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P1,P4-Diadenosine 5′-Tetraphosphate Modulates l -Arginine and l -Citrulline Uptake by Bovine Aortic Endothelial Cells
Archives of Biochemistry and Biophysics Vol. 375, No. 1, March 1, pp. 124 –130, 2000 doi:10.1006/abbi.1999.1643, available online at http://www.idealibrary.com on
P 1,P 4-Diadenosine 5⬘-Tetraphosphate Modulates L-Arginine and L-Citrulline Uptake by Bovine Aortic Endothelial Cells Richard H. Hilderman,* ,† ,‡ ,1 Terrie E. Casey,* and Luminita H. Pojoga† ,‡ *Department of Microbiology and Molecular Medicine, †Department of Biological Sciences, and the ‡Vascular Research Laboratory, Greenville Hospital System and the South Carolina Experiment Station, Clemson University, Clemson, South Carolina 29634-1903
Received August 3, 1999, and in revised form November 16, 1999
Nucleoside (5⬘) oligophospho–(5⬘) nucleosides containing adenosine and guanosine are novel classes of extracellular signal molecules that are stored in platelets and released into the blood following stress (1– 4). These ␣,-dinucleotides contain two nucleoside moieties linked via their 5⬘ positions by a chain of phosphates. Three types of ␣,-dinucleotides found in platelets are adenosine dinucleotides (Ap x A; x ⫽ 3–6), guanosine dinucleotides (Gp x G; x ⫽ 3–6), and adenosine/guanosine dinucleotides (Ap x G; x ⫽ 3–6) (1– 4). The physiological roles of the ␣,-dinucleotides are not clear; however, studies using whole animals and organ models suggest that adenosine dinucleotides are vasoregulators (5–9), guanosine dinucleotides are modulators of growth in vascular smooth muscle cells (4), and
adenosine/guanosine dinucleotides act as vasoconstrictors (4). P 1 ,P 4 -Diadenosine 5⬘-tetraphosphate (Ap 4A) 2 is one of the most abundant and is the best characterized ␣,-dinucleotide. Among the better characterized targets for extracellular Ap 4A are cells of the vascular system. Ap 4A has been shown to modulate blood vessel tone (5–9), induce the release of nitric oxide (NO) from endothelial cells (10), prime respiratory burst and regulate apoptosis in neutrophils (11), and inhibit ADPinduced platelet aggregation (2, 12). Additionally, Ap 4A has been shown to activate glycogen phosphorylase in hepatocytes (13), elicit smooth-muscle contractions in the vas deferens (14) and urinary bladder (15), and, in other tissues, promote catecholamine release (16). The biological effects of Ap 4A on cells have been attributed to its interaction with cell surface receptors. The presence of membrane receptors for Ap 4A has been demonstrated in several tissues where it binds to various purinoceptor subtypes and induces calcium mobilization (17–19). However, how Ap 4A interacts with endothelial cells, a critical physiological target, is not clear. Our goal is to characterize the interaction of Ap 4A with bovine aortic endothelial cells (BAEC). As a step in this direction, we have demonstrated that Ap 4A binds to a heterogeneous population of receptors on the BAEC cell surface (20). Competition ligand binding studies demonstrated that Ap 4A interacts with a receptor that has a higher affinity for Ap 4A, Ap 3A, and Ap 2A but significantly lower affinity for Ap 5A and Ap 6A. Gp x G and Ap x G dinucleotides and synthetic P 2 antagonists and agonists do not interact with the higher affinity Ap 4A receptor. Competition binding studies
1 To whom correspondence and reprint requests should be addressed. Fax: 864-656-1127. E-mail: [email protected].
2 Abbreviations used: Ap 4A, P 1 ,P 4 -diadenosine 5⬘-tetraphosphate; BAEC, bovine aortic endothelial cells; L-Cit, L-citrulline.
We have previously demonstrated that P 1,P 4-diadenosine 5ⴕ-tetraphosphate (Ap 4A) interacts with highaffinity and low-affinity binding sites on the bovine aortic endothelial cell (BAEC) surface. In this report we demonstrate that Ap 4A interaction with the lower affinity site modulates L-arginine (L-Arg) and L-citrulline (L-Cit) uptake by BAEC. Competition uptake studies demonstrate that L-Arg and L-Cit uptake occurs through a common transporter system that is sensitive to Ap 4A. Evidence is also presented that is consistent with Ap 4A modulating L-Arg uptake by increasing the affinity of L-Arg for the transporter. © 2000 Academic Press
Key Words: endothelial cells; Ap 4A; transport; arginine; citrulline.
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0003-9861/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
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also demonstrated that Ap 4A binds, at a lower affinity, to a second class of binding sites which also bind the other Ap x A, Ap x G, and Gp x G dinucleotides and synthetic P 2 antagonists and agonists (20). Since stimulation of endothelial cells by bradykinin or ATP results in a rapid increase of L-arginine (L-Arg) uptake and NO release (21), we investigated whether Ap 4A modulates L-Arg and L-citrulline (L-Cit) uptake in BAEC. In this report we present evidence that demonstrates that Ap 4A modulates the uptake of L-Arg and L-Cit by interacting with the lower affinity Ap 4 A binding site on the BAEC cell surface. Evidence is also presented that demonstrates that Ap 4A modulates LArg and L-Cit uptake through a common transporter system. MATERIALS AND METHODS Materials. BAEC were supplied by Dr. Robert Auerbach of the University of Wisconsin. Minimal Essential Medium (MEM) was purchased from GIBCO. Penicillin and streptomycin were purchased from Sigma. Heat-inactivated fetal bovine calf serum (FBS) was purchased from HyClone. L-[2,3,4- 3H]Arg, L-[ureido- 14C]Cit, and En 3Hance Spray were purchased from New England Nuclear. All nucleotides were purchased from Sigma or RBI. All other reagents were of analytical reagent grade or better. Cell culture. BAEC (1.5 ⫻ 10 5) were seeded in 35-mm gelatincoated Petri dishes and grown in medium, pH 7.4, consisting of MEM, 10% (v/v) FBS, 0.044 mol/L NaHCO 3, penicillin (100 units/ml), and streptomycin (100 g/ml). Cell cultures were maintained at 37°C in a humidified atmosphere of 95% air–5% CO 2. All experiments were performed with passage 10 cells. Measurement of L-Arg and L-Cit transport. L-Arg and L-Cit transport studies were performed as previously described (22). All experiments were performed using cells in early growth phase ((2.5–5) ⫻ 10 4 cells/cm 2) in 35-mm gelatin-coated Petri dishes. Endothelial cells were washed three times with Krebs–Henseleit buffer [10 mM Hepes (pH 7.4), 120 mM NaCl, 4.6 mM KCl, 1.5 mM CaCl 2, 0.5 mM MgCl 2, 1.5 mM NaH 2PO 4, 0.7 mM Na 2HPO 4, 10 mM glucose] and were equilibrated for 15 min at 37°C in 0.9 ml of the same buffer. Uptake studies were performed in a final volume of 1.0 ml and were initiated by the addition of either 39 nM L-[ 3H]Arg (5500 – 6000 cpm/pmol) or 3.9 M L-[ 14C]Cit (50 – 60 cpm/pmol). The samples were incubated for 30 min at 37°C and then washed three times with ice-cold Krebs– Henseleit buffer. Washing three times with ice-cold Krebs–Henseleit buffer containing either 10 mM L-Arg or 10 mM L-Cit had no effect on the total amount of radiolabel recovered (data not shown). These data are consistent with the washing procedure removing all the radiolabeled L-Arg or L-Cit from the cell surface. After washing with the Krebs–Henseleit buffer, the samples were digested with 1.0 ml of 0.1 M NaOH and the alkaline solution transferred into plastic liquid scintillation vials and mixed with 10 ml of a toluene-based/Triton X-100 (2:1) scintillation fluid prior to counting in a Beckman LS3133P liquid scintillation counter. Blanks were obtained by adding radiolabeled amino acid to the cells and immediately washing the cells with ice-cold Krebs–Henseleit buffer prior to digesting with NaOH and counting. All experiments were performed in triplicate and repeated at least three times. In some of the L-[ 3H]Arg uptake experiments, D-[ 14C]mannitol was additionally present during the incubation as an extracellular tracer. The recovery of D-[ 14C]mannitol was less than 0.05%. Metabolism of L-Arg and L-Cit. L-Arg and L-Cit metabolism studies were performed using cells in early growth phase ((2.5–5) ⫻ 10 4 cells/cm 2) in 35-mm gelatin-coated Petri dishes. Endothelial cells
FIG. 1. Time course of L-Arg and L-Cit uptake by BAEC. BAEC were seeded in 35-mm gelatin-coated Petri dishes and grown, and uptake studies were performed as described under Materials and Methods. BAEC were incubated at 37°C in the presence of 3.9 M 3 14 L-[ H]Arg (60 –70 cpm/pmol (■) or L-[ C]Cit (50 – 60 cpm/pmol) (䊐). At the time points indicated, cells were washed three times with ice-cold Krebs–Henseleit buffer, digested in 0.1 M NaOH, and counted as described under Materials and Methods. Data are an average of three different experiments performed in triplicate. Error bars are shown as standard deviations.
