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    Flores et al 2002 l arginina Flores et al 2002 l arginina Document Transcript

    • Rapid Stimulation of L-Arginine Transport by D-Glucose Involves p42/44mapk and Nitric Oxide in Human Umbilical Vein Endothelium Carlos Flores, Susana Rojas, Claudio Aguayo, Jorge Parodi, Giovanni Mann, Jeremy D. Pearson, Paola Casanello, Luis SobreviaAbstract—D-Glucose infusion and gestational diabetes induce vasodilatation in humans and increase L-arginine transport and nitric oxide (NO) synthesis in human umbilical vein endothelial cells. High D-glucose (25 mmol/L, 2 minutes) induced membrane hyperpolarization and an increase of L-arginine transport (Vmax 6.1 0.7 versus 4.4 0.1 pmol/ g protein per minute) with no change in transport affinity (Km 105 9 versus 111 16 mol/L). L-[3H]Citrulline formation and intracellular cGMP, but not intracellular Ca2 , were increased by high D-glucose. The effects of D-glucose were mimicked by levcromakalim (ATP-sensitive K channel blocker), paralleled by p42/p44mapk and Ser1177– endothelial NO synthase phosphorylation, inhibited by NG-nitro-L-arginine methyl ester (L-NAME; NO synthesis inhibitor), gliben- clamide (ATP-sensitive K channel blocker), KT-5823 (protein kinase G inhibitor), PD-98059 (mitogen-activated protein kinase kinase 1/2 inhibitor), and wortmannin (phosphatidylinositol 3-kinase inhibitor), but they were unaffected by calphostin C (protein kinase C inhibitor). Elevated D-glucose did not alter superoxide dismutase activity. Our findings demonstrate that the human fetal endothelial L-arginine/NO signaling pathway is rapidly activated by elevated D-glucose via NO and p42/44mapk. This could be determinant in pathologies in which rapid fluctuations of plasma D-glucose may occur and may underlie the reported vasodilatation in early stages of diabetes mellitus. (Circ Res. 2003;92:64-72.) Key Words: humans endothelium glucose arginine nitric oxide tivated protein (MAP) kinases (p42/44mapk).5,14,15 p42/44mapkT he cationic amino acid L-arginine is the substrate for nitric oxide (NO) synthesis via endothelial NO synthase(eNOS)1 and is taken up primarily by the Na -independent activation may itself be dependent on PKC activation and NO synthesis.5,14 However, the effect of short-term incubationhigh-affinity (Km 100 to 400 mol/L) systems y /CAT-1 with elevated D-glucose on the endothelial L-arginine/NOand y /CAT-2B (where CAT indicates cationic amino acid pathway has not been investigated.4,11,16,17transporter) in human umbilical vein endothelial cells The present study shows that a 2-minute incubation with(HUVECs).2,3 L-Arginine transport and NO synthesis (L- 25 mmol/L D-glucose increases L-arginine transport and NOarginine/NO pathway) are increased in HUVECs from pa- synthesis in HUVECs. The underlying cellular mechanismstients with gestational diabetes.2 Interestingly, long-term involve phosphorylation of eNOS at Ser1177 via phosphatidyl-incubation (24 hours) of HUVECs from normal pregnancies inositol 3-kinase (PI3-k) and activation of eNOS and p42/with elevated D-glucose mimics the effect of gestational p44mapk by D-glucose.18diabetes on the L-arginine/NO pathway.4 In addition, elevatedD-glucose for 24 hours4,5 or 5 days6 increases eNOS geneexpression. A recent report shows that D-glucose infusion Materials and Methodsinduces vasodilatation in humans,7 and in animal models, anelevation of plasma D-glucose results in rapid (seconds to Cell Cultureminutes) vasodilatation.8 –10 Therefore, rapid fluctuations in Human umbilical vein endothelium was isolated (collagenase diges-the D-glucose level are crucial in maintaining human fetal tion 0.25 mg/mL) and cultured (37°C, 5% CO2, confluent passage 2) in medium 199 containing 5 mmol/L D-glucose, 10% newborn calfendothelial function.2–5,11 serum, 10% fetal calf serum, 3.2 mmol/L L-glutamine, 100 mol/L D-Glucose activates protein kinase C (PKC), an enzyme L-arginine, and 100 U/mL penicillin-streptomycin (primary cultureinvolved with long-term stimulation of the L-arginine/NO medium).2– 4 Before an experiment (24 hours), the incubation me-pathway,5,12–14 and (within 1 hour) p42 and p44 mitogen-ac- dium was changed to serum-free medium 199. Original received July 26, 2002; revision received October 31, 2002; accepted November 11, 2002. From the Cellular and Molecular Physiology Laboratory (C.F., S.R., C.A., J.P., P.C., L.S.), Department of Physiology, Faculty of Biological Sciences,and the Department of Obstetrics and Gynaecology (P.C.), Faculty of Medicine, University of Concepción, Concepción, Chile, and King’s CollegeLondon (G.M., J.D.P.), Guy’s Campus, London, UK. Correspondence to Dr L. Sobrevia, Cellular and Molecular Physiology Laboratory (CMPL), Department of Physiology, Faculty of Biological Sciences,University of Concepción, PO Box 160-C, Concepción, Chile. E-mail lsobrev@udec.cl © 2003 American Heart Association, Inc. Circulation Research is available at http://www.circresaha.org DOI: 10.1161/01.RES.0000048197.78764.D6 64
