAEC treated with PTX demonstrated that long-term treatment of PAEC with this toxin led to a decrease in the activity of PKC-{alpha} without significant change in the activities of PKC-{epsilon} (novel isoform of PKC) and PKC-{zeta} (atypical isoforms of PKC; Fig. 5). In bovine PAEC, it was shown (27) that short-term treatment with PTX (30 min) induced a stronger activation of PKC than a 2-h treatment. It is reasonable to suggest that long-term stimulation of PKC with PTX, as with long-term treatment with the phorbol ether PMA, will deplete some isoforms of PKC. Such a mechanism could explain the decrease in PKC-{alpha} activity under the persistent activation by PTX (18–24 h). However, our Western blot analysis showed that PTX did not change the expression of PKC isoforms in PAEC (Fig. 5B), suggesting that the decrease in PKC-{alpha} activity is not derived from changes in its expression but is related to other, as yet, unknown mechanisms.

Recently, we have shown (18) that classical isoforms of PKC are involved in the regulation of CAT-1 transport activity in PAEC. It is known that classical isoforms of PKC target to caveolae under an activation (22, 30). Therefore, we suggested that translocation of activated PKC-{alpha} to caveolae stimulates local phosphorylation of the CAT-1 transporter, which leads to inhibition of its transport activity in PAEC. In contrast, depletion of PKC-{alpha} induced by the long-term treatment with PMA promotes dephosphorylation of the CAT-1 transporter and activation of the transporter (18). Activation of L-arginine transport and the decrease in PKC-{alpha} activity after long-term treatment with PTX fit well with this proposed mechanism that PTX affects L-arginine uptake in PAEC through downregulation of PKC-{alpha} activity.

Detection of DAF-FM fluorescence in PAEC showed that PTX increased NO production and that an activation of PKC by PMA attenuated the effects of PTX on NO production (Fig. 7). This result correlates with our data on the effects of PTX and PMA on L-arginine transport in PAEC (Fig. 6), suggesting that transport of extracellular L-arginine and NO production are interrelated. Together with the activation of NO production, PTX also increased L-[3H]citrulline production from extracellular L-[3H]arginine (Fig. 8). Increased L-[3H]citrulline production under the action of PTX might be related to two intracellular processes: the activation of delivery of extracellular L-[3H]arginine and/or the activation of eNOS activity. PAEC contain ~1 mM intracellular L-arginine (3). Therefore, the delivery of extracellular L-arginine will be a rate-limiting factor for NO synthesis only if eNOS cannot use the intracellular pool of L-arginine. Our data demonstrate that PTX increases L-[3H]citrulline production without affecting eNOS activity in PAEC (Fig. 8), suggesting that the PTX-induced changes in L-citrulline production were the result of activated L-arginine delivery in PAEC. These results support the concept that NO production by endothelial cells can be regulated not only through eNOS activity but also through the regulation of CAT-1-mediated L-arginine transport.

In conclusion, our results show that long-term treatment of PAEC with PTX activates CAT-1-mediated L-arginine uptake through downregulation of PKC-{alpha}. PTX-induced downregulation of PKC-{alpha} does not affect the activity of eNOS. The PTX-induced increase in L-arginine transport results in increased NO production in PAEC, supporting the idea that extracellular L-arginine can modulate NO synthesis independent of the intracellular pool of L-arginine.


    ACKNOWLEDGMENTS
 
We thank Humberto Herrera and Nicole Allred for excellent technical assistance.

GRANTS

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).


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. Zharikov, Div. of Pulmonary Medicine, UF College of Medicine, P. O. Box 100225, Gainesville, FL 32610-0225 (E-mail: zharikov{at}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.


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