were washed three times with Krebs–Henseleit buffer and were equilibrated for 15 min at 37°C in 0.9 ml of the same buffer. Metabolism studies were performed in a final volume of 1.0 ml and these experiments were initiated by the addition of 39 nM L-[ 3H]Arg ((1.5– 2.0) ⫻ 10 4 cpm/pmol) or 3.9 M L-[ 14C]Cit (200 –250 cpm/pmol). The samples were incubated for 5 or 30 min at 37°C, washed three times with ice-cold Krebs–Henseleit buffer, and lysed in 0.2 ml of distilled H 2O. Cell extracts were obtained from the Petri dishes by scraping followed by centrifuging the extracts at 10,000g for 10 min. Fifty microliters of the supernatant was spotted onto TLC plates (Whatman silica gel AL SIL G/UV). The plates were developed in chloroform/methanol/ammonium hydroxide/water, 0.5:4.5:2.0:1.0 (vol/vol), over a distance of 16 cm as previously described (23). After development, plates were dried and autoradiography was carried out at ⫺80°C for 24 – 48 h using Kodak X-OMATAR X-ray film and Cronex Lighting Plus intensifying screens after spraying TLC plates with En 3Hance Spray. Densitometer tracings of autoradiographs were obtained using a BioRad G5-710 calibrated imaging densitometer. Percent of radioactive material was calculated from the relative percentage of total absorbance units ( A ⫻ mm 2).
RESULTS
Time course of L-Arg and L-Cit uptake by BAEC. Uptake of L-Arg and L-Cit into cultured BAEC was linear for at least 120 min (Fig. 1). After 30 min, uptake of 3.9 M L-Arg and 3.9 M L-Cit was 286 ⫾ 20 and 73 ⫾ 6 pmol/10 6 cells, respectively. These results demonstrate that the uptake of L-Arg by BAEC is about 4-fold greater than L-Cit uptake. Because of the different uptake affinities for L-Arg and L-Cit, all subsequent
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experiments were performed with 39 nM L-[ 3H]Arg and 3.9 M L-[ 14C]Cit (see also Fig. 4). In all subsequent experiments, uptake was measured over a time period of 30 min. Effect of Ap 4A on L-Arg and L-Cit uptake by BAEC. The ability of Ap 4A to enhance the uptake of both L-Arg and L-Cit is presented in Fig. 2. Variable amounts of nonradiolabeled Ap 4A and a fixed amount of either 3 14 L-[ H]Arg or L-[ C]Cit were incubated with BAEC for 30 min at 37°C. Ap 4A enhanced in a dose-dependent manner the uptake of both L-Arg (Fig. 2A) and L-Cit (Fig. 2B), but only at high Ap 4A concentrations. Ap 4A had no effect on L-Arg or L-Cit uptake until the concentration of Ap 4A reached 10 M. At 100 M, Ap 4A enhanced L-Arg and L-Cit uptake by 48 and 38%, respectively, over controls that did not contain Ap 4A. These data suggest that Ap 4A modulates the uptake of L-Arg and L-Cit. To determine whether Ap 4A modulates the metabolism of L-Arg and L-Cit, we incubated L-[ 3H]Arg or 14 L-[ C]Cit with BAEC for 5 and 30 min at 37°C in the presence or absence of Ap 4A. The cells were then lysed, centrifuged, and spotted on TLC plates and the plates were developed and analyzed by autoradiography. As shown in Figs. 3A and 3C, L-[ 3H]Arg is rapidly metabolized, with only 29.0 and 17.2% of the radiolabel remaining as L-Arg (R f value of 0.45) after 5 and 30 min of incubation, respectively. The remaining radiolabeled material remains at the origin or migrates as an unknown component with an R f value of 0.80. One hundred micromolar Ap 4A does not affect the relative amount of radiolabeled material at the origin, comigrating with L-[ 3H]Arg or the component moving with an R f value of 0.80 (Fig. 3C). After 5 min of incubation, in the presence or absence of Ap 4A, none of the radiolabeled material comigrates with L-[ 14C]Cit (R f value of 0.86) but is completely metabolized into the component with an R f value of 0.80 (Fig. 3B). These data are consistent with Ap 4A not affecting the metabolism of intracellular L-Arg. Effect of suramin on L-Arg and L-Cit uptake by BAEC. Suramin inhibits Ap 4A binding to the lower affinity site but does not inhibit Ap 4A binding to the higher affinity Ap 4A receptor (20). Thus we determined the effect of suramin on Ap 4A modulation of L-Arg uptake (Table I). Ap 4A enhanced the uptake of L-Arg 1.42-fold whereas suramin had no effect on L-Arg uptake. In the presence of suramin, Ap 4A modulation of L-Arg uptake decreased from 1.42- to 1.03-fold. Similar results were obtained when L-[ 14C]Cit was used in place of L-[ 3H]Arg (data not shown). In addition, dinucleotides that interact with only the lower affinity Ap 4A binding site (20) also enhanced the uptake of L-Arg and L-Cit (data not shown). These results further support the notion that Ap 4A modulates L-Arg and
FIG. 2. Effect of Ap 4A on L-Arg and L-Cit uptake by BAEC. BAEC were seeded in 35-mm gelatin-coated Petri dishes and grown, and uptake studies were performed as described under Materials and Methods. (A) BAEC in a final volume of 1.0 ml were incubated at 37°C for 30 min with 39 nM L-[ 3H]Arg (5500 – 6000 cpm/pmol) and varying concentrations of nonradiolabeled Ap 4A. After the incubation the cells were washed with ice-cold Krebs–Henseleit buffer, digested with 0.1 M NaOH, and counted as described under Materials and Methods. These data are expressed as a relative percentage of controls (picomoles of L-[ 3H]Arg uptake in the absence of Ap 4A). (B) BAEC in a final volume of 1.0 ml were incubated at 37°C for 30 min with 3.9 M L-[ 14C]Cit (50 – 60 cpm/pmol) and varying concentrations of nonradiolabeled Ap 4A. After the incubation the cells were washed with ice-cold Krebs–Henseleit buffer, digested with 0.1 M NaOH, and counted as described under Materials and Methods. These data are expressed as a relative percentage of controls (picomoles of L-[ 14C]Cit uptake in the absence of Ap 4A). Data are an average of four different experiments performed in triplicate. L-Cit