    • Flores et al D-Glucose Acutely Activates L-Arginine/NO Pathway 65L-Arginine Transport Intracellular Ca2L-Arginine transport (1 Ci/mL, 37°C, 1 minute) was determined in Cells on glass coverslips were loaded (30 minutes, 23°C) with thecells preincubated (15 seconds to 5 minutes) with Krebs solution acetoxymethyl derivative of fluo 3 (5 mol/L). Coverslips were(mmol/L: NaCl 131, KCl 5.6, NaHCO3 25, NaH2PO4 1, HEPES 20, transferred to an experimental bath with Krebs solution containing 5CaCl2 2.5, and MgCl2 1 [pH 7.4, 37°C]) containing 5 or 25 mmol/L or 25 mmol/L D-glucose, and Ca2 was imaged using a Zeiss LSMD-glucose, 25 mmol/L L-glucose, or 5 mmol/L D-glucose plus 410 confocal microscope.420 mmol/L D-mannitol (osmotic controls).2– 4 L-Arginine transportwas also determined in Krebs solution in which NaCl was replaced Western Blotsby equimolar concentrations of choline chloride2– 4 or in cells After pretreatment with 10 mol/L PD-98059 (30 minutes), 100incubated (30 minutes) with KCl (5.5 to 131 mmol/L), with NaCl mol/L SNAP (2 to 5 minutes), or 30 nmol/L wortmannin (30decreased equivalently, or with 131 mmol/L KCl for 2, 4, 10, 20, or minutes), the cells were incubated with 5 or 25 mmol/L D-glucose (230 minutes. In trans-stimulation experiments, cells were preincu- minutes). Cell protein extracts were probed with a primary poly-bated (2 hours) with primary culture medium containing 10 mmol/L clonal mouse antiphosphorylated (1:1000) or nonphosphorylatedL-lysine. Cell-associated radioactivity and data analyses were per- (1:1500) p44/p42mapk, rabbit anti-eNOS (1:2500) or anti-phosphory-formed as described.2– 4 lated Ser1177– eNOS (1:2500) antibodies, and horseradish peroxidase– conjugated goat secondary antibodies as described.3,5 Primary poly- L-Arginine transport was assayed in cells preincubated (30 min- clonal mouse anti-actin (1:2000) served as the internal control.utes) with 100 mol/L NG-nitro-L-arginine methyl ester (L-NAME, Proteins were detected by enhanced chemiluminescence and quan-eNOS inhibitor),2,3 10 mol/L PD-98059 (MAP kinase kinase 1/2 tified by densitometry (Ultroscan XL enhanced laser densitometer,[MEK1/2] inhibitor), 19 100 mol/L S-nitroso-N-acetyl- L , D - LKB Instruments).3,5penicillamine (SNAP, NO donor), 1 mol/L KT-5823 (proteinkinase G [PKG] inhibitor),20 100 nmol/L calphostin C (PKC inhib- Semiquantitative PCRitor),21 10 mol/L glibenclamide (ATP-sensitive K [K ATP] channel Extracted mRNA (Dynal) was reversed-transcribed into cDNA usingblocker),22 1 mol/L levcromakalim (K ATP activator),23 or 30 oligo(dT18) plus random hexamers (10-mer) and M-MLV reversenmol/L wortmannin (PI3-k inhibitor).24 transcriptase (Promega) for 1 hour at 37°C.3 Polymerase chain reactions (PCRs) were performed in 20- L samples (2 L of 10Membrane Potential PCR buffer, 0.8 L of 50 mmol/L Mg2 , 0.4 L dNTPs, 13.6 L[3H]Tetraphenylphosphonium ([3H]TPP ) influx (46 nmol/L, 0.5 RNase-free H2O, 0.2 L Taq DNA polymerase, and 0.5 mol/L Ci/mL, 15 to 120 seconds), a membrane potential–sensitive sequence-specific oligonucleotide primers for human CAT-1, CAT-probe,2,3,25 was determined in Krebs solution containing 5 or 2A, or CAT-2B). Samples were incubated (4 minutes, 95°C),25 mmol/L D-glucose, 100 mol/L L-arginine, and 5.5 or followed by 35 cycles of 30 seconds at 95°C, 30 seconds at 57°C, 30131 mmol/L KCl.2,3 Resting membrane potential (Em, whole-cell seconds at 72°C, and a final extension for 7 minutes at 72°C. -Actinpatch clamp) was recorded using an EPC-7 amplifier (List Medical) expression was used as a reference value. Reverse transcriptionas described.2 Em was measured for at least 1 minute in the presence (RT)-PCR products were sequenced in both directions by Taqof 5 or 25 mmol/L D-glucose. Only recordings with 0.1-mV dideoxy terminator cycle sequencing (automated DNA sequencervariations were considered. Em was also determined in cells prein- 373A, Applied Biosystems).3cubated for 2 hours with 10 mmol/L L-lysine. Oligonucleotide