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. 2004 Jul 22;560(Pt 1):111–122. doi: 10.1113/jphysiol.2004.068288

Role of adenosine transport in gestational diabetes-induced l-arginine transport and nitric oxide synthesis in human umbilical vein endothelium

Gustavo Vásquez 1, Felipe Sanhueza 1, Rodrigo Vásquez 1, Marcelo González 1, Rody San Martín 1, Paola Casanello 1,2, Luis Sobrevia 1
PMCID: PMC1665196  PMID: 15272035

Abstract

Gestational diabetes is associated with increased l-arginine transport and nitric oxide (NO) synthesis, and reduced adenosine transport in human umbilical vein endothelial cells (HUVEC). Adenosine increases endothelial l-arginine/NO pathway via A2 purinoceptors in HUVEC from normal pregnancies. It is unknown whether the effect of gestational diabetes is associated with activation of these purinoceptors or altered expression of human cationic amino acid transporter 1 (hCAT-1) or human equilibrative nucleoside transporter 1 (hENT1), or endothelial NO synthase (eNOS) in HUVEC. Cells were isolated from normal or gestational diabetic pregnancies and cultured up to passage 2. Gestational diabetes increased hCAT-1 mRNA expression (2.4-fold) and activity, eNOS mRNA (2.3-fold), protein level (2.1-fold), and phosphorylation (3.8-fold), but reduced hENT1 mRNA expression (32%) and activity. Gestational diabetes increased extracellular adenosine (2.7 μm), and intracellular l-arginine (1.9 mm) and l-citrulline (0.7 mm) levels compared with normal cells (0.05 μm, 0.89 mm, 0.35 mm, respectively). Incubation of HUVEC from normal pregnancies with 1 μm nitrobenzylthioinosine (NBMPR) mimicked the effect of gestational diabetes, but NBMPR was ineffective in diabetic cells. Gestational diabetes and NBMPR effects involved eNOS, PKC and p42/44mapk activation, and were blocked by the A2a purinoceptor antagonist ZM-241385. Thus, gestational diabetes increases the l-arginine/NO pathway involving activation of mitogen-activated protein (MAP) kinases, protein kinase C (PKC) and NO cell signalling cascades following activation of A2a purinoceptors by extracellular adenosine. A functional relationship is proposed between adenosine transport and modulation of l-arginine transport and NO synthesis in HUVEC, which could be determinant in regulating vascular reactivity in diabetes mellitus.

Gestational diabetes is associated with endothelial dysfunction (Sobrevia & Mann, 1997; Michiels, 2003). The vasodilator nitric oxide (NO) is generated by the endothelium from l-arginine via the constitutive, Ca2+/calmodulin-dependent NO synthase (eNOS) (Moncada et al. 1991). NO synthesis is increased (Smulders et al. 1994) or reduced (Calver et al. 1992; Elliott et al. 1993) in diabetic subjects, and NO synthesis and membrane transport of l-arginine are increased in human umbilical vein endothelial cells (HUVEC) from gestational diabetic pregnancies (Sobrevia et al. 1995), or in HUVEC exposed to elevated extracellular d-glucose (Flores et al. 2003). The cell signalling mechanisms involved in the acute stimulation of l-arginine transport by high d-glucose include activation of eNOS and the 44 kDa and 42 kDa mitogen-activated protein (MAP) kinases (p42/44mapk) in HUVEC (Flores et al. 2003). However, the mechanisms involved in the modulation of the human endothelial l-arginine/NO pathway by gestational diabetes have not been investigated (Sobrevia & Mann, 1997; Michiels, 2003; Fleming & Busse, 2003).

Adenosine transport is reduced in HUVEC from gestational diabetic pregnancies (Sobrevia et al. 1994). Adenosine acts as a vasodilator in several vascular beds (Sobrevia & Mann, 1997), and a reduced removal of extracellular adenosine could result in increased extracellular adenosine concentrations altering its several biological effects (Griffith & Jarvis, 1996; van Mil et al. 2002; Dinenno, 2003). Adenosine is removed from the extracellular space by the equilibrative nucleoside transporters 1 (ENT1) in HUVEC (Montecinos et al. 2000; Parodi et al. 2002); however, the involvement of this membrane transporter, and its regulation, in gestational diabetic endothelium has not been reported (Baldwin et al. 2004).

We and others have suggested that adenosine stimulates the l-arginine/NO pathway via activation of A2a purinoceptors in HUVEC from normal pregnancies (Sobrevia et al. 1997; Wyatt et al. 2002; Sohn et al. 2003). However, nothing is known regarding the effect of adenosine in HUVEC from gestational diabetic pregnancies (Michiels, 2003; Baldwin et al. 2004). We have studied whether inhibition of adenosine transport is linked to gestational diabetic-increased l-arginine/NO pathway in HUVEC, and whether A2 purinoceptors are involved in this effect of adenosine. Our results show that gestational diabetes increased the human endothelial l-arginine/NO pathway and reduced adenosine transport associated with higher expression of the human cationic amino acid transporter 1 (hCAT-1) mRNA, and eNOS, and with lower expression of hENT1 mRNA, respectively. Gestational diabetes increases extracellular adenosine level and activates A2a purinoceptors stimulating the l-arginine/NO pathway. This effect of gestational diabetes involves eNOS, protein kinase C (PKC) and p42/44mapk activation.

Methods

Gestational diabetic patients and newborns

Umbilical cords were collected after delivery from full-term normal or gestational diabetic pregnancies (Ethics Committee approval and patient informed consent were obtained). Patients were normotensive and exhibited elevated glycosylated haemoglobin A1c (Table 1). Patients with basal glycaemia <90 mg dl−1 (i.e. overnight starvation) and >140 mg dl−1 at 2 h after an oral glucose load (75 g) were diagnosed as having gestational diabetes. Umbilical cord vein blood d-glucose concentration was similar in normal or gestational diabetic subjects, but newborns from diabetic pregnancies exhibited higher birth weight and ponderal index compared with normal pregnancies (Table 1).