uptake by interacting with the lower affinity binding site on BAEC. Characterization of the Ap 4A modulation of L-Arg and L-Cit uptake by BAEC. To characterize the Ap 4A modulation of L-Arg and L-Cit uptake, we determined
Ap 4A MODULATES ARGININE AND CITRULLINE UPTAKE
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FIG. 3. Effect of Ap 4A on L-Arg and L-Cit metabolism by BAEC. BAEC were seeded in 35-mm gelatin-coated Petri dishes and grown, and uptake studies were performed as described under Materials and Methods. (A) BAEC in a final volume of 1.0 ml were incubated in the presence or absence of Ap 4A at 37°C for either 5 or 30 min with 39 nM L-[ 3H]Arg (1.5–2.0 ⫻ 10 4 cpm/pmol). After the incubation the samples were analyzed by TLC and autoradiography as described under Materials and Methods. Lane 1, 39 nM L-[ 3H]Arg (R f value of 0.45); lane 2, 39 nM L-[ 3H]Arg incubated in Krebs–Henseleit buffer for 30 min at 37°C and then mixed with cell lysates prior to TLC; lane 3, 39 nM 3 3 L-[ H]Arg incubated with BAEC for 5 min at 37°C prior to TLC; lane 4, 39 nM L-[ H]Arg incubated with BAEC in the presence of 100 M Ap 4A for 5 min at 37°C prior to TLC; lane 5, 39 nM L-[ 3H]Arg incubated with BAEC for 30 min at 37°C prior to TLC; lane 6, 39 nM L-[ 3H]Arg incubated with BAEC in the presence of 100 M Ap 4A for 30 min at 37°C prior to TLC. (B) BAEC in a final volume of 1.0 ml were incubated in the presence or absence of Ap 4A at 37°C for either 5 or 30 min with 3.9 M L-[ 14C]Cit (200 –250 cpm/pmol). After the incubation the samples were analyzed by TLC and autoradiography as described under Materials and Methods. Lane 1, 3.9 M L-[ 14C]Cit (R f value of 0.86); lane 2, 3.9 M L-[ 14C]Cit incubated in Krebs–Henseleit buffer for 30 min at 37°C and then mixed with cell lysates prior to TLC; lane 3, 3.9 M 14 14 L-[ C]Cit incubated with BAEC for 5 min at 37°C prior to TLC; lane 4, 3.9 M L-[ C]Cit incubated with BAEC in the presence of 100 M Ap 4A for 5 min at 37°C prior to TLC; lane 5, 3.9 M L-[ 14C]Cit incubated with BAEC for 30 min at 37°C prior to TLC; lane 6, 3.9 M L-[ 14C]Cit incubated with BAEC in the presence of 100 M Ap 4A for 30 min at 37°C prior to TLC. (C) Densitometer tracing of the L-Arg autoradiograph in (A) using a BioRad G5-710 calibrated imaging densitometer.
the effect of increasing concentrations of L-Arg and L-Cit on Ap 4 A-induced uptake. Variable amounts of radiolabeled L-Arg or L-Cit were incubated with BAEC for 30 min at 37°C in the presence or absence of Ap 4A. As shown in Fig. 4, the Ap 4A modulation of both L-Arg and L-Cit uptake decreases with increasing concentrations of L-Arg and L-Cit. At 39 nM L-Arg the ratio of 3 3 L-[ H]Arg uptake in the presence of Ap 4 A/L-[ H]Arg uptake in the absence of Ap 4A was 1.46. Ap 4A modulation decreased with increasing concentrations of L-
Arg and reached a plateau at 390 M L-Arg, where the ratio was 0.64. Ap 4A modulation of L-Cit uptake also shows a similar inhibition with increasing concentrations of L-Cit (Fig. 4). At 1.9 M L-Cit the ratio of L-[ 14C]Cit uptake in the presence of Ap 4A/L-[ 14C]Cit uptake in the absence of Ap 4A was 1.50. At 390 M L-Cit the ratio decreased to 0.80. These data suggest that the Ap 4A modulation of L-Arg and L-Cit uptake is dependent on the extracellular concentration of L-Arg and L-Cit, with the maxi-
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HILDERMAN, CASEY, AND POJOGA TABLE I
Effect of Suramin on L-Arg Uptake by BAEC a Addition
pmol/10 6 cells
Ratio b
None 100 M Ap 4A 500 M suramin 100 M Ap 4A ⫹ 500 M suramin
7.32 ⫾ 0.62 10.40 ⫾ 0.92 6.86 ⫾ 0.57
1.00 1.42 0.92
7.69 ⫾ 0.72
1.03
a
Experiments were performed as described in the legend of Fig. 2 except cells were preincubated with 500 M suramin for 10 min at 37°C prior to the addition of Ap 4A and L-[ 3H]Arg. Data are an average of three different experiments performed in triplicate. b Total picomoles of L-Arg uptake in the presence of Ap 4A or suramin divided by total picomoles of L-Arg uptake in the absence of Ap 4A or suramin.
mum modulatory effect occurring at low concentrations. Specificity of Ap 4A modulation of L-Arg and L-Cit uptake. To determine whether the Ap 4A modulation of L-Arg and L-Cit uptake occurs through the same transporter, homologous and heterologous competition uptake studies were performed using L-[ 14C]Cit and
FIG. 4. Effect of increasing concentrations of L-Arg and L-Cit on Ap 4A-induced uptake. BAEC were seeded in 35-mm gelatin-coated Petri dishes, and grown, and uptake studies were performed as described under Materials and Methods. BAEC in a final volume of 1.0 ml were incubated at 37°C for 30 min with varying concentrations of L-[ 3H]Arg or L-[ 14C]Cit. These incubations were performed in the presence and absence of 100 M Ap 4A. After incubation the cells were washed with ice-cold Krebs–Henseleit buffer, digested with 0.1 M NaOH, and counted as described under Materials and Methods. The data are expressed as ratio of uptake in the presence of Ap 4A/ uptake in the absence of Ap 4A. Data are an average of three different experiments performed in triplicate.