primers were as follows: hCAT-1 (sense) 5 -CCAGTACTTCCGACGAGTTAGA-3 , hCAT-1 (antisense) 5 -CATCCACACAGCAAACCGGACC-3 , hCAT-2A (sense) 5 -PKC Activity TATCCCGATTTTTTTGCTGTGTGC-3 , hCAT-2A (antisense)PKC activity (32P incorporation from [ -32P]ATP into a synthetic 5 -TGCAGTCAACGTGGCAGCAACT-3 , hCAT-2B (sense) 5 -PKC substrate peptide analogue5) was determined in cells exposed to TCCCAATGCCTCGTGTAATCTA-3 , hCAT-2B (antisense)5 or 25 mmol/L D-glucose (2 minutes) after pretreatment with 100 5 -GCATGCTGAAGCCCTGTCTCTGC-3 , -actin (sense) 5 -nmol/L phorbol 12-myristate 13-acetate (5 minutes, PKC activator), AACCGCGAGAAGATGACCCAGATCATCTTT-3 , and -actin100 nmol/L 4 -phorbol 12,13-didecanoate (5 minutes, less active (antisense) 5 -AGCAGCCGTGGCCATCTCTTGCTCGAAGTC-3 .phorbol 12-myristate 13-acetate analogue), 100 nmol/L calphostin C Expected size products were as follows: hCAT-1, 450 bp; hCAT-2A,(30 minutes), 10 mol/L PD-98059 (30 minutes), 100 mol/L 690 bp; hCAT-2B, 360 bp; and -actin, 350 bp.SNAP (5 minutes), 100 mol/L L-NAME (30 minutes), or 30nmol/L wortmannin (30 minutes). SOD Activity and -Tocopherol Experiments Cells were homogenized in buffer containing 50 mmol/L Tris-(hy-cGMP Determination droxymethyl)-aminomethane, 100 mmol/L potassium chloride,Cells preincubated (30 minutes) in Krebs solution (37°C) containing 0.02% Triton X-100, 100 mmol/L sodium pyrophosphate, andL -arginine (100 mol/L) and 3-isobutyl-1-methylxanthine 100 mmol/L sodium fluoride (pH 7.4), which was supplemented with(0.5 mmol/L, phosphodiesterase inhibitor),2,3 in the absence or trypsin inhibitors (4 mg/mL aprotinin, 1 mg/mL benzamidine, 5presence of L-NAME (100 mol/L), were exposed to 5 or g/mL leupeptin, and 200 mol/L sodium orthovanadate). Aliquots25 mmol/L D-glucose for the last 2 minutes of the 30-minute (1 mg protein/mL) were incubated (25°C, 2 minutes) with potassium phosphate buffer (50 mmol/L, pH 10.2) containing adrenochromeincubation period with 3-isobutyl-1-methylxanthine. cGMP was (200 mol/L) and epinephrine (10 mol/L), and absorbance wasdetermined in HCl-cell extracts by radioimmunoassay.2,3 measured at 480 nm. Superoxide dismutase (SOD) activity was calculated from the inhibition curve for epinephrine auto-oxidationL-Citrulline Assay versus protein concentration. Basal absorbance (100% activity) wasCells were incubated with L-[3H]arginine (100 mol/L, 4 Ci/mL, the reaction in the absence of cell extracts.26 Cells were also30 minutes, 37°C) in the absence or presence of L-NAME (100 preincubated (30 minutes) with -tocopherol (500 g/mL, 1% mol/L). Cells were exposed to 5 or 25 mmol/L D-glucose for 30 ethanol).27minutes or for the last 2, 4, 10, or 20 minutes of this incubationperiod. Some experiments were performed in cells exposed only to MaterialsL-[3H]arginine (4 Ci/mL) and 5 or 25 mmol/L D-glucose. Formic Sera, agarose, and buffers were from GIBCO Life Technologies.acid– digested cells (200 L) were passed through a sodium ion form Collagenase type II (Clostridium histolyticum) was from Boehring-of the cation ion-exchange resin Dowex 50W (50X8-200), and er-Mannheim, and Bradford protein reagent was from Bio-RadL-[3H]citrulline concentration was determined in the H2O eluate.3 Laboratories. L-NAME and SNAP were from Calbiochem. Ethidium
    • 66 Circulation Research January 10/24, 2003bromide, Dowex (50WX8-400), and all other reagents were fromSigma Chemical Co. L-[2,3-3H]Arginine (36.1 Ci/mmol), D-[1-14 C]mannitol (49.3 mCi/mmol), [ -32P]ATP, and [3H]TPP (37Ci/mmol) were from NEN. 3 ,5 -cGMP-TME was from ICN. Anti-bodies were from Cell Signaling, New England Biolabs.Statistical AnalysisValues are mean SEM, where n indicates the number of differentcell cultures (4 to 8 replicates per experiment). Statistical analyseswere carried out on raw data using the Peritz F multiple-meanscomparison test.28 A Student t test was applied for unpaired data, anda value of P 0.05 was considered statistically significant. ResultsL-Arginine TransportElevated D-glucose (2 minutes), but not L-glucose orD-mannitol, stimulated L-arginine transport (half-maximaleffect [K1/2] 13 2 mmol/L D-glucose) (Figure 1A). Basaltransport rates increased significantly after 30 seconds ofexposure to elevated D-glucose (K1/2 25 5 seconds), withmaximal rates achieved within 1 minute and sustained over 5minutes (Figure 1B). Subsequent experiments were per-formed using 25 mmol/L D-glucose for 2 minutes. D-Glu-cose–stimulated L-arginine transport decreased to basal val-ues within 5 minutes after