Table 1.

Clinical characteristics of patients and newborns

Normal Gestational diabetes
Maternal age (years) 32 ± 2 (27–37)    29.9 ± 2.8 (23–34)    
Maternal height (cm) 155 ± 8 (149–167)   156 ± 7 (151–166)   
Gestational age (weeks) 38.1 ± 0.3 (37.6–38.5) 38.2 ± 0.3 (37.7–38.6) 
Maternal systolic pressure (mmHg) 112 ± 5 (108–114)   109 ± 3 (105–113)   
Haemoglobin A1c (%) 3.4 ± 0.5 (3.1–4.1)  9.8 ± 0.3* (7.9–11.5)
Glycaemia basal (mg dl−1) 96 ± 5 (91–101)   78 ± 4* (71–87)   
Glycaemia 2 h post glucose (mg dl−1) 107 ± 5 (97–113)    157 ± 9* (141–167)  
Fetal sex (male/female) 14/14 13/15
Birth weight (g) 3432 ± 56 (3088–3670) 4841 ± 67* (4201–5334)
Ponderal index (g cm−3× 100) 3.11 ± 0.2 (2.97–3.15) 3.87 ± 0.2* (3.61–4.01)
Umbilical vein d-glucose (mm) 2.5 ± 0.2 (2.3–2.7)  2.9 ± 0.4 (2.3–3.5)  
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Glycaemia was determined at basal conditions (overnight starvation) and 2 h after an oral load with glucose as described in Methods. Mean ± s.e.m. (range) or numbers.

*

P < 0.05 versus normal. (n = 28).

Cell culture

Endothelium isolated by collagenase (0.25 mg ml−1) digestion from human umbilical veins (HUVEC) was cultured (37°C, 5% CO2) in medium 199 (M199) containing 5 mmd-glucose, 10% new born calf serum, 10% fetal calf serum, 3.2 mml-glutamine, 100 μml-arginine, and 100 U ml−1 penicillin–streptomycin (primary culture medium). Cells were grown for 8–10 days to reach passage 2 confluent cultures when experiments were performed. Twenty-four hours before an experiment, the incubation medium was changed to sera-free M199 (Casanello & Sobrevia, 2002; González et al. 2004). In a separate set of experiments, HUVEC were cultured for 6 h (primary culture) in sera-free culture medium, and adenosine and l-arginine transport, and NO synthesis were measured (see below). The effect of gestational diabetes on membrane transport of adenosine and l-arginine, and NO synthesis were similar to experiments performed on cells in passage 2 (not shown).

Transport assays

l-Arginine transport (100 μm, 1 min, 37°C) was assayed in Krebs containing (mm): NaCl, 131; KCl, 5.6; NaHCO3, 25; NaH2PO4, 1; d-glucose, 5; Hepes, 20; CaCl2, 2.5; MgCl2, 1 (pH 7.4, 37°C), and 2 μCi ml−1l-[3H]arginine as described (Casanello & Sobrevia, 2002; González et al. 2004). Total adenosine transport (10 μm, 20 s, 37°C) was determined in Krebs containing 4 μCi ml−1[3H]adenosine, in the absence or presence (30 min) of the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR, 1 μm) (Sobrevia et al. 1994). The difference between total transport and transport in the presence of 1 μm NBMPR was considered ENT1 (NBMPR-sensitive)-mediated transport (Griffith & Jarvis, 1996; Baldwin et al. 2004). Cell monolayers were rinsed (200 μl well−1 ice-cold PBS) and radioactivity determined as described (Casanello & Sobrevia, 2002; González et al. 2004). Data were analysed using the computer programs Enzfitter and Ultra Fit (Elsevier, Biosoft).

Transport was assayed in cells pre-incubated (30 min) with NG-nitro-l-arginine methyl ester (l-NAME, 100 μm, eNOS inhibitor), PD-98059 [10 μm, MAP kinase kinase 1/2 (MEK1/2) inhibitor], phorbol 12-myristate 13-acetate (PMA, 100 nm, PKC activator), calphostin C (100 nm, PKC inhibitor), 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine hydrochloride (CGS-21680, 100 nm, 2 min, A2 purinoceptor agonist), 4-(2-[7-amino-2-[furyl][1,2,4]triazolo[2,3-alpha][1,3,5,]triazin-5-yl amino]ethyl)phenol (ZM-241385, 100 nm, A2a purinoceptor selective antagonist) (Sobrevia et al. 1997; Preston et al. 2004), N6-cyclopentyladenosine (CPA, 100 nm, 2 min, A1 purinoceptor agonist) or 8-cyclopentyl-1,3-dipropilxanthine (DPCPX, 100 nm, A1 purinoceptor antagonist).

PKC activity

PKC activity was estimated by 32P incorporation from [γ-32P]ATP into a synthetic PKC substrate peptide analogue in membrane or cytosol fractions isolated from endothelial cells (Flores et al. 2003). Cells were exposed (30 min) to 1 μm NBMPR, calphostin C, ZM-241385, PD-98059 or l-NAME.

l-[3H]Citrulline assay

Cells were incubated with Krebs containing 100 μml-arginine and 4 μCi ml−1l-[3H]arginine (30 min, 37°C) in the absence or presence of 1 μm NBMPR, ZM-241385 or l-NAME. Digested cells (95% formic acid) were passed through a cation ion-exchange resin Dowex-50W (50 × 8-200) and l-[3H]citrulline was determined in the H2O eluate (Casanello & Sobrevia, 2002; González et al. 2004).