FIG. 5. Inhibition of L-Cit uptake by nonradiolabeled L-Arg and L-Cit. BAEC were seeded in 35-mm gelatin-coated Petri dishes, and grown, and uptake studies were performed as described under Materials and Methods. BAEC in a final volume of 1.0 ml were incubated at 37°C for 30 min with 3.9 M L-[ 14C]Cit (50 – 60 cpm/pmol) and varying concentrations of nonradiolabeled L-Cit (■), varying concentrations of nonradiolabeled L-Arg (F), varying concentrations of nonradiolabeled L-Cit plus 100 M Ap 4A (䊐), and varying concentrations of nonradiolabeled L-Arg plus 100 M Ap 4A (E). After the incubation the cells were washed with ice-cold Krebs–Henseleit buffer, digested with 0.1 M NaOH, and counted as described under Materials and Methods. These data are expressed as a relative percentage of controls (picomoles of L-[ 14C]Cit uptake in the absence of nonradiolabeled L-Cit and L-Arg). Data are an average of three different experiments performed in triplicate.
nonradiolabeled L-Cit and L-Arg in the presence and absence of Ap 4A. The ability of nonradiolabeled L-Cit to inhibit L-[ 14C]Cit uptake is shown in Fig. 5. Variable amounts of nonradiolabeled L-Cit and a fixed amount of 14 L-[ C]Cit in the presence and absence of 100 M Ap 4 A were incubated with BAEC for 30 min at 37°C. In the presence of Ap 4A, nonradiolabeled L-Cit is a more effective competitor at L-Cit concentrations less than 100 M. These data are consistent with L-Cit being transported into BAEC by two transporter systems: an Ap 4A-sensitive and an Ap 4A-resistant system. Figure 5 also shows the ability of nonradiolabeled 14 L-Arg to inhibit L-[ C]Cit uptake. Nonradiolabeled LArg competes more effectively with L-[ 14C]Cit than does nonradiolabeled L-Cit. However, L-Arg inhibited only about 40% of the L-[ 14C]Cit uptake, reaching a plateau at 100 M. In the presence of Ap 4A, nonradiolabeled L-Arg is a more effective competitor, but also only inhibiting about 40% of the L-[ 14C]Cit uptake and reaching a plateau at 10 M. These data suggest that L-Arg and L-Cit uptake occurs through the same Ap 4A-sensitive transporter system.
Ap 4A MODULATES ARGININE AND CITRULLINE UPTAKE
DISCUSSION
Past work from this laboratory has demonstrated that Ap 4A induces the release of NO from cultured BAEC (10). In our characterization of the interaction of Ap 4A with endothelial cells, we have demonstrated that Ap 4A interacts with a heterogeneous population of receptors on BAEC (20). Ap 4A interacts with a putative P 4 purinoceptor which has a higher affinity for only Ap 4A, Ap 3A, and Ap 2A. Ap 4A also interacts with a lower affinity to a second class of binding sites. The results reported in this paper demonstrate that Ap 4A modulates the uptake of L-Arg and L-Cit by interacting with the lower affinity binding site. Our results also demonstrate that Ap 4A modulates L-Arg and L-Cit uptake through a common transporter system. Ap 4A modulates the uptake of L-Arg and L-Cit in a dose-dependent manner (Fig. 2) but only at Ap 4A concentrations higher than required for Ap 4A to interact with its higher affinity receptor (20, 24). Suramin, which competes with Ap 4A binding only at the lower affinity site (20), blocks the modulation of L-Arg and L-Cit uptake (Table I). These results are consistent with Ap 4A interacting with the lower affinity site to enhance the uptake of L-Arg and L-Cit. Since Ap 4A does not affect the intracellular metabolism of L-Arg (Figs. 3A and 3C), these data are consistent with Ap 4A modulating the L-Arg uptake by increasing its transport but not affecting its metabolism. These data also suggest that Ap 4A modulates L-Arg by increasing its affinity for the transporter. On the other hand, essentially all of the L-[ 14C]Cit is metabolized after 5 min in the presence and absence of Ap 4A (Fig. 3B); thus it is not possible to determine whether Ap 4A modulates L-Cit uptake by affecting its metabolism and/or transport. L-[2,3,4- 3H]Arg is metabolized into a component with an R f value of 0.80 and into a component that does not move from the origin (Fig. 3A) whereas L-[ureido- 14C]Cit is metabolized only into a component with an R f value of 0.80 (Fig. 3C). At present, we do not know the identity of these components but it is reasonable to postulate that the radiolabeled material at the origin is L-Arg incorporated into proteins. Studies are in progress to identify these components. To our knowledge the mechanism by which L-Cit is transported into endothelial cells has not been determined. The data presented in this paper are consistent with L-Cit being transported into BAEC by at least two transporter systems: an Ap 4A-sensitive and an Ap 4Aresistant system (Fig. 5). Homologous and heterologous competition uptake studies demonstrate that both L-Arg uptake and L-Cit uptake occur through the Ap 4A-sensitive system (Fig. 5). In the presence of increasing concentrations of L-Arg and L-Cit, BAEC overcome the Ap 4A modulatory effect
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on L-Arg and L-Cit uptake (Fig. 4). Other investigators have demonstrated that L-Arg levels in plasma decrease under stressful conditions such as sepsis (25). These data are consistent with the stress molecule, Ap 4A, only modulating L-Arg uptake at low extracellular concentrations. At the present time, it is not known whether Ap 4A modulation is limited to L-Arg and L-Cit or whether Ap 4A modulates the uptake of other amino acids by endothelial cells. In addition, the physiological role of this Ap 4A modulation of L-Arg and L-Cit uptake by BAEC is not known. Investigators have demonstrated L-Arg and L-Cit are required for nitric oxide synthase to generate NO (26 –29). Other investigators have shown that bradykinin, ATP, interleukin-1, and necrosis factor-␣ stimulate an increase of L-Arg uptake and NO release by endothelial cells (21, 30). These data suggest that the Ap 4A-induced uptake of L-Arg and L-Cit by BAEC may enhance NO synthesis. Studies are in progress to determine not only the effect of Ap 4A-induced uptake of L-Arg and L-Cit on NO synthesis but also whether Ap 4A induces the uptake of other amino acids. ACKNOWLEDGMENTS This research was supported in part by NSF MCB-9816681, USDA NRICG 96-35204-3669, and Greenville Hospital System/Clemson University Cooperative and South Carolina Experiment Station Grant SC01630. The authors thank Dr. James Zimmerman of Clemson University for critically evaluating the manuscript.