reexposure of cells to 5 mmol/LD-glucose (Figure 1B). RT-PCR analysis detected onlyhCAT-1 and hCAT-2B mRNA in HUVECs (Figure 1C). Elevated D-glucose had no effect on the nonsaturablecomponent (KD) of overall L-arginine transport but increasedVmax, with no change in apparent Km (Figure 2A, Table 1).Cell incubation with L-lysine (10 mmol/L, 2 hours) increased6.5-fold the L-arginine transport in 5 mmol/L D-glucose(Figure 2B). However, L-arginine transport was increased2.9-fold in cells exposed to 25 mmol/L D-glucose (last 2minutes of the 2-hour incubation with L-lysine) (Figure 2B).D-Glucose stimulation and trans-stimulation by L-lysine ofL-arginine transport was unaltered in Na -free Krebs solution(not shown).TPP Influx and Membrane PotentialElevated D-glucose increased TPP influx (1.8-fold) andcaused membrane hyperpolarization (Table 2). TPP influxand L-arginine transport were inhibited by KCl-inducedmembrane depolarization. Glibenclamide (K ATP channelblocker) blocked D-glucose–increased L-arginine transport Figure 1. Effect of D-glucose on L-arginine transport and CATand changes in TPP influx and Em (Table 2). Levcromakalim expression. A, L-Arginine transport (100 mol/L, 1 minute, 37°C)(K ATP channel activator) hyperpolarized the plasma mem- in HUVECs incubated (2 minutes) with increasing concentrationsbrane and increased L-arginine transport and TPP influx only of D-glucose ( ), L-glucose ( ), or 5 mmol/L D-glucose plus 5 toin 5 mmol/L D-glucose; effects were blocked by gliben- 20 mmol/L D-mannitol (▫). *P 0.05 and **P 0.04 vs 5 or 7.5 mmol/L D-glucose and corresponding values in L-glucoseclamide (Table 2). The effects of D-glucose were also blocked and D-glucose D-mannitol. B, Time course of effect ofby wortmannin (not shown). Preloading cells with L-lysine D-glucose on L-arginine transport (as in panel A) in cells incu-did not alter Em ( 66.1 0.3 mV, P 0.05; n 29 cells) bated for 0 to 5 minutes in 5 mmol/L D-glucose ( ), 25 mmol/L D-glucose ( ), or 5 mmol/L D-glucose 20 mmol/L D-mannitol (▫).compared with control cells ( 67.5 0.5 mV, P 0.05; n 45 High D-glucose– containing Krebs solution was replaced (arrows)cells). L-Arginine transport was inhibited by KCl with half- by 5 mmol/L D-glucose, and cells were incubated for 10 min-maximal inhibition at 12 2 and 17 4 mmol/L KCl for 5 and utes. *P 0.04 vs all other values. Values are mean SEM (n 13). C, RT-PCR for mRNA from cells in 5 mmol/L D-glucose.25 mmol/L D-glucose, respectively. The time needed to mRNA was reversed-transcribed into cDNA, and PCR was per-induce half-maximal inhibition of transport with 131 mmol/L formed for human CAT-1 (lane 2, 449 bp), CAT-2A (lane 3, 690KCl was similar in cells in 5 mmol/L D-glucose (6.5 0.6 bp), or CAT-2B (lane 4, 357 bp). Lane 5 is -actin (350 bp), andminutes) compared with 25 mmol/L D-glucose (7.2 0.6 lane 1 is DNA ladder (100 to 2000 bp). Data are representative of 14 cell cultures.minutes).
    • Flores et al D-Glucose Acutely Activates L-Arginine/NO Pathway 67 lation at Ser1177, cGMP, and L-[3H]citrulline formation. Sim- ilar results were found in cells exposed to 25 mmol/L D-glucose for the last 4, 10, or 20 minutes of the 30-minute incubation period with L-[3H]arginine (not shown). Intracel- lular Ca2 in cells incubated with 25 mmol/L D-glucose for 2 minutes (42 7 nmol/L) was not statistically different (P 0.05, n 125 cells) from values in cells incubated with 5 mmol/L D-glucose (35 5 nmol/L). L-NAME also inhibited the effect of D-glucose on L-arginine transport Vmax (Table 1), TPP influx, and Em (Table 2); however, L-NAME did not alter TPP influx or Em in 5 mmol/L D-glucose. Other experiments show that SNAP (NO donor) induces TPP influx, L-arginine transport, and membrane hyperpolarization only in 5 mmol/L D-glucose (Table 2) and that dibutyryl cGMP (dbcGMP) increased L-arginine transport (Figure 4A) and TPP influx (Figure 4B). The effects of D-glucose and dbcGMP were blocked by KT-5823, a PKG inhibitor (Table 2). PKC and MAP Kinase Involvement PKC activity was unaltered at up to 5 minutes of incubation with high D-glucose (Figure 5A) or after the addition of SNAP (not shown). Furthermore, calphostin C (PKC inhibi- tor) had no effect on D-glucose–increased L-arginine transport (Figure 5B), TPP influx, or NO synthesis (not shown). However, longer incubation with elevated D-glucose ( 10Figure 2. Effect of D-glucose on kinetic parameters and trans- minutes) increased membrane PKC activity (not shown),stimulation of L-arginine transport. A, Saturable