High-performance liquid chromatography

Intracellular l-citrulline and l-arginine were analysed by high-performance liquid chromatography (HPLC) (Casanello & Sobrevia, 2002; González et al. 2004). In brief, cell extractions with 96% methanol (30 min) were exposed to three cycles of freeze–thawing and centrifuged (1500 r.p.m., 2 min). Supernatants were evaporated to dryness under a stream of nitrogen gas and resuspended in 100 μl methanol. Aliquots of samples (20 μl) or standards were injected onto a Hypersil Ultratechsphere ODS-5 μm reversed-phase HPLC column (Jones Chromatography) in a Kontron 400 Series gradient HPLC system (Kontron Instruments Ltd). Amino acid concentrations were calculated from the peak areas by reference to the area of the internal standard, l-homoserine (Casanello & Sobrevia, 2002; González et al. 2004).

Adenosine in 200 μl of culture medium collected after 30 min or 24 h of incubation with 1 μm NBMPR was mixed with 10 μl of 0.5 m acetate-buffer, 10 μl of 1 μm internal standard, and 10 μl of 50% aqueous chloroacetaldehyde (Andresen et al. 1999). Samples were incubated (80°C, 1 h), and centrifuged (14 000 r.p.m., 4 min). Aliquots (80 μl) were injected into an ISCO (Lincoln, NE) HPLC system (pump model 2350, gradient programmer model 2360, 4.6 × 250 mm C18 reverse-phase column, 5 μm particle size, ChemResearch Data Management System, Lincoln, NE). The mobile phase was 10 mm citrate-buffer with 4.5% acetonitrile and was run isocratically at 1 ml min−1. Fluorescence detection was achieved at an excitation wavelength of 275 nm and an emission wavelength of 420 nm using a Waters M-470 fluorescence detector. The ratio of the area under the adenosine peaks to the area under the internal standard peak was compared with a standard curve (Andresen et al. 1999).

Western blotting

Proteins were separated by polyacrylamide gel (8%) electrophoresis, transferred to Immobilon-P polyvinylidene difluoride membranes and probed with primary polyclonal rabbit anti-eNOS (1: 1500), anti-phosphorylated eNOS at serine1177 (Ser1177–P∼eNOS; 1: 250), anti-p42/44mapk (1: 1500), anti-phosphorylated p42/44mapk (1: 1500), or anti-α-actin (1: 2000) antibodies (Casanello & Sobrevia, 2002; González et al. 2004). Membranes were washed (×6) in Tris buffered saline Tween (TBST, 50 mm Tris/HCl, 150 mm NaCl, 0.02% v/v Tween 20, pH 7.4), and incubated (1 h) in TBST/0.2% BSA containing horseradish peroxidase-conjugated goat anti-rabbit antibody. Proteins were detected using enhanced chemiluminescence detection reagents and quantified by densitometry using an Ultrascan XL enhanced laser densitometer (LKB Instruments) (Casanello & Sobrevia, 2002; González et al. 2004).

Total RNA and reverse transcription

Total RNA was isolated from HUVEC exposed (30 min or 24 h) to culture medium with or without 1 μm NBMPR using a QIAGEN RNeasy kit. RNA quality and integrity were insured by gel visualization and spectrophotometric analysis (OD260/280), quantified at 260 nm and precipitated to obtain 4 μg μl−1. Aliquots of 1 μg of total RNA were reversed transcribed into cDNA using oligo (dT18) plus random hexamers (10-mers) and avian Moloney murine leukaemia virus-reverse transcriptase (M-MLV, Promega, USA) (Casanello & Sobrevia, 2002; González et al. 2004).

Real-time RT-PCR

Real-time PCR was performed using a LightCycler™ rapid thermal cycler system (Roche Diagnostics, Lewes, UK). Reactions were done in 10 μl volume using 0.5 μm primers, and dNTPs, Taq DNA polymerase and reaction buffer provided in the Quanti-Tect SYBR Green PCR Master Mix (QIAGEN, Crawley, U.K). Real-time assays included a 95°C denaturation step (15 s), followed by annealing (20 s) at 54°C (hCAT-1), 54°C (eNOS), 58°C (hENT1) or 52°C (28S), and a final extension at 72°C for variable times (hCAT-1, 10 s; eNOS, 17 s; hENT1, 15 s; 28S, 10 s). Detection of the fluorescent product was carried out at the end of each cycle after an additional 3 s step to 3°C below the product melting temperature (Tm). Product specificity was confirmed by agarose gel electrophoresis (1.5% v/v) and melting curve analysis. The product Tm values were 79.17°C (hCAT-1), 86.43°C (eNOS), 79.5°C (hENT1) and 82.88°C (28S).

To prepare standards, hCAT-1, eNOS, hENT1 and 28S PCR products were separated by agarose gel electrophoresis (1.8% v/v) and visualized by staining with ethidium bromide (EtBr, 0.5 μg ml−1). Products were removed from the gel, spin column, purified (Qiaquick gel extraction kit, Qiagen, Crawley, U.K), and quantified by densitometry with reference to the molecular weight marker HpaII digest of pBluescript II 1 (SK+). Standards were prepared by 10-fold serial dilutions in tRNA to obtain 101–109 copies/2 μl. Gene expression was quantified using a 2 μl sample of a 10-fold dilution of HUVEC cDNA. Assays included a tRNA or HUVEC RNA blank in addition to HUVEC cDNA samples and standards.

Oligonucleotide primers were: hCAT-1 (sense) 5′-GAGTTAGATCCAGCAGACCA-3′, hCAT-1 (anti-sense) 5′-TGTTCACAATTAGCCCAGAG-3′, eNOS (sense) 5′-CCAGCTAGCCAAAGTCACCAT-3′, eNOS (anti-sense) 5′-GTCTCGGAGCCATACAGGATT-3′, hENT1 (sense) 5′-TCTCCAACTCTCAGCCCACCAA-3′, hENT1 (anti-sense) 5′-CCTGCGATGCTGGACTTGACCT-3′, 28S (sense) 5′-TTGAAAATCCGGGGGAGAG-3′, 28S (anti-sense) 5′-ACATTGTTCCAACATGCCAG-3′. Expected size products were hCAT-1 148 bp, eNOS 354 bp, hENT1 151 bp and 28S 100 bp.