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22. Schmidt, K., Klatt, P., and Mayer, B. (1993) Mol. Pharmacol. 44, 615– 621. 23. Sessa, W. C., Hecker, M., Mitchell, J. A., and Vane, J. A. (1990) Proc. Natl. Acad. Sci. USA 87, 8607– 8611. 24. Hilderman, R. H., and Fairbank, A. T. (1999) Biochimie 81, 255–260. 25. Clowes, G. H. A., Heideman, M., Lindberg, B., Randall, H. T., Hirsch, E. F., Cha, C. J., and Martin, H. (1980) Surgery 88, 531–540. 26. Palmer, R. M. J., Ashton, D. S., and Moncada, S. (1988) Nature 333, 664 – 666. 27. Chen, F. Y., and Lee, T. J. F. (1995) J. Pharmacol. Exp. Ther. 273, 895–901. 28. Yu, J. G., O’Brien, W. E., and Lee, T. J. F. (1997) J. Cereb. Blood Flow Metab. 17, 884 – 893. 29. Wu, G., and Meininger, C. J. (1993) Am. J. Physiol. 265, H1967– H1971. 30. Durante, W., Liao, L., Iftikhar, I., O’Brien, W. E., and Schafer, A. I. (1996) Circ. Res. 78, 1075–1082.
P 1,P 4-Diadenosine 5⬘-Tetraphosphate Modulates L-Arginine and L-Citrulline Uptake by Bovine Aortic Endothelial Cells Richard H. Hilderman,* ,† ,‡ ,1 Terrie E. Casey,* and Luminita H. Pojoga† ,‡ *Department of Microbiology and Molecular Medicine, †Department of Biological Sciences, and the ‡Vascular Research Laboratory, Greenville Hospital System and the South Carolina Experiment Station, Clemson University, Clemson, South Carolina 29634-1903
Received August 3, 1999, and in revised form November 16, 1999
Nucleoside (5⬘) oligophospho–(5⬘) nucleosides containing adenosine and guanosine are novel classes of extracellular signal molecules that are stored in platelets and released into the blood following stress (1– 4). These ␣,-dinucleotides contain two nucleoside moieties linked via their 5⬘ positions by a chain of phosphates. Three types of ␣,-dinucleotides found in platelets are adenosine dinucleotides (Ap x A; x ⫽ 3–6), guanosine dinucleotides (Gp x G; x ⫽ 3–6), and adenosine/guanosine dinucleotides (Ap x G; x ⫽ 3–6) (1– 4). The physiological roles of the ␣,-dinucleotides are not clear; however, studies using whole animals and organ models suggest that adenosine dinucleotides are vasoregulators (5–9), guanosine dinucleotides are modulators of growth in vascular smooth muscle cells (4), and
adenosine/guanosine dinucleotides act as vasoconstrictors (4). P 1 ,P 4 -Diadenosine 5⬘-tetraphosphate (Ap 4A) 2 is one of the most abundant and is the best characterized ␣,-dinucleotide. Among the better characterized targets for extracellular Ap 4A are cells of the vascular system. Ap 4A has been shown to modulate blood vessel tone (5–9), induce the release of nitric oxide (NO) from endothelial cells (10), prime respiratory burst and regulate apoptosis in neutrophils (11), and inhibit ADPinduced platelet aggregation (2, 12). Additionally, Ap 4A has been shown to activate glycogen phosphorylase in hepatocytes (13), elicit smooth-muscle contractions in the vas deferens (14) and urinary bladder (15), and, in other tissues, promote catecholamine release (16). The biological effects of Ap 4A on cells have been attributed to its interaction with cell surface receptors. The presence of membrane receptors for Ap 4A has been demonstrated in several tissues where it binds to various purinoceptor subtypes and induces calcium mobilization (17–19). However, how Ap 4A interacts with endothelial cells, a critical physiological target, is not clear. Our goal is to characterize the interaction of Ap 4A with bovine aortic endothelial cells (BAEC). As a step in this direction, we have demonstrated that Ap 4A binds to a heterogeneous population of receptors on the BAEC cell surface (20). Competition ligand binding studies demonstrated that Ap 4A interacts with a receptor that has a higher affinity for Ap 4A, Ap 3A, and Ap 2A but significantly lower affinity for Ap 5A and Ap 6A. Gp x G and Ap x G dinucleotides and synthetic P 2 antagonists and agonists do not interact with the higher affinity Ap 4A receptor. Competition binding studies
1 To whom correspondence and reprint requests should be addressed. Fax: 864-656-1127. E-mail: [email protected].
2 Abbreviations used: Ap 4A, P 1 ,P 4 -diadenosine 5⬘-tetraphosphate; BAEC, bovine aortic endothelial cells; L-Cit, L-citrulline.
We have previously demonstrated that P 1,P 4-diadenosine 5ⴕ-tetraphosphate (Ap 4A) interacts with highaffinity and low-affinity binding sites on the bovine aortic endothelial cell (BAEC) surface. In this report we demonstrate that Ap 4A interaction with the lower affinity site modulates L-arginine (L-Arg) and L-citrulline (L-Cit) uptake by BAEC. Competition uptake studies demonstrate that L-Arg and L-Cit uptake occurs through a common transporter system that is sensitive to Ap 4A. Evidence is also presented that is consistent with Ap 4A modulating L-Arg uptake by increasing the affinity of L-Arg for the transporter. © 2000 Academic Press
Key Words: endothelial cells; Ap 4A; transport; arginine; citrulline.