L-arginine trans- confirming our previous observations in HUVECs.5 In con-port (1 minute, 37°C) in HUVECs incubated (2 minutes) in trast, high D-glucose induced p42/p44mapk phosphorylation5 mmol/L D-glucose ( ) or 25 mmol/L D-glucose ( ). B,L-Arginine transport (100 mol/L, 1 minute, 37°C) in cells prein- (Figure 6A), an effect blocked by PD-98059 and mimickedcubated (2 hours) in medium 199 in the absence (control) or by brief exposure (2 minutes) to SNAP (Figure 6B). Inter-presence of 10 mmol/L L-lysine. Transport assays were per- estingly, the effect of D-glucose on p42/44mapk phosphoryla-formed in cells exposed to 5 mmol/L D-glucose (open bars) or tion was blocked by wortmannin (Figure 6C). PD-98059 also25 mmol/L D-glucose (filled bars) for the last 2 minutes of the2-hour incubation period with L-lysine. Values are mean SEM blocked the stimulatory effects of elevated D-glucose and(n 16). *P 0.04 vs all other values. SNAP on TPP influx, L-arginine transport, and Em (Table 2).NO Involvement SOD Activity and -Tocopherol EffectElevated D-glucose increased eNOS phosphorylation at SOD activity in cells in 5 mmol/L D-glucose (5.5 0.6 U/mL)Ser1177 (Figure 3A), L-[3H]citrulline (Figure 3B), and cGMP was not significantly altered (P 0.05, n 5) by 25 mmol/Laccumulation (Figure 3C). L-NAME inhibited the effect of D-glucose (6 1 U/mL). Extracellular SOD or -tocopherol 3D-glucose on cGMP and L-[ H]citrulline formation but did not did not block (P 0.05, n 4 to 8) the effect of D-glucose onalter eNOS-Ser1177 phosphorylation (not shown). However, L-arginine transport (5.6 0.3 and 5.8 0.6 pmol/ g proteinwortmannin inhibited D-glucose–induced eNOS phosphory- per minute, respectively), L-citrulline formation (3.1 0.2 and TABLE 1. D-Glucose Effect on L-Arginine Transport in HUVECs Vmax /Km, pmol/ g KD, pmol/ g Vmax, pmol/ g Protein per Minute Protein per Minute Km, mol/L Protein per Minute per mol/L per mol/L 5 mmol/L D-Glucose 105 9 4.4 0.1 0.044 0.011 0.002 0.0007 PD-98059 123 12 4.2 0.2 0.034 0.012 0.003 0.0012 L-NAME 97 16 3.8 0.3 0.039 0.016 0.003 0.0009 25 mmol/L D-Glucose 111 16 6.1 0.7* 0.055 0.012* 0.003 0.0011 PD-98059 133 22 3.7 0.3† 0.028 0.009† 0.004 0.0012 L-NAME 125 22 3.6 0.5† 0.029 0.007† 0.003 0.0008 L-Arginine transport (100 mol/L, 37°C) in cells preincubated (30 minutes) with 5 mmol/L D-glucose, in absence or presence of 100 mol/L PD-98059 or 100 mol/L L-NAME, and exposed (2 minutes) to Krebs containing 5 or 25 mmol/L D-glucose. Values are mean SEM, n 6. *P 0.05, †P 0.05 vs 5 and 25 mmol/L D-glucose, respectively.
    • 68 Circulation Research January 10/24, 2003 TABLE 2. D-Glucose Effect on L-Arginine Transport, [3H]TPP Influx, and Em in HUVECs L-Arginine Transport, pmol/ g [3H]TPP Influx, pmol/mg Protein per Minute Protein per Minute Em, mV 5 mmol/L D-Glucose Control 1.8 0.3 2.1 0.3 67.2 0.5 KCl 0.2 0.1* 0.1 0.04* 8.1 0.5* Glibenclamide 1.3 0.3 1.5 0.3 63.9 0.6 Levcromakalim 4.6 0.3* 5.6 0.3* 74.2 0.3* Levcromakalim glibenclamide 1.6 0.2† 1.4 0.6† 64.5 0.2† PD-98059 2.1 0.1 1.7 0.2 64.2 0.5 L-NAME 1.6 0.1 1.5 0.3 65.1 0.3 SNAP 5.1 0.3* 3.9 0.4* 76.2 1.0* SNAP PD-98059 2.1 0.3† 1.9 0.5† 65.3 1.0† KT-5823 1.9 0.5 1.9 0.3 66.8 0.9 25 mmol/L D-Glucose Control 4.0 0.5* 3.7 0.3* 77.1 0.2* KCl 0.3 0.2*‡ 0.3 0.03*‡ 6.2 0.5*‡ Glibenclamide 2.3 0.4‡ 2.4 0.4‡ 62.7 0.5‡ Levcromakalim 4.3 0.4* 4.7 0.2* 75.1 0.3* Levcromakalim glibenclamide 2.2 0.3†‡ 1.8 0.3†‡ 61.9 0.3†‡ PD-98059 0.9 0.3‡ 1.7 0.1‡ 69.9 1.0‡ L-NAME 1.4 0.1‡ 1.8 0.2‡ 65.7 0.7‡ SNAP 4.1 0.4* 3.2 0.2* 78.2 1.0* SNAP PD-98059 1.1 0.5†‡ 1.9 0.2†‡ 65.7 1.0†‡ KT-5823 1.9 0.5‡ 1.9 0.3‡ 65.3 0.5‡ L-Arginine transport (100 mol/L), 3H TPP influx (46 nmol/L), and Em (whole-cell patch clamp) in cells preincubated (30 minutes) with 5 mmol/L D-glucose and 5.5 mmol/L (control) or 131 mmol/L KCl. Cells in 5.5 mmol/L KCl were preincubated with 10 mol/L glibenclamide (5 minutes), 1 mol/L levcromakalim (5 minutes), 10 mol/L PD-98059 (30 minutes), 100 mol/L L-NAME (30 minutes), 10 mol/L KT-5823 (30 minutes), or 100 mol/L SNAP (2 minutes) and exposed (2 minutes) to 5 or 25 mmol/L D-glucose. Values are mean SEM, n 17. *P 0.05 vs control in 5 mmol/L D-glucose; †P 0.05 vs corresponding values in SNAP or levcromakalim; ‡P 0.05 vs control in 25 mmol/L D-glucose.2.8 0.4 pmol/ g protein per 30 minutes, respectively), TPP confirmed here, is Na independent and inhibited by mem-influx (4.4 0.2 and 4.1 0.3 pmol/mg protein per minute, brane depolarization.2,3,32 Because of their similar kineticrespectively), or