Materials

Newborn and fetal calf serum, agarose and buffers were from Gibco Life Technologies. Collagenase Type II (Clostridium histolyticum) was from Boehringer Mannheim (FRG) and Bradford protein reagent from Bio-Rad Laboratories, Herts (UK). Ethidium bromide and Dowex-50 (50 × 8–200) were from Sigma. l-NAME was from Calbiochem (La Jolla, USA. l-[2,3,-3H]-Arginine (36.1 Ci mmol−1) and [3H]adenosine (37 Ci mmol−1) were from NEN, Dreieich (Germany). eNOS antibodies were from Cell Signalling, New England Biolabs (UK), and α-actin antibodies from Santa Cruz Biotechnology (USA).

Statistics

Values are means ± s.e.m., where n indicates the number of different cell cultures with 4–8 replicate measurements per experiment. Statistical analyses were carried out on raw data using the Peritz F multiple means comparison test (Harper, 1984). Student's t test was applied for unpaired data and P < 0.05 was considered statistically significant.

Results

l-Arginine and adenosine transport

l-Arginine transport in gestational diabetic cells was higher compared with normal cells (Fig. 1A). Gestational diabetes-induced increase of l-arginine transport was blocked by calphostin C (PKC inhibitor), PD-98059 (MEK-1/2 inhibitor) or l-NAME (eNOS inhibitor), but these molecules did not alter l-arginine transport in normal cells. Adenosine transport was reduced in diabetic compared with normal cells, an effect also blocked by calphostin C or PD-98059 (Fig. 1B). l-NAME also blocked the effect of gestational diabetes and increased adenosine transport compared with basal transport in normal cells.

Figure 1. Effect of gestational diabetes on l-arginine and adenosine transport.

Figure 1

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A, transport of 100 μml-arginine (2 μCi ml−1, 1 min, 37°C) in HUVEC from normal (□) or gestational diabetic (▪) pregnancies in the absence (control) or presence (30 min) of calphostin C, PD-98059 or NG-nitro-l-arginine methyl ester (l-NAME). B, transport of 10 μm adenosine (4 μCi ml−1, 20 s, 37°C) as in A. Mean ± s.e.m., *P < 0.05 versus all other values (n = 14).

The nucleoside transport inhibitor NBMPR increased l-arginine transport only in normal cells, but did not alter the stimulatory effect of gestational diabetes (Fig. 2A). Calphostin C, PD-98059 or L-NAME blocked the effects of gestational diabetes and NBMPR. Adenosine transport was inhibited by NBMPR in normal cells, but was unaltered in gestational diabetes. Calphostin C, PD-98059 and l-NAME were ineffective in the presence of NBMPR in both cell types. hCAT-1 mRNA expression was increased by gestational diabetes and in normal cells exposed to NBMPR (Fig. 3A). However, hENT1 mRNA was significantly reduced by gestational diabetes or NBMPR (Fig. 3B).

Figure 2. Effect of NBMPR on l-arginine and adenosine transport.

Figure 2

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A, transport of 100 μml-arginine (2 μCi ml−1, 1 min, 37°C) in HUVEC from normal (□) or gestational diabetic (▪) pregnancies in the absence (control) or presence (30 min) of nitrobenzylthioinosine (NBMPR), calphostin C, PD-98059 or NG-nitro-l-arginine methyl ester (l-NAME). B, transport of 10 μm adenosine (4 μCi ml−1, 20 s, 37°C) as in A. Mean ± s.e.m., *P < 0.05 versus all other values (n = 12).

Figure 3. hCAT-1, hENT1 and eNOS mRNA expression.

Figure 3

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Real-time RT-PCR for A, hCAT-1 (148 bp), B, hENT1 (151 bp) or C eNOS (354 bp) mRNA from HUVEC isolated from normal (□) or gestational diabetic (▪) pregnancies. Cells were incubated (30 min or 24 h) in the absence or presence of nitrobenzylthioinosine (NBMPR). 28S mRNA (100 bp) was the internal reference. Values are given as the ratio of the number of copies for hCAT-1, hENT1 or eNOS mRNA over the number of copies for 28S mRNA, where correction factors 104 (hCAT-1 and hENT1) or 105 (eNOS) were applied. Mean ± s.e.m., *P < 0.05 versus all other values (n = 15).

Involvement of A2a purinoceptors

In absence of NBMPR, adenosine and CGS-21680 (A2 purinoceptor agonist) increased l-arginine transport in normal cells, an effect blocked by ZM-241385 (A2a purinoceptor antagonist) (Table 2). However, ZM-241385 did not alter basal l-arginine transport in normal cells. Gestational diabetes-increased l-arginine transport was unaltered by adenosine or CGS-21680. However, ZM-241385 blocked the effect of gestational diabetes in the absence or presence of adenosine or CGS-21680. NBMPR increased l-arginine transport only in normal cells in the absence of adenosine or CGS-21680. ZM-241385 blocked the effect of NBMPR in normal cells in the absence or presence of adenosine or CGS-21680 (Table 2). l-Arginine transport was unaltered by CPA (A1 purinoceptor agonist) or DPCPX (A1 purinoceptor antagonist) in the absence or presence of NBMPR in both cell types (not shown), confirming previous observations (Sobrevia et al. 1997). Therefore, all subsequent experiments were performed using ZM-241385, adenosine or CGS-21680.

