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. 2000 Dec 15;529(Pt 3):777–790. doi: 10.1111/j.1469-7793.2000.00777.x

Regulation of adenosine transport by D-glucose in human fetal endothelial cells: involvement of nitric oxide, protein kinase C and mitogen-activated protein kinase

Viviana P Montecinos *, Claudio Aguayo *, Carlos Flores *, Amanda W Wyatt *, Jeremy D Pearson *, Giovanni E Mann *, Luis Sobrevia *
PMCID: PMC2270237  PMID: 11118505

Abstract

  1. The effects of elevated D-glucose on adenosine transport were investigated in human cultured umbilical vein endothelial cells isolated from normal pregnancies.

  2. Elevated D-glucose resulted in a time- (8-12 h) and concentration-dependent (half-maximal at 10 ± 2 mM) inhibition of adenosine transport, which was associated with a reduction in the Vmax for nitrobenzylthioinosine (NBMPR)-sensitive (es) saturable nucleoside with no significant change in Km. D-Fructose (25 mM), 2-deoxy-D-glucose (25 mM) or D-mannitol (20 mM) had no effect on adenosine transport.

  3. Adenosine transport was inhibited following incubation of cells with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA; 100 nM, 30 min to 24 h). D-Glucose-induced inhibition of transport was abolished by calphostin C (100 nM, an inhibitor of PKC), and was not further reduced by PMA.

  4. Increased PKC activity in the membrane (particulate) fraction of endothelial cells exposed to D-glucose or PMA was blocked by calphostin C but was unaffected by NG-nitro-L-arginine methyl ester (L-NAME; 100 μM, an inhibitor of nitric oxide synthase (NOS)) or PD-98059 (10 μM, an inhibitor of mitogen-activated protein kinase kinase 1).

  5. D-Glucose and PMA increased endothelial NOS (eNOS) activity, which was prevented by calphostin C or omission of extracellular Ca2+ and unaffected by PD-98059.

  6. Adenosine transport was inhibited by S-nitroso-N-acetyl-l,D-penicillamine (SNAP; 100 μM, an NO donor) but was increased in cells incubated with L-NAME. The effect of SNAP on adenosine transport was abolished by PD-98059.

  7. Phosphorylation of mitogen-activated protein kinases p44mapk (ERK1) and p42mapk (ERK2) was increased in endothelial cells exposed to elevated D-glucose (25 mM for 30 min to 24 h) and the NO donor SNAP (100 μM, 30 min). The effect of D-glucose was blocked by PD-98059 or L-NAME, which also prevented the inhibition of adenosine transport mediated by elevated D-glucose.

  8. Our findings provide evidence that D-glucose inhibits adenosine transport in human fetal endothelial cells by a mechanism that involves activation of PKC, leading to increased NO levels and p42-p44mapk phosphorylation. Thus, the biological actions of adenosine appear to be altered under conditions of sustained hyperglycaemia.

Breakdown of ATP is increased in endothelial cells from animals with experimental diabetes mellitus (Kahn et al. 1994), which could lead to an accumulation of adenosine extracellularly in diabetes and/or hyperglycaemia (Cassar et al. 1998). Adenosine is a potent vasoactive nucleoside and its plasma and tissue levels are regulated by an efficient endothelial transport system (∼300 molecules transporter−1 s−1 in human umbilical vein endothelium; Sobrevia et al. 1994), which modulates several of its biological actions (for review see Baldwin et al. 1999). We have previously shown that saturable adenosine transport is mediated via a Na+-independent and nitrobenzylthioinosine (NBMPR)-sensitive (es) equilibrative nucleoside transporter (Griffiths et al. 1997; Baldwin et al. 1999), which is inhibited by nucleosides but unaffected by nucleobases in human umbilical vein (HUVECs; Sobrevia et al. 1994) and bovine adrenal medulla (BAMECs; Sen et al. 1996) endothelial cells. Adenosine transport and the number of es transporters are reduced in HUVECs isolated from gestational diabetic pregnancies (Sobrevia et al. 1994), a disease that is characterised by sustained elevated plasma D-glucose levels (Dornhorst & Beard, 1993).