changes in Em ( 76 0.3 and 78 0.3 mV, properties, CAT-1 and CAT-2B are hard to distinguish at therespectively). functional level.31 Our results show that both high-affinity hCAT-1 (Km 100 to 200 mol/L) and hCAT-2B (Km 200 Discussion to 400 mol/L), but not the low-affinity hCAT-2A trans-The present study establishes that D-glucose induces a rapid porter (Km 2 to 5 mmol/L), are present in HUVECs,concentration-dependent stimulation of L-arginine transport confirming previous reports.3,33 CAT-1 is more sensitive thanin HUVECs. This effect requires NO synthesis associated CAT-2B to trans-stimulation by cationic amino acids.3,31,34with increased phosphorylation of eNOS at Ser1177 and acti- When we preloaded HUVECs with L-lysine, L-arginine trans-vation of p42/p44mapk and PI3-k, and it is independent of PKC port was increased by 7-fold in 5 mmol/L D-glucose.and intracellular Ca2 changes. These findings provide the However, the L-lysine trans-stimulatory effect was less ef-first evidence that short-term hyperglycemia activates the fective ( 3-fold) in cells exposed for 2 minutes to 25 mmol/LL -arginine/NO signaling pathway in human fetal D-glucose. Because L-arginine transport is trans-stimulatedendothelium. by 9.8-fold or 1.8-fold in Xenopus oocytes injected with L-Arginine transport is mediated by systems y /CATs,2,3,12 hCAT-1 or hCAT-2B mRNA, respectively, 34 trans-y L,29,30 and b0, 12 in HUVECs, with the first likely predom- stimulation in HUVECs in 5 mmol/L D-glucose may beinating at the physiological concentration of extracellular preferentially mediated by hCAT-1. The reduced trans-L-arginine. The cDNAs for four potential human y trans- stimulation of transport in high D-glucose may result from aporters (hCAT-1, hCAT-2B, hCAT-2A, and hCAT-4) have state of maximal activity of L-arginine transporters alreadybeen sequenced.31 L-Arginine transport in HUVECs occurs induced by L-lysine; therefore, high D-glucose could notwith relatively high affinity (Km 80 to 100 mol/L) and, as further increase L-arginine transport. In addition, the possi-
    • Flores et al D-Glucose Acutely Activates L-Arginine/NO Pathway 69 Figure 4. cGMP involvement in the effect of D-glucose on L-arginine transport and [3H]TPP influx. HUVECs preincubated (30 minutes) with 5 mmol/L D-glucose in the absence (control) or presence of KT-5823 or dbcGMP were exposed (2 minutes) to 5 mmol/L D-glucose (open bars) or 25 mmol/L D-glucose (filled bars) in the presence of KT-5823 and/or dbcGMP, and L-arginine transport (100 mol/L, 1 minute, 37°C) (A) and [3H]TPP influx (46 nmol/L, 1 minute, 37°C) (B) were determined. Values are mean SEM (n 12). *P 0.05 vs all other values. Vmax for L-arginine transport without altering the apparent Km. As noted above, L-arginine transport is sensitive to changes in extracellular K and Em. Because high D-glucose induced membrane hyperpolarization, stimulation of L-arginine trans- port could result from changes in Em. The stimulatory effectFigure 3. Effect of D-glucose on eNOS activity. A, Immunoblot of D-glucose on TPP influx and L-arginine transport wasfor total eNOS and phosphorylated eNOS at Ser1177 (eNOS P- blocked by glibenclamide, a K ATP channel blocker. In addi-Ser1177) in HUVECs preincubated (30 minutes) with 5 mmol/LD-glucose in the absence or presence of wortmannin and then tion, levcromakalim (K ATP activator)23 mimics D-glucose–exposed (2 minutes) to 5 or 25 mmol/L D-glucose (see Materials induced changes in Em, L-arginine transport, and TPP influx.and Methods). Top panel shows densitometry ratios. Data are K ATP channels are expressed in the endothelium35 and arerepresentative of 6 cell cultures. B, L-[3H]Citrulline formationfrom L-[3H]arginine (4 Ci/mL, 37°C) in cells preincubated (30 activated by D-glucose36; thus, the effects of D-glucose mayminutes) with Krebs solution in the absence (control) or pres- involve changes in the activity of glibenclamide-sensitiveence of L-NAME and exposed for the last 2 minutes to Krebs K ATP channels.solution containing 5 mmol/L D-glucose (open bars) or D-Glucose (24 hours) also increases eNOS expression4 and25 mmol/L D-glucose (filled bars). C, Intracellular cGMP undersame experimental conditions as in panel B. Values are activity2 in HUVECs. In the present study, eNOS activity wasmean SEM (n 17). *P 0.05 vs all other values. increased in HUVECs exposed for 1 to 5 minutes to high D-glucose, an effect associated with increased phosphoryla-bility that L-lysine–stimulated L-arginine transport was due to tion of eNOS at Ser1177, a residue known to be associated withmembrane hyperpolarization is unlikely because Em was eNOS activation.37 The rapid eNOS stimulatory effect ofunaltered in L-lysine–preloaded cells. D-glucose was not further increased by longer incubations Long-term incubation (24 hours) of HUVECs with high periods with D-glucose, which could be due to a maximal andD-glucose increases Vmax for L-arginine transport.4 The pres- sustained activation of eNOS acutely induced by elevatedent study shows that acute (2-minute) D-glucose increases D-glucose. Because D-glucose did not alter basal intracellular