Table 2.

l-Arginine transport in HUVEC from gestational diabetes

Conditions Normal Gestational diabetes
Without NBMPR
 Control 2.9 ± 0.3  4.6 ± 0.3*
 Adenosine 4.4 ± 0.3* 4.5 ± 0.2*
 CGS-21680 4.3 ± 0.3* 3.9 ± 0.2*
 ZM-241385 2.7 ± 0.2  2.4 ± 0.3†
 Adenosine + ZM-241385 2.8 ± 0.2  2.3 ± 0.4†
 CGS-21680 + ZM-241385 2.3 ± 0.2  2.5 ± 0.3†
With NBMPR
 Control 4.1 ± 0.2* 4.8 ± 0.4*
 Adenosine 4.0 ± 0.2* 4.4 ± 0.4*
 CGS-21680 4.4 ± 0.4* 4.4 ± 0.4*
 ZM-241385 2.3 ± 0.5† 2.5 ± 0.5†
 Adenosine + ZM-241385 2.2 ± 0.4† 2.7 ± 0.3†
 CGS-21680 + ZM-241385 2.6 ± 0.3† 2.9 ± 0.5†
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Human umbilical vein endothelial cells (HUVEC) were incubated in Krebs with or without nitrobenzylthionosine (NBMPR, 1 μm, 30 min), in the absence (control) or presence of adenosine (10 μm, 2 min), CGS-21680 (100 nm, 2 min), or ZM-241385 (100 nm, 30 min) (see Methods). Values are mean ± s.e.m., n = 16.

*

P < 0.05 versus control in normal cells without NBMPR

†

P < 0.05 versus corresponding control values.

Intracellular l-arginine and l-citrulline

Intracellular l-citrulline and l-arginine were higher (P < 0.05, n = 6) in gestational diabetic (0.71 ± 0.19 and 1.9 ± 0.2 mm, respectively) compared with normal cells (0.35 ± 0.21 and 0.89 ± 0.2 mm, respectively). Neither l-citrulline (0.67 ± 0.1 μm) nor l-arginine (1.7 ± 0.3 mm) levels were altered in gestational diabetic cells incubated with NBMPR. However, NBMPR increased l-citrulline (0.6 ± 0.1 μm) and l-arginine (1.6 ± 0.1 mm) in normal cells. ZM-241385 blocked the effect of gestational diabetes on l-citrulline and l-arginine levels (0.28 ± 0.3 and 1.16 ± 0.2 mm, respectively) or the effect of NBMPR in normal cells (0.21 ± 0.4 and 1.02 ± 0.2 mm, respectively).

Extracellular adenosine

Extracellular adenosine concentration in cultures of gestational diabetic cells (2.7 ± 0.6 μm) was higher (P < 0.05, n = 6) than in normal cells (0.05 ± 0.02 μm). NBMPR did not alter extracellular adenosine in gestational diabetic cells (30 min: 2.3 ± 0.5 μm, 24 h: 2.5 ± 0.6 μm, P > 0.05), but NBMPR increased adenosine concentration in normal cells (30 min: 1.8 ± 0.6 μm; 24 h: 2.1 ± 0.4 μm).

PKC activity

Gestational diabetes increased PKC activity in plasma membrane fractions compared with normal cells (Table 3). NBMPR did not alter PKC activity in gestational diabetes, but increased PKC activity in normal cells. The effect of NBMPR in normal cells, and gestational diabetes was blocked by ZM-241385 or calphostin C (Table 3). Neither PD-98059 nor l-NAME altered PKC activity in either cell type (not shown).

Table 3.

Effect of gestational diabetes on PKC activity

Conditions Normal Gestational diabetes
Cytosol Membrane Cytosol Membrane
Control 155 ± 25  27 ± 9  57 ± 15* 188 ± 22‡
NBMPR  19 ± 12* 189 ± 23†  65 ± 14* 191 ± 23‡
NBMPR + ZM-241385 143 ± 27  45 ± 14 154 ± 12†  56 ± 12†
ZM-241385 156 ± 36  38 ± 8 172 ± 31†  62 ± 11†
NBMPR + calphostin C 191 ± 41  29 ± 12 149 ± 32†  47 ± 7†
Calphostin C 143 ± 23  32 ± 11 163 ± 27†  38 ± 12†
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PKC activity in cytosol and membrane fractions prepared from human umbilical vein endothelial cells exposed for 30 min to Krebs. Experiments were performed in the absence (control) or presence (30 min) of NBMPR (1 μm), calphostin C (100 nm), or ZM-241385 (100 nm). PKC activity is given in pmol (mg protein)−1 min−1 (mean ± s.e.m., n = 6).

*

P < 0.05 versus values in cytosol fraction in normal

†

P < 0.05 versus corresponding control

‡

P < 0.05 versus control in membrane fraction in normal.

eNOS activity and expression

l-[3H]Citrulline formation was increased in gestational diabetic compared with normal cells (Fig. 4A). NBMPR increased l-[3H]citrulline formation in normal cells, but did not alter gestational diabetes-increased l-[3H]citrulline formation. The effect of NBMPR and gestational diabetes was blocked by ZM-241385. l-NAME inhibited l-[3H]citrulline formation in both cell types in the absence or presence of NBMPR.

Figure 4. Effect of gestational diabetes on eNOS activity.

Figure 4

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A, l-[3H]citrulline formation from l-[3H]arginine (4 μCi ml−1, 30 min, 37°C) in HUVEC from normal (□) and gestational diabetic (▪) pregnancies (see Methods). Cells were incubated in the absence (−) or presence (+) of nitrobenzylthioinosine (NBMPR), NG-nitro-l-arginine methyl ester (l-NAME) or ZM-241385. B, Western blot for total eNOS or eNOS phosphorylated at Ser1177 (P∼eNOS) in the absence or presence of NBMPR or ZM-241385. α-Actin was the internal reference. Representative blot of other five cell cultures. Lower panel shows P∼eNOS/total eNOS protein ratios. Mean ± s.e.m., *P < 0.05 versus all other values (n = 12).

Total eNOS protein abundance was higher in gestational diabetic compared with normal cells (Fig. 4B). NBMPR increased total eNOS protein level in normal cells, but did not alter gestational diabetic-increased eNOS protein level. NBMPR and gestational diabetes effects were inhibited by ZM-241385. eNOS phosphorylation was also increased in gestational diabetic compared with normal cells. NBMPR increased eNOS phosphorylation only in normal cells. NBMPR and gestational diabetes-increased eNOS phosphorylation was blocked by ZM-241385. Gestational diabetes also increased eNOS mRNA expression compared with normal cells, an effect mimicked by NBMPR only in normal cells (Fig. 3C).