It is known that hyperglycaemia in diabetes leads to a higher activity of protein kinase C (PKC) and nitric oxide (NO) synthesis (Koya & King, 1998; reviewed in Poston & Taylor, 1995; Sobrevia & Mann, 1997). Elevated D-glucose for periods up to 24 h has been reported to lead to phosphorylation and activation of mitogen-activated protein kinases p42 (p42mapk, ERK2) and p44 (p44mapk, ERK1) in mesangial cells isolated from rat glomeruli (Haneda et al. 1997) and rat aorta smooth muscle cells (Natarajan et al. 1999). Hyperglycaemia-induced phosphorylation of p42- p44mapk seems to be dependent on the activation of PKC (Haneda et al. 1997) and higher NO levels (Parenti et al. 1998; Hood & Granger, 1998). Moreover, endothelial NO synthase (eNOS) activity has been shown to involve the activation of PKC in HUVECs (H. Li et al. 1998).

In the present study we have investigated the effects of elevated extracellular D-glucose on adenosine transport in HUVECs isolated from normal pregnancies. To elucidate the underlying cellular mechanisms, we examined whether the effects of elevated D-glucose on adenosine transport are altered following inhibition of PKC, eNOS and mitogen-activated protein kinase (MAPK) activities. Our findings establish that elevated D-glucose inhibits adenosine transport in human fetal endothelium. This effect of D-glucose involves NO, and activation of PKC and MAPK signalling pathways. Since activation of A2a-purinoceptors by adenosine stimulates NO synthesis in human endothelium (Sexl et al. 1997; Sobrevia et al. 1997; J. Li et al. 1998), accumulation of adenosine extracellularly following inhibition of adenosine transport by D-glucose may contribute to diabetes- and hyperglycaemia-induced synthesis of NO in human endothelial cells. A part of this study has been reported in abstract form (Montecinos et al. 1998).

METHODS

Endothelial cell culture

Venous endothelial cells were isolated by collagenase (0.25 mg ml−1) digestion from umbilical cords obtained from full-term normal pregnancies with vaginal deliveries. Informed written consent was given from the hospital for the use of the umbilical cords. Endothelial cells were cultured in primary culture medium 199 (M199) containing 5 mM D-glucose, 10 % fetal calf serum, 10 % newborn calf serum, 3.2 mM L-glutamine, 100 i.u. ml−1 penicillin- streptomycin and 0.03 mg ml−1 gentamicin at 37°C in a 5 % CO2 atmosphere. Confluent second passage cells were resuspended (104 cells ml−1) in primary culture medium containing 0.01 mg ml−1 endothelial cell growth supplement (ECGS) and plated into 24-well plates. Twenty-four hours prior to an experiment, the incubation medium was changed to one free from ECGS (Sobrevia et al. 1994).

Adenosine transport

Confluent third passage monolayers (∼2.5 × 104 cells per well) were rinsed with warmed (37°C) Krebs solution (mM): NaCl, 131; KCl, 5.6; NaHCO3, 25; NaH2PO4, 1; CaCl2, 2.5; MgCl2, 1; D-glucose, 5; Hepes, 20 (pH 7.4). Triplicate monolayer wells were then preincubated for 30 min at 22°C in the same Krebs solution or Krebs solution containing the adenosine transport inhibitor NBMPR (10 μM). In some experiments, sodium in the Krebs solution was replaced by N-methylglucamine-HCl or choline chloride. After removal of the preincubation solution, inward fluxes of [3H]adenosine at 22°C were determined by the addition of 200 μl of Krebs solution containing [3H]adenosine (1-4 μCi) and D-[14C]mannitol (0.05-0.4 μCi, an extracellular marker). As described previously (Sobrevia et al. 1994; Sen et al. 1996), no significant difference was observed in the extracellular volume distribution of D-[14C]mannitol. The kinetics of adenosine transport were measured under similar conditions in cells incubated with increasing concentrations of adenosine (0-500 μM) for periods of 5 s at 22°C in Krebs solution.

To assess the specificity of adenosine transport, the nucleosides formycin B, guanosine and uridine, the nucleobases adenine, hypoxanthine and guanine, or the well-characterised inhibitor of facilitated-diffusion nucleoside transport dilazep (Sobrevia et al. 1994) were used as potential inhibitors of adenosine transport.

Uptake of adenosine (5-20 s) was terminated by removal of the Krebs solution 1 s before the addition of 250-500 μl of ice-cold Krebs solution containing 10 μM NBMPR. The cell monolayer was rinsed with a further three washes of ice-cold stop solution. Radioactivity associated with the monolayers at time zero was determined by exposing the cells simultaneously to radiolabelled solution and ice-cold stop solution. Radioactivity in formic acid cell digests was determined by liquid scintillation counting, and uptake values were corrected for 3H in the extracellular space and expressed as picomoles per 106 cells. Kinetic data were analysed using the computer programs Enzfitter and Ultra Fit (Elsevier, Biosoft) and fitted best by a Michaelis-Menten equation.