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also demonstrated that Ap 4A binds, at a lower affinity, to a second class of binding sites which also bind the other Ap x A, Ap x G, and Gp x G dinucleotides and synthetic P 2 antagonists and agonists (20). Since stimulation of endothelial cells by bradykinin or ATP results in a rapid increase of L-arginine (L-Arg) uptake and NO release (21), we investigated whether Ap 4A modulates L-Arg and L-citrulline (L-Cit) uptake in BAEC. In this report we present evidence that demonstrates that Ap 4A modulates the uptake of L-Arg and L-Cit by interacting with the lower affinity Ap 4 A binding site on the BAEC cell surface. Evidence is also presented that demonstrates that Ap 4A modulates LArg and L-Cit uptake through a common transporter system. MATERIALS AND METHODS Materials. BAEC were supplied by Dr. Robert Auerbach of the University of Wisconsin. Minimal Essential Medium (MEM) was purchased from GIBCO. Penicillin and streptomycin were purchased from Sigma. Heat-inactivated fetal bovine calf serum (FBS) was purchased from HyClone. L-[2,3,4- 3H]Arg, L-[ureido- 14C]Cit, and En 3Hance Spray were purchased from New England Nuclear. All nucleotides were purchased from Sigma or RBI. All other reagents were of analytical reagent grade or better. Cell culture. BAEC (1.5 ⫻ 10 5) were seeded in 35-mm gelatincoated Petri dishes and grown in medium, pH 7.4, consisting of MEM, 10% (v/v) FBS, 0.044 mol/L NaHCO 3, penicillin (100 units/ml), and streptomycin (100 g/ml). Cell cultures were maintained at 37°C in a humidified atmosphere of 95% air–5% CO 2. All experiments were performed with passage 10 cells. Measurement of L-Arg and L-Cit transport. L-Arg and L-Cit transport studies were performed as previously described (22). All experiments were performed using cells in early growth phase ((2.5–5) ⫻ 10 4 cells/cm 2) in 35-mm gelatin-coated Petri dishes. Endothelial cells were washed three times with Krebs–Henseleit buffer [10 mM Hepes (pH 7.4), 120 mM NaCl, 4.6 mM KCl, 1.5 mM CaCl 2, 0.5 mM MgCl 2, 1.5 mM NaH 2PO 4, 0.7 mM Na 2HPO 4, 10 mM glucose] and were equilibrated for 15 min at 37°C in 0.9 ml of the same buffer. Uptake studies were performed in a final volume of 1.0 ml and were initiated by the addition of either 39 nM L-[ 3H]Arg (5500 – 6000 cpm/pmol) or 3.9 M L-[ 14C]Cit (50 – 60 cpm/pmol). The samples were incubated for 30 min at 37°C and then washed three times with ice-cold Krebs– Henseleit buffer. Washing three times with ice-cold Krebs–Henseleit buffer containing either 10 mM L-Arg or 10 mM L-Cit had no effect on the total amount of radiolabel recovered (data not shown). These data are consistent with the washing procedure removing all the radiolabeled L-Arg or L-Cit from the cell surface. After washing with the Krebs–Henseleit buffer, the samples were digested with 1.0 ml of 0.1 M NaOH and the alkaline solution transferred into plastic liquid scintillation vials and mixed with 10 ml of a toluene-based/Triton X-100 (2:1) scintillation fluid prior to counting in a Beckman LS3133P liquid scintillation counter. Blanks were obtained by adding radiolabeled amino acid to the cells and immediately washing the cells with ice-cold Krebs–Henseleit buffer prior to digesting with NaOH and counting. All experiments were performed in triplicate and repeated at least three times. In some of the L-[ 3H]Arg uptake experiments, D-[ 14C]mannitol was additionally present during the incubation as an extracellular tracer. The recovery of D-[ 14C]mannitol was less than 0.05%. Metabolism of L-Arg and L-Cit. L-Arg and L-Cit metabolism studies were performed using cells in early growth phase ((2.5–5) ⫻ 10 4 cells/cm 2) in 35-mm gelatin-coated Petri dishes. Endothelial cells
FIG. 1. Time course of L-Arg and L-Cit uptake by BAEC. BAEC were seeded in 35-mm gelatin-coated Petri dishes and grown, and uptake studies were performed as described under Materials and Methods. BAEC were incubated at 37°C in the presence of 3.9 M 3 14 L-[ H]Arg (60 –70 cpm/pmol (■) or L-[ C]Cit (50 – 60 cpm/pmol) (䊐). At the time points indicated, cells were washed three times with ice-cold Krebs–Henseleit buffer, digested in 0.1 M NaOH, and counted as described under Materials and Methods. Data are an average of three different experiments performed in triplicate. Error bars are shown as standard deviations.
were washed three times with Krebs–Henseleit buffer and were equilibrated for 15 min at 37°C in 0.9 ml of the same buffer. Metabolism studies were performed in a final volume of 1.0 ml and these experiments were initiated by the addition of 39 nM L-[ 3H]Arg ((1.5– 2.0) ⫻ 10 4 cpm/pmol) or 3.9 M L-[ 14C]Cit (200 –250 cpm/pmol). The samples were incubated for 5 or 30 min at 37°C, washed three times with ice-cold Krebs–Henseleit buffer, and lysed in 0.2 ml of distilled H 2O. Cell extracts were obtained from the Petri dishes by scraping followed by centrifuging the extracts at 10,000g for 10 min. Fifty microliters of the supernatant was spotted onto TLC plates (Whatman silica gel AL SIL G/UV). The plates were developed in chloroform/methanol/ammonium hydroxide/water, 0.5:4.5:2.0:1.0 (vol/vol), over a distance of 16 cm as previously described (23). After development, plates were dried and autoradiography was carried out at ⫺80°C for 24 – 48 h using Kodak X-OMATAR X-ray film and Cronex Lighting Plus intensifying screens after spraying TLC plates with En 3Hance Spray. Densitometer tracings of autoradiographs were obtained using a BioRad G5-710 calibrated imaging densitometer. Percent of radioactive material was calculated from the relative percentage of total absorbance units ( A ⫻ mm 2).
RESULTS
Time course of L-Arg and L-Cit uptake by BAEC. Uptake of L-Arg and L-Cit into cultured BAEC was linear for at least 120 min (Fig. 1). After 30 min, uptake of 3.9 M L-Arg and 3.9 M L-Cit was 286 ⫾ 20 and 73 ⫾ 6 pmol/10 6 cells, respectively. These results demonstrate that the uptake of L-Arg by BAEC is about 4-fold greater than L-Cit uptake. Because of the different uptake affinities for L-Arg and L-Cit, all subsequent
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experiments were performed with 39 nM L-[ 3H]Arg and 3.9 M L-[ 14C]Cit (see also Fig. 4). In all subsequent experiments, uptake was measured over a time period of 30 min. Effect of Ap 4A on L-Arg and L-Cit uptake by BAEC. The ability of Ap 4A to enhance the uptake of both L-Arg and L-Cit is presented in Fig. 2. Variable amounts of nonradiolabeled Ap 4A and a fixed amount of either 3 14 L-[ H]Arg or L-[ C]Cit were incubated with BAEC for 30 min at 37°C. Ap 4A enhanced in a dose-dependent manner the uptake of both L-Arg (Fig. 2A) and L-Cit (Fig. 2B), but only at high Ap 4A concentrations. Ap 4A had no effect on L-Arg or L-Cit uptake until the concentration of Ap 4A reached 10 M. At 100 M, Ap 4A enhanced L-Arg and L-Cit uptake by 48 and 38%, respectively, over controls that did not contain Ap 4A. These data suggest that Ap 4A modulates the uptake of L-Arg and L-Cit. To determine whether Ap 4A modulates the metabolism of L-Arg and L-Cit, we incubated L-[ 3H]Arg or 14 L-[ C]Cit with BAEC for 5 and 30 min at 37°C in the presence or absence of Ap 4A. The cells were then lysed, centrifuged, and spotted on TLC plates and the plates were developed and analyzed by autoradiography. As shown in Figs. 3A and 3C, L-[ 3H]Arg is rapidly metabolized, with only 29.0 and 17.2% of the radiolabel remaining as L-Arg (R f value of 0.45) after 5 and 30 min of incubation, respectively. The remaining radiolabeled material remains at the origin or migrates