Involvement of p42/44mapk

Gestational diabetes increased p42/44mapk phosphorylation but did not change total p42/44mapk protein level compared with normal cells (Fig. 5). p42/44mapk phosphorylation was increased by NBMPR only in normal cells. The effects of gestational diabetes and NBMPR were blocked by PD-98059 and ZM-241385 in the absence or presence of NBMPR.

Figure 5. Effect of gestational diabetes on p42/44mapk phosphorylation.

Figure 5

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HUVEC from normal or gestational diabetic pregnancies were incubated (30 min) in the absence (−) or presence (+) of nitrobenzylthioinosine (NBMPR), ZM-241385 or PD-98059. Total protein was Western blotted for total or phosphorylated p42/44mapk. Representative blot of seven other cell cultures.

Discussion

This study establishes for the first time that increased l-arginine transport and NO synthesis (i.e. l-arginine/NO pathway) in human umbilical vein endothelial cells (HUVEC) from gestational diabetes is associated with increased human cationic amino acid transporter 1 (hCAT-1) mRNA, and eNOS mRNA and protein expression. This study also demonstrates that gestational diabetes-decreased adenosine transport is associated with reduced hENT1 mRNA expression. Reduced hENT1 expression could, in part, explain the extracellular adenosine accumulation detected in culture medium from gestational diabetic cells. Elevated extracellular adenosine could activate A2a purinoceptors stimulating the l-arginine/NO pathway. Cell signalling mechanisms implicated in the effect of gestational diabetes involve eNOS, PKC and p42/44mapk activation. Thus, a functional relationship is established between adenosine transport, and l-arginine transport and NO synthesis leading to modulation of umbilical vein tone, as this could be determinant for normal fetal growth and development (Mildenberger et al. 2003).

Gestational diabetes is a syndrome that occurs during pregnancy and is associated with imbalanced d-glucose metabolism leading to endothelial dysfunction and abnormal regulation of vascular tone (Sobrevia & Mann, 1997). One important local mechanism involved in the control of vascular tone is endothelial NO synthesis (Moncada et al. 1991; Michiels, 2003), a process that could require membrane transport of l-arginine (Zharikov et al. 2004), the substrate for eNOS (Moncada et al. 1991; Sobrevia & Mann, 1997). Since human umbilical vein is not innervated (Fox & Khong, 1990), local NO release by the umbilical vein endothelium modulates the umbilical vein tone, maintaining blood flow from the placenta to the fetus (Mildenberger et al. 2003). The latter is mainly determined by the difference between the fetal aortic blood pressure and umbilical vein blood pressure, highlighting the importance of local modulation of the umbilical vein vascular tone for appropriate fetal growth and development (Mildenberger et al. 2003). Gestational diabetes increases the NO level in the umbilical vein blood (von Mandach et al. 2003) and increases l-arginine transport and eNOS activity in cultures of HUVEC (Sobrevia et al. 1995). In contrast, gestational diabetes reduces membrane transport of the endogenous purine nucleoside adenosine in HUVEC (Sobrevia et al. 1994). Adenosine is a vasodilator in most vascular beds (Sobrevia & Mann, 1997) and increases l-arginine transport and NO synthesis via A2a purinoceptor activation in HUVEC (Sobrevia et al. 1997; Wyatt et al. 2002; Sohn et al. 2003). Since biological effects of adenosine depend on its extracellular concentration (Griffith & Jarvis, 1996; Sobrevia & Mann, 1997), the effect of gestational diabetes on the l-arginine/NO pathway could result from extracellular accumulation of adenosine leading to activation of adenosine purinoceptors in HUVEC (Sobrevia et al. 1997; Sobrevia & Mann, 1997).

Extracellular adenosine concentration in normal umbilical cord blood (0.1–2 μm) (Maguire et al. 1998) is similar to that in adult human cubital vein (0.1–0.4 μm) (Ontyd & Schrader, 1984) or peripheral venous blood (∼0.04 μm) (Drumm et al. 2004). Since adenosine transport is significantly inhibited by NBMPR in HUVEC (Sobrevia et al. 1994; Montecinos et al. 2000; Parodi et al. 2002), and this cell type does not express ecto-nucleosidases (Sobrevia et al. 1994; Yegutkin et al. 2001), an increase of extracellular adenosine level is likely. This was confirmed in gestational diabetic HUVEC where adenosine was ∼2.7 μm compared with ∼0.05 μm in normal HUVEC. Interestingly, when normal HUVEC were exposed to the adenosine transport inhibitor NBMPR, a similar increase of extracellular adenosine was detected. Since l-arginine transport and NO synthesis were increased in normal HUVEC exposed to NBMPR, but this inhibitor did not further increase the gestational diabetes-stimulated l-arginine transport and NO synthesis, it is likely that the effect of gestational diabetes on l-arginine transport and NO synthesis could result from abnormally elevated levels of extracellular adenosine in this disease. Gestational diabetes- and NBMPR-increased l-arginine transport and NO synthesis were blocked by the selective A2a purinoceptor antagonist ZM-241385 (Sobrevia et al. 1997; Preston et al. 2004) in HUVEC. Thus, the effect of gestational diabetes could result from A2a purinoceptor activation by extracellular accumulated adenosine in this pathology. This suggestion agrees with a similar mechanism reported for adenosine-mediated endothelium-derived NO-dependent vasodilatation in humans under hypoxia (van Mil et al. 2002; Dinenno, 2003), but since adenosine concentration has not been addressed in diabetic subjects (reviewed by Baldwin et al. 2004), our findings cannot be compared with other studies in humans.

