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. 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