as an unknown component with an R f value of 0.80. One hundred micromolar Ap 4A does not affect the relative amount of radiolabeled material at the origin, comigrating with L-[ 3H]Arg or the component moving with an R f value of 0.80 (Fig. 3C). After 5 min of incubation, in the presence or absence of Ap 4A, none of the radiolabeled material comigrates with L-[ 14C]Cit (R f value of 0.86) but is completely metabolized into the component with an R f value of 0.80 (Fig. 3B). These data are consistent with Ap 4A not affecting the metabolism of intracellular L-Arg. Effect of suramin on L-Arg and L-Cit uptake by BAEC. Suramin inhibits Ap 4A binding to the lower affinity site but does not inhibit Ap 4A binding to the higher affinity Ap 4A receptor (20). Thus we determined the effect of suramin on Ap 4A modulation of L-Arg uptake (Table I). Ap 4A enhanced the uptake of L-Arg 1.42-fold whereas suramin had no effect on L-Arg uptake. In the presence of suramin, Ap 4A modulation of L-Arg uptake decreased from 1.42- to 1.03-fold. Similar results were obtained when L-[ 14C]Cit was used in place of L-[ 3H]Arg (data not shown). In addition, dinucleotides that interact with only the lower affinity Ap 4A binding site (20) also enhanced the uptake of L-Arg and L-Cit (data not shown). These results further support the notion that Ap 4A modulates L-Arg and
FIG. 2. Effect of Ap 4A on L-Arg and L-Cit uptake by BAEC. BAEC were seeded in 35-mm gelatin-coated Petri dishes and grown, and uptake studies were performed as described under Materials and Methods. (A) BAEC in a final volume of 1.0 ml were incubated at 37°C for 30 min with 39 nM L-[ 3H]Arg (5500 – 6000 cpm/pmol) and varying concentrations of nonradiolabeled Ap 4A. After the incubation the cells were washed with ice-cold Krebs–Henseleit buffer, digested with 0.1 M NaOH, and counted as described under Materials and Methods. These data are expressed as a relative percentage of controls (picomoles of L-[ 3H]Arg uptake in the absence of Ap 4A). (B) BAEC in a final volume of 1.0 ml were incubated at 37°C for 30 min with 3.9 M L-[ 14C]Cit (50 – 60 cpm/pmol) and varying concentrations of nonradiolabeled Ap 4A. After the incubation the cells were washed with ice-cold Krebs–Henseleit buffer, digested with 0.1 M NaOH, and counted as described under Materials and Methods. These data are expressed as a relative percentage of controls (picomoles of L-[ 14C]Cit uptake in the absence of Ap 4A). Data are an average of four different experiments performed in triplicate. L-Cit
uptake by interacting with the lower affinity binding site on BAEC. Characterization of the Ap 4A modulation of L-Arg and L-Cit uptake by BAEC. To characterize the Ap 4A modulation of L-Arg and L-Cit uptake, we determined
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FIG. 3. Effect of Ap 4A on L-Arg and L-Cit metabolism by BAEC. BAEC were seeded in 35-mm gelatin-coated Petri dishes and grown, and uptake studies were performed as described under Materials and Methods. (A) BAEC in a final volume of 1.0 ml were incubated in the presence or absence of Ap 4A at 37°C for either 5 or 30 min with 39 nM L-[ 3H]Arg (1.5–2.0 ⫻ 10 4 cpm/pmol). After the incubation the samples were analyzed by TLC and autoradiography as described under Materials and Methods. Lane 1, 39 nM L-[ 3H]Arg (R f value of 0.45); lane 2, 39 nM L-[ 3H]Arg incubated in Krebs–Henseleit buffer for 30 min at 37°C and then mixed with cell lysates prior to TLC; lane 3, 39 nM 3 3 L-[ H]Arg incubated with BAEC for 5 min at 37°C prior to TLC; lane 4, 39 nM L-[ H]Arg incubated with BAEC in the presence of 100 M Ap 4A for 5 min at 37°C prior to TLC; lane 5, 39 nM L-[ 3H]Arg incubated with BAEC for 30 min at 37°C prior to TLC; lane 6, 39 nM L-[ 3H]Arg incubated with BAEC in the presence of 100 M Ap 4A for 30 min at 37°C prior to TLC. (B) BAEC in a final volume of 1.0 ml were incubated in the presence or absence of Ap 4A at 37°C for either 5 or 30 min with 3.9 M L-[ 14C]Cit (200 –250 cpm/pmol). After the incubation the samples were analyzed by TLC and autoradiography as described under Materials and Methods. Lane 1, 3.9 M L-[ 14C]Cit (R f value of 0.86); lane 2, 3.9 M L-[ 14C]Cit incubated in Krebs–Henseleit buffer for 30 min at 37°C and then mixed with cell lysates prior to TLC; lane 3, 3.9 M 14 14 L-[ C]Cit incubated with BAEC for 5 min at 37°C prior to TLC; lane 4, 3.9 M L-[ C]Cit incubated with BAEC in the presence of 100 M Ap 4A for 5 min at 37°C prior to TLC; lane 5, 3.9 M L-[ 14C]Cit incubated with BAEC for 30 min at 37°C prior to TLC; lane 6, 3.9 M L-[ 14C]Cit incubated with BAEC in the presence of 100 M Ap 4A for 30 min at 37°C prior to TLC. (C) Densitometer tracing of the L-Arg autoradiograph in (A) using a BioRad G5-710 calibrated imaging densitometer.
the effect of increasing concentrations of L-Arg and L-Cit on Ap 4 A-induced uptake. Variable amounts of radiolabeled L-Arg or L-Cit were incubated with BAEC for 30 min at 37°C in the presence or absence of Ap 4A. As shown in Fig. 4, the Ap 4A modulation of both L-Arg and L-Cit uptake decreases with increasing concentrations of L-Arg and L-Cit. At 39 nM L-Arg the ratio of 3 3 L-[ H]Arg uptake in the presence of Ap 4 A/L-[ H]Arg uptake in the absence of Ap 4A was 1.46. Ap 4A modulation decreased with increasing concentrations of L-
Arg and reached a plateau at 390 M L-Arg, where the ratio was 0.64. Ap 4A modulation of L-Cit uptake also shows a similar inhibition with increasing concentrations of L-Cit (Fig. 4). At 1.9 M L-Cit the ratio of L-[ 14C]Cit uptake in the presence of Ap 4A/L-[ 14C]Cit uptake in the absence of Ap 4A was 1.50. At 390 M L-Cit the ratio decreased to 0.80. These data suggest that the Ap 4A modulation of L-Arg and L-Cit uptake is dependent on the extracellular concentration of L-Arg and L-Cit, with the maxi-
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HILDERMAN, CASEY, AND POJOGA TABLE I
Effect of Suramin on L-Arg Uptake by BAEC a Addition
pmol/10 6 cells
Ratio b
None 100 M Ap 4A 500 M suramin 100 M Ap 4A ⫹ 500 M suramin
7.32 ⫾ 0.62 10.40 ⫾ 0.92 6.86 ⫾ 0.57
1.00 1.42 0.92
7.69 ⫾ 0.72
1.03
a
Experiments were performed as described in the legend of Fig. 2 except cells were preincubated with 500 M suramin for 10 min at 37°C prior to the addition of Ap 4A and L-[ 3H]Arg. Data are an average of three different experiments performed in triplicate. b Total picomoles of L-Arg uptake in the presence of Ap 4A or suramin divided by total picomoles of L-Arg uptake in the absence of Ap 4A or suramin.
mum modulatory effect occurring at low concentrations. Specificity of Ap 4A modulation of L-Arg and L-Cit uptake. To determine whether the Ap 4A modulation of L-Arg and L-Cit uptake occurs through the same transporter, homologous and heterologous competition uptake studies were performed using L-[ 14C]Cit and
FIG. 4. Effect of increasing concentrations of L-Arg and L-Cit on Ap 4A-induced uptake. BAEC were seeded in 35-mm gelatin-coated Petri dishes, and grown, and uptake studies were performed as described under Materials and Methods. BAEC in a final volume of 1.0 ml were incubated at 37°C for 30 min with varying concentrations of L-[ 3H]Arg or L-[ 14C]Cit. These incubations were performed in the presence and absence of 100 M Ap 4A. After incubation the cells were washed with ice-cold Krebs–Henseleit buffer, digested with 0.1 M NaOH, and counted as described under Materials and Methods. The data are expressed as ratio of uptake in the presence of Ap 4A/ uptake in the absence of Ap 4A. Data are an average of three different experiments performed in triplicate.