Since l-arginine transport and hCAT-1 mRNA expression were increased in normal HUVEC exposed to NBMPR, it is feasible that the effect of NBMPR on arginine transport could result from increased hCAT-1 expression, a membrane transporter known to be expressed in HUVEC (Casanello & Sobrevia, 2002; Flores et al. 2003; González et al. 2004). However, l-arginine transport was unaltered by NBMPR in gestational diabetic HUVEC. The lack of effect of NBMPR could be due to maximal stimulation of the l-arginine/NO pathway induced by gestational diabetes. hCAT-1 mRNA expression was increased by gestational diabetes, thus a higher number of l-arginine membrane transporters could be available at the plasma membrane in this cell type, a suggestion supported by previous reports showing that gestational diabetes increases the maximal velocity (Vmax), but not the apparent Michaelis-Menten constant (Km) for l-arginine transport in HUVEC (Sobrevia et al. 1995).

Gestational diabetes-increased NO synthesis was due to increased eNOS activity since l-[3H]citrulline formation from l-[3H]arginine and eNOS phosphorylation at Ser1177, a residue associated with eNOS activation in HUVEC (Flores et al. 2003; González et al. 2004) and most cell types (Fleming & Busse, 2003) were higher than in normal HUVEC. Increased NO synthesis could also result from higher eNOS expression since eNOS mRNA and protein level were higher in gestational diabetic compared with normal HUVEC. Gestational diabetes-increased eNOS expression and activity were unaltered by NBMPR, but NBMPR increased eNOS expression and phosphorylation in normal cells. The effect of NBMPR on eNOS phosphorylation is likely to be due to activation of A2a purinoceptors since it was blocked by ZM-241385. Since all these results were paralleled by reduced adenosine transport, this phenomenon could be determinant in the effect of gestational diabetes or NBMPR in normal cells.

Nucleoside transport is mediated by the Na+-independent, equilibrative nucleoside transporter 1 (system es ENT1) in HUVEC (Sobrevia et al. 1994; Montecinos et al. 2000; Parodi et al. 2002), a membrane transporter sensitive to <1 μm NBMPR (Baldwin et al. 2004). Since adenosine transport inhibition by gestational diabetes was similar to 1 μm NBMPR-induced maximal inhibition in normal HUVEC, and considering that the apparent Km for adenosine transport in normal and gestational diabetic HUVEC (Km∼60–80 μm) (Sobrevia et al. 1994) was in the same range as ENT1-mediated transport in other cell types (Griffith & Jarvis, 1996; Pastor-Anglada et al. 2001; Baldwin et al. 2004), it is likely that the effect of gestational diabetes could result from reduced ENT1-mediated transport. Gestational diabetes also reduced human ENT1 (hENT1) mRNA expression, supporting this possibility. In addition, the possibility that reduced adenosine transport is due to reduced hENT1 mRNA expression is supported by the findings that NBMPR did not further inhibit hENT1 mRNA expression in gestational diabetic HUVEC, but inhibited hENT1 mRNA expression in normal HUVEC. Since a reduction in the level of hENT1 mRNA does not necessarily mean a lower hENT1 protein level, and inhibition of adenosine transport was higher (∼63) in proportion to the reduced mRNA level (∼51%) detected in gestational diabetic HUVEC, it is feasible that the effect of gestational diabetes on adenosine transport could not exclusively be due to a reduced hENT1 expression, but also to a lower hENT1-mediated transport activity.

Diabetes mellitus is associated with PKC activation (Michiels, 2003). NBMPR activated PKC and p42/44mapk in normal HUVEC, but did not alter the gestational diabetes-increased PKC activity and p42/44mapk phosphorylation. NBMPR stimulation of PKC activity was blocked by ZM-241385 and calphostin C (PKC inhibitor). Thus, gestational diabetes stimulates PKC and p42/44mapk via activation of A2a purinoceptors, as reported for the effect of adenosine in bovine corneal (Zhang et al. 2002) and coronary (Zhou et al. 1996) endothelial cells. PKC and p42/44mapk activation induce downregulation of adenosine transport in HUVEC, since transport in gestational diabetic cells or in normal cells exposed to NBMPR was blocked by calphostin C and PD-98059 (MEK1/2 inhibitor). Inhibition of adenosine transport by PKC activation has been reported in HUVEC (Montecinos et al. 2000), bovine adrenal endothelial cells (Sen et al. 1993) and T84 intestinal epithelial cells (Mun et al. 1998). Adenosine and its analogue CGS-21680 increase p42/44mapk phosphorylation in HUVEC (Wyatt et al. 2002), therefore supporting the possibility that gestational diabetes-induced inhibition of adenosine transport could be due to activation of p42/44mapk.

The increased l-arginine transport exhibited by gestational diabetic HUVEC was blocked when cells were incubated with calphostin C, suggesting the involvement of PKC in the effect of gestational diabetes. PKC stimulation in gestational diabetes was abolished by ZM-241385, suggesting that PKC activation could result from activation of A2a purinoceptors. However the role of PKC as modulator of l-arginine transport is contradictory since l-arginine transport has been shown to be either increased in HUVEC (Flores et al. 2003; González et al. 2004), human intestinal epithelial Caco-2 cells (Pan et al. 2002), and rabbit alveolar macrophages (Racke et al. 1998), reduced in pig pulmonary aortic endothelial cells (Krotova et al. 2003; Zharikov et al. 2004), EA.hy926 endothelial cells or Xenopus laevis oocytes expressing hCAT-1 (Gräf et al. 2001), or unaltered in rat aortic smooth muscle cells (Baydoun et al. 1999).