    • 70 Circulation Research January 10/24, 2003Figure 5. Lack of effect of calphostin C in the effect ofD-glucose on L-arginine transport. A, PKC activity in cytosolic(open symbols) and membrane (filled symbols) fractions fromHUVECs preincubated (30 minutes) with Krebs solution without( , ) or with 100 nmol/L calphostin C (▫, ). Cells were incu-bated (30 seconds) in 25 mmol/L D-glucose in the absence orpresence of calphostin C. B, Time-course effect of 25 mmol/LD-glucose on L-arginine transport (100 mol/L, 1 minute, 37°C)(as in panel A) in Krebs solution without ( ) or with 100 nmol/Lcalphostin C ( ). Values are mean SEM (n 18). *P 0.05 vs allother values.Ca2 , it is likely that rapid eNOS activation by D-glucose isCa2 independent, supporting recent observations of Ca2 - Figure 6. NO and PI3-k involvement in effect of D-glucose onindependent eNOS activation in HUVECs.37,38 D-Glucose– p42/44mapk phosphorylation. A, Immunoblot for phosphorylated p44mapk (p44 P) and p42mapk (p42 P) and nonphosphorylatedinduced phosphorylation of eNOS at Ser1177, L-arginine trans- p44mapk (p44) or p42mapk (p42) in HUVECs preincubated (30 min-port, and TPP influx were blocked by wortmannin, utes) with 5 mmol/L D-glucose in the absence or presence ofsuggesting that the PI3-k pathway could be involved in the PD-98059 and then exposed (2 minutes) to 5 or 25 mmol/L D-glucose. B, Effect of SNAP on p42/p42mapk in HUVECs ineffects of D-glucose. L-Arginine transport could be determi- 5 mmol/L D-glucose. C, Effect of wortmannin on p42/p42mapk innant for eNOS activity11; however, the possibility that the HUVECs (as in panel A). Data are representative of similarD-glucose–induced increase of L-citrulline production was results in 17 cell cultures.due to elevated L-arginine transport seems unlikely, inasmuchas D-glucose–induced NO synthesis was unaltered in the synthase.2 NO causes membrane hyperpolarization in theabsence of extracellular L-arginine. endothelium,3,36 and NO (from SNAP) has been shown to L-NAME blocked D-glucose–increased L-arginine trans- cause comparable increases in TPP influx and L-arginineport and NO synthesis in HUVECs. This inhibitor does not transport and to cause membrane hyperpolarization to thosealter basal L-arginine transport in the endothelium2– 4,39; thus, caused by D-glucose, although SNAP treatment did notNO most likely mediates changes in L-arginine transport, as further enhance the effects of high D-glucose, in HUVECs.suggested in bovine aortic endothelium.40 This result is These findings support the hypothesis that NO acutely mod-similar to that found in HUVECs from patients with gesta- ulates L-arginine transport by a mechanism that involvestional diabetes; L-arginine transport in these cells is increased membrane hyperpolarization.concomitantly with membrane hyperpolarization and NO NO-altered K channel activity may occur by both indirectsynthesis, and this increase is inhibited by blocking NO mechanisms via cGMP and direct NO action on channels.36
    • Flores et al D-Glucose Acutely Activates L-Arginine/NO Pathway 71Our results show that the D-glucose increases in L-arginine fellowships. We thank the midwives of the Hospital Regional-transport and TPP influx were mimicked by dbcGMP and Concepción (Chile) labor ward for the supply of umbilical cords and Isabel Jara for secretarial assistance.blocked by the PKG inhibitor KT-5823. In addition, D-glucose–induced membrane hyperpolarization was alsoblocked by KT-5823. Thus, modulation of ion channel References 1. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, patho-activity (and hence, L-arginine transport) could be due to the physiology and pharmacology. Pharmacol Rev. 1991;43:109 –142.activation of PKG downstream from NO synthesis. 