FIG. 5. Inhibition of L-Cit uptake by nonradiolabeled L-Arg and L-Cit. BAEC were seeded in 35-mm gelatin-coated Petri dishes, and grown, and uptake studies were performed as described under Materials and Methods. BAEC in a final volume of 1.0 ml were incubated at 37°C for 30 min with 3.9 M L-[ 14C]Cit (50 – 60 cpm/pmol) and varying concentrations of nonradiolabeled L-Cit (■), varying concentrations of nonradiolabeled L-Arg (F), varying concentrations of nonradiolabeled L-Cit plus 100 M Ap 4A (䊐), and varying concentrations of nonradiolabeled L-Arg plus 100 M Ap 4A (E). After the incubation the cells were washed with ice-cold Krebs–Henseleit buffer, digested with 0.1 M NaOH, and counted as described under Materials and Methods. These data are expressed as a relative percentage of controls (picomoles of L-[ 14C]Cit uptake in the absence of nonradiolabeled L-Cit and L-Arg). Data are an average of three different experiments performed in triplicate.
nonradiolabeled L-Cit and L-Arg in the presence and absence of Ap 4A. The ability of nonradiolabeled L-Cit to inhibit L-[ 14C]Cit uptake is shown in Fig. 5. Variable amounts of nonradiolabeled L-Cit and a fixed amount of 14 L-[ C]Cit in the presence and absence of 100 M Ap 4 A were incubated with BAEC for 30 min at 37°C. In the presence of Ap 4A, nonradiolabeled L-Cit is a more effective competitor at L-Cit concentrations less than 100 M. These data are consistent with L-Cit being transported into BAEC by two transporter systems: an Ap 4A-sensitive and an Ap 4A-resistant system. Figure 5 also shows the ability of nonradiolabeled 14 L-Arg to inhibit L-[ C]Cit uptake. Nonradiolabeled LArg competes more effectively with L-[ 14C]Cit than does nonradiolabeled L-Cit. However, L-Arg inhibited only about 40% of the L-[ 14C]Cit uptake, reaching a plateau at 100 M. In the presence of Ap 4A, nonradiolabeled L-Arg is a more effective competitor, but also only inhibiting about 40% of the L-[ 14C]Cit uptake and reaching a plateau at 10 M. These data suggest that L-Arg and L-Cit uptake occurs through the same Ap 4A-sensitive transporter system.
Ap 4A MODULATES ARGININE AND CITRULLINE UPTAKE
DISCUSSION
Past work from this laboratory has demonstrated that Ap 4A induces the release of NO from cultured BAEC (10). In our characterization of the interaction of Ap 4A with endothelial cells, we have demonstrated that Ap 4A interacts with a heterogeneous population of receptors on BAEC (20). Ap 4A interacts with a putative P 4 purinoceptor which has a higher affinity for only Ap 4A, Ap 3A, and Ap 2A. Ap 4A also interacts with a lower affinity to a second class of binding sites. The results reported in this paper demonstrate that Ap 4A modulates the uptake of L-Arg and L-Cit by interacting with the lower affinity binding site. Our results also demonstrate that Ap 4A modulates L-Arg and L-Cit uptake through a common transporter system. Ap 4A modulates the uptake of L-Arg and L-Cit in a dose-dependent manner (Fig. 2) but only at Ap 4A concentrations higher than required for Ap 4A to interact with its higher affinity receptor (20, 24). Suramin, which competes with Ap 4A binding only at the lower affinity site (20), blocks the modulation of L-Arg and L-Cit uptake (Table I). These results are consistent with Ap 4A interacting with the lower affinity site to enhance the uptake of L-Arg and L-Cit. Since Ap 4A does not affect the intracellular metabolism of L-Arg (Figs. 3A and 3C), these data are consistent with Ap 4A modulating the L-Arg uptake by increasing its transport but not affecting its metabolism. These data also suggest that Ap 4A modulates L-Arg by increasing its affinity for the transporter. On the other hand, essentially all of the L-[ 14C]Cit is metabolized after 5 min in the presence and absence of Ap 4A (Fig. 3B); thus it is not possible to determine whether Ap 4A modulates L-Cit uptake by affecting its metabolism and/or transport. L-[2,3,4- 3H]Arg is metabolized into a component with an R f value of 0.80 and into a component that does not move from the origin (Fig. 3A) whereas L-[ureido- 14C]Cit is metabolized only into a component with an R f value of 0.80 (Fig. 3C). At present, we do not know the identity of these components but it is reasonable to postulate that the radiolabeled material at the origin is L-Arg incorporated into proteins. Studies are in progress to identify these components. To our knowledge the mechanism by which L-Cit is transported into endothelial cells has not been determined. The data presented in this paper are consistent with L-Cit being transported into BAEC by at least two transporter systems: an Ap 4A-sensitive and an Ap 4Aresistant system (Fig. 5). Homologous and heterologous competition uptake studies demonstrate that both L-Arg uptake and L-Cit uptake occur through the Ap 4A-sensitive system (Fig. 5). In the presence of increasing concentrations of L-Arg and L-Cit, BAEC overcome the Ap 4A modulatory effect
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on L-Arg and L-Cit uptake (Fig. 4). Other investigators have demonstrated that L-Arg levels in plasma decrease under stressful conditions such as sepsis (25). These data are consistent with the stress molecule, Ap 4A, only modulating L-Arg uptake at low extracellular concentrations. At the present time, it is not known whether Ap 4A modulation is limited to L-Arg and L-Cit or whether Ap 4A modulates the uptake of other amino acids by endothelial cells. In addition, the physiological role of this Ap 4A modulation of L-Arg and L-Cit uptake by BAEC is not known. Investigators have demonstrated L-Arg and L-Cit are required for nitric oxide synthase to generate NO (26 –29). Other investigators have shown that bradykinin, ATP, interleukin-1, and necrosis factor-␣ stimulate an increase of L-Arg uptake and NO release by endothelial cells (21, 30). These data suggest that the Ap 4A-induced uptake of L-Arg and L-Cit by BAEC may enhance NO synthesis. Studies are in progress to determine not only the effect of Ap 4A-induced uptake of L-Arg and L-Cit on NO synthesis but also whether Ap 4A induces the uptake of other amino acids. ACKNOWLEDGMENTS This research was supported in part by NSF MCB-9816681, USDA NRICG 96-35204-3669, and Greenville Hospital System/Clemson University Cooperative and South Carolina Experiment Station Grant SC01630. The authors thank Dr. James Zimmerman of Clemson University for critically evaluating the manuscript.
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