We previously reported that adenosine (Montecinos et al. 2000; Wyatt et al. 2002; Parodi et al. 2002) and l-arginine (Casanello & Sobrevia, 2002; Flores et al. 2003) transport are downregulated and upregulated, respectively, by exogenous NO in normal HUVEC. Interestingly, our results show that the effect of gestational diabetes on adenosine or l-arginine transport, is blocked by l-NAME (eNOS inhibitor), suggesting that NO was involved in the effect of gestational diabetes. Similar results were found in normal HUVEC exposed to NBMPR. The possibility that increased l-arginine transport in gestational diabetes is due to higher levels of NO agrees with reports showing that NO increases l-arginine transport in bovine aortic endothelium (Ogonowski et al. 2000), or HUVEC (Casanello & Sobrevia, 2002; Flores et al. 2003)), and is supported by the higher NO level detected in human umbilical vein blood from gestational diabetic subjects (von Mandach et al. 2003). Since NO synthesis in gestational diabetes depends on activation of PKC, and NO also induces p42/44mapk phosphorylation in normal HUVEC (Flores et al. 2003; González et al. 2004), it is more than likely that NO release results from A2a purinoceptor activation, thus involving these molecules in the effect of gestational diabetes on the l-arginine/NO pathway in HUVEC.

Our results also show that the phenotype of HUVEC is maintained for several generations in vitro. This is similar to our earlier observations in this cell type (Sobrevia et al. 1994) and in HUVEC from pregnancies affected by intrauterine growth restriction (Casanello & Sobrevia, 2002). Therefore, it is feasible that an adverse or abnormal intrauterine environment could lead to alterations in fetal endothelial physiology. The fact that these cells exhibit reduced adenosine transport maintained after several passages in culture could also be a sign of what has happened during fetal growth and development. It is now well documented that several adult diseases (for example, diabetes mellitus type II or hypertension) may have their origin in intrauterine life (i.e. ‘fetal programming’ or ‘fetal plasticity’) (Barker, 2002). Adults born from gestational diabetic pregnancies (i.e. diabetes of the mother) exhibit a higher risk of developing type II diabetes mellitus or of developing gestational diabetes in future pregnancies (Aerts & Van Assche, 2003), thus suggesting that some alterations may have occurred in these individuals during gestation. The latter observation is even more interesting considering that in this study HUVEC isolated from gestational diabetes were cultured in medium containing normal concentrations of d-glucose (i.e. ∼5 mm), a molecule reported to be involved in endothelial dysfunction in diabetes mellitus (Sobrevia & Mann, 1997; Mann et al. 2003). Despite the inhibitory effect of d-glucose on adenosine transport described in HUVEC (Montecinos et al. 2000; Parodi et al. 2002), we speculate that alterations in the activity and expression of hENT1, and the l-arginine/NO signalling pathway in HUVEC from gestational diabetes could be due to changes other than abnormal levels of d-glucose, since the concentration of this hexose in umbilical vein blood from both normal and gestational diabetic pregnancies was unaltered (∼3 mmd-glucose).

In conclusion, this study demonstrates that stimulation of l-arginine transport and NO synthesis by gestational diabetes is paralleled by reduced adenosine transport in HUVEC. A summary of our findings is shown in Fig. 6. The effect of gestational diabetes on the l-arginine/NO pathway may result from an increased extracellular adenosine level, due to low adenosine uptake as a consequence of reduced hENT1 mRNA expression. Accumulation of extracellular adenosine could activates A2a purinoceptors leading to increased expression of hCAT-1 mRNA and eNOS mRNA and protein, and increased l-arginine transport activity and NO synthesis. The effect of gestational diabetes on adenosine and l-arginine transport involves activation of PKC and p42/44mapk, and increased NO levels. Thus, we hypothesize the establishment of a functional link between adenosine transport and the l-arginine/NO pathway, governing the normal function of human fetal endothelium from gestational diabetic pregnancies. These results also highlight the physiological effects of purinoceptors in the umbilical vein endothelium in pathologies where alterations of blood flow from the mother to the fetus via the umbilical vein may occur, altering the normal supply of nutrients to the developing fetus, such as in intrauterine growth restriction (Casanello & Sobrevia, 2002), fetal hypoxia (Mildenberger et al. 2003) or gestational diabetes (Sobrevia et al. 1994; Sobrevia et al. 1997). Finally, our findings also demonstrate that gestational diabetes induces alterations in the phenotype of human fetal endothelium, an effect that is maintained after several cell generations in culture.

Figure 6. Role of adenosine in the modulation of the l-arginine/NO signalling pathway in human umbilical vein endothelium from gestational diabetes.

Figure 6

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Gestational diabetes is associated with reduced adenosine transport through the human equilibrative nucleoside transporter 1 (hENT1) leading to an increase (vertical arrows) of extracellular concentrations of this nucleoside. Adenosine then activates the A2a subtype of the adenosine receptors in the plasma membrane of human umbilical vein endothelial cells (HUVEC). Following the stimulation of A2a purinoceptors by adenosine, protein kinase C (PKC) and the 44 kDa and 42 kDa mitogen-activated protein kinases (p42/44mapk) are activated stimulating (+)l-arginine transport through the human cationic amino acid transporter-1 (hCAT-1) and the activity of endothelial nitric oxide synthase (eNOS) which becomes phosphorylated (∼P) at Ser1177. The increase in eNOS activity leads to increased l-arginine conversion to l-citrulline and nitric oxide (NO) which then activates hCAT-1 to contribute to the stimulatory effect of adenosine on l-arginine transport. NO, PKC and p42/44mapk reduce (−) adenosine transport mediated by hENT1. The inhibitory effect of gestational diabetes on adenosine transport is also associated with reduced gene transcription for hENT1, but increased gene transcription for hCAT-1 and eNOS. Since well-characterized antibodies against hENT1 and hCAT-1 are not widely available, question marks indicate the possibility that hCAT-1 and/or hENT1 protein levels were also changed in gestational diabetes.

Acknowledgments

This study was supported by Fondo Nacional de Ciencia y Tecnología (FONDECYT 1030781 and 1030607). We thank Miss Alexandra Almeida for her excellent secretarial assistance, and the midwives of Hospital Clínico of the Pontificia Universidad Católica de Chile labour ward for supply of umbilical cords.

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