2. Sobrevia L, Cesare P, Yudilevich DL, Mann GE. Diabetes-induced acti- PKC activity is increased in subjects with diabetes mellitus vation of system y and nitric oxide synthase in human endothelial cells association with membrane hyperpolarization. J Physiol (Lond). 1995;or in endothelium chronically exposed to high 489:183–192.D-glucose.12,16,17 Activation of diacylglycerol/phorbol ester– 3. Casanello P, Sobrevia L. Intrauterine growth retardation is associatedsensitive PKC isozymes activates eNOS and the NO- with reduced activity and expression of the cationic amino acid transportdependent increased p42/p44 mapk phosphorylation in systems y /hCAT-1 and y /hCAT-2B, and lower activity of nitric oxide synthase in human umbilical vein endothelial cells. Circ Res. 2002;91:HUVECs exposed for 24 hours to high D-glucose.5 However, 127–134.25 mmol/L D-glucose for 1 to 5 minutes did not alter PKC 4. Sobrevia L, Nadal A, Yudilevich DL, Mann GE. Activation of L-arginineactivity in this cell type, suggesting that the rapid D-glucose transport (system y ) and nitric oxide synthase by elevated glucose andeffect on L-arginine transport was PKC independent. Because insulin in human endothelial cells. J Physiol (Lond). 1996;490:775–781. mapk 5. Montecinos VP, Aguayo C, Flores C, Wyatt AW, Pearson JD, Mann GE,D-glucose induces a rapid (2-minute) p42/p44 phosphor- Sobrevia L. Regulation of adenosine transport by D-glucose in humanylation and because inhibition of the p42/p44mapk phosphory- fetal endothelial cells: involvement of nitric oxide, protein kinase C andlation by PD-98059 also inhibits the D-glucose increase in mitogen-activated protein kinase. J Physiol (Lond). 2000;529:777–790. 6. Cosentino F, Hishikawa K, Katusic ZS, Lüscher TF. High glucoseTPP influx and L-arginine transport, it is likely that p42/ increases nitric oxide synthase expression and superoxide anion gen-p44mapk activation is involved in this pathway. Activation of eration in human aortic endothelial cells. Circulation. 1997;96:25–28.p42/44mapk requires PI3-k activity in HUVECs.41 Our results 7. Beckman JA, Goldfine AB, Gordon MB, Garrett LA, Creager MA.show that D-glucose–induced p42/44mapk phosphorylation is Inhibition of protein kinase C prevents impaired endothelium-dependent vasodilation caused by hyperglycemia in humans. Circ Res. 2002;90:blocked by wortmannin, suggesting that the D-glucose effect 107–111.requires PI3-k activity in HUVECs. SNAP-increased p42/ 8. Duckrow RB. Decreased cerebral blood flow during acute hypergly-p44mapk phosphorylation and L-arginine transport were caemia. Brain Res. 1995;703:145–150.blocked by PD-98059, complementing results showing that 9. Wascher TC, Bachernegg M, Kickenweiz A, Stark G, Stark U, Toplak H, Graier WF. Involvement of the L-arginine-nitric oxide pathway inNO, via cGMP, causes rapid p42/p44mapk phosphorylation in hyperglycaemia-induced coronary artery dysfunction of isolated guineathe endothelium.4,42 Because D-glucose–induced and NO- pig hearts. Eur J Clin Invest. 1996;26:707–712.induced membrane hyperpolarization are blocked by PD- 10. Cipolla MJ, Porter JM, Osol G. High glucose concentration dilate cerebral98059, p42/p44mapk activation could modulate ion channel arteries and diminish myogenic tone through an endothelial mechanism. Stroke. 1997;28:405– 411.activity and L-arginine transport in HUVECs. 11. Sobrevia L, Mann GE. Dysfunction of the nitric oxide signalling pathway Elevated D-glucose leads to overproduction of oxygen- in diabetes and hyperglycaemia. Exp Physiol. 1997;82:1–30.derived free radicals in several cell types.11,16,17 We found 12. Pan M, Wasa M, Lind DS, Gertler J Abbott W, Souba WW. TNF-that SOD activity in HUVECs was unaltered by 25 mmol/L stimulated arginine transport by human vascular endothelium requires activation of protein kinase C. Ann Surg. 1995;221:590 – 601.D-glucose and that SOD or -tocopherol did not block the 13. Racké K, Hey C, Mössner J, Hammermann R, Stichnote C, Wessler I.effects of D-glucose, suggesting that short-term incubation Activation of L-arginine transport by protein kinase C in rabbit, rat andwith elevated D-glucose would not generate enough oxygen- mouse alveolar macrophages. J Physiol (Lond). 1998;551:813– 825. 14. Haneda M, Araki S, Togawa M, Sugimoto T, Isono M, Kikkawa R.derived free radicals to induce changes in the L-arginine/NO Mitogen-activated protein kinase cascade is activated in glomeruli ofpathway in HUVECs. diabetic rats and glomerular mesangial cells cultured under high glucose The present study has established that high D-glucose conditions. 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