Binding of nitrobenzylthioinosine

Triplicate HUVEC monolayers cultured in 5 or 25 mM D-glucose (24 h) were prepared for [3H]NBMPR equilibrium binding studies, following two rinses with Krebs solution and subsequently a 15 min incubation at 22°C in 5 or 25 mM D-glucose-containing Krebs solution, in the presence or absence of 10 μM NBMPR (see Sobrevia et al. 1994; Sen et al. 1996; Aguayo & Sobrevia, 2000). The monolayers were then incubated with 800-1500 μl of [3H]NBMPR or [3H]NBMPR plus 10 μM NBMPR for 30 min at 22°C in the presence of the corresponding D-glucose concentration. At the end of the incubations, 200 μl of the supernatant was retained for radioactivity determination and the cells were rapidly rinsed three times with ice-cold phosphate-buffered saline (PBS). Radioactivity associated with the monolayer was determined as described above for the transport assays. Specific binding was defined as the difference in the binding in the presence and absence of 10 μM NBMPR.

Modulation of adenosine transport

To assess the effect of potential modulators of endothelial adenosine transport, monolayers were cultured in M199 containing specified concentrations of D-glucose (5-40 mM, 1-24 h), 2-deoxy-D-glucose (25 mM, 24 h), D-fructose (25 mM, 24 h) or 5 mM D-glucose plus appropriate osmotic concentrations of D-mannitol (0-35 mM, 1-24 h). Transport of adenosine (10 μM) was then measured at 22°C during the last 20 s of the specified incubation period (see Sobrevia et al. 1994; Sen et al. 1996).

To assess the involvement of PKC in adenosine transport, endothelial cells were exposed to phorbol 12-myristate 13-acetate (PMA; 100 nM, 30 min to 24 h), the less active PMA analogue 4α-phorbol 12,13-didecanoate (4α-PDD; 100 nM, 30 min to 24 h) or the PKC inhibitor calphostin C (100 nM, 30 min) (Kobayashi et al. 1989). Transport (20 s) was determined after incubation of endothelial cells for the specified periods in Krebs solution in the presence or absence of NBMPR (10 μM, 30 min).

To examine the involvement of MAPK signalling cascades in the inhibitory action of D-glucose on adenosine transport, endothelial cells were incubated with 10 μM PD-98059 (30 min to 24 h), an inhibitor of MAPK kinase 1 (MEK-1; Lazar et al. 1995), the upstream regulator of p44mapk and p42mapk (Crews & Erikson, 1992). The involvement of tyrosine kinases was examined further by screening the effects of 10 μM genistein (30 min to 24 h; a general inhibitor of tyrosine kinases; see May et al. 1996) or its less active analogue daidzein (10 μM, 30 min to 24 h).

The effect of NO on adenosine transport was assayed by exposing endothelial cell monolayers to the spontaneous NO donor S-nitroso-N-acetyl-l,D-penicillamine (SNAP; 100 μM, 30 min to 24 h) or in cells preincubated with the eNOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 10 nM to 100 μM, 30 min to 24 h). Cells were incubated with these agents in the absence or presence of elevated D-glucose or D-mannitol (i.e. 30 min to 24 h) and transport assays were performed as described above.

Determination of endothelial protein kinase C activity

PKC activity was assayed using the method described by Castro et al. (1998). Endothelial cells were harvested in a lysis solution containing 20 mM Tris-HCl, 2 mM EDTA, 0.5 mM EGTA, 5 mM β-mercaptoethanol, 250 mM sucrose, 0.1 mM phenylmethylsulfonylfluoride, 0.02 mM leupeptin, 1 mM benzamidine, 2 μg ml−1 soybean trypsin inhibitor and 10 μg ml−1 aprotinin (pH 7.4). Cells were lysed by sonication at 4°C and the lysate was centrifuged at 100000 g (60 min, 4°C). The resulting supernatant was considered the cytosolic (soluble) fraction. The pellet was resuspended in lysis buffer containing 1 % Triton X-100, incubated for 60 min at 4°C with continuous mixing, and then centrifuged at 100000 g for 60 min at 4°C. This supernatant was considered the membrane (particulate) fraction.

Endothelial PKC activity was determined by measuring 32P incorporation from [γ-32P]ATP into a synthetic PKC substrate peptide analogue, corresponding to a fragment of glycogen synthase (GS) (Castro et al. 1998). The reaction mixture (100 μl) consisted of 25 μM GS peptide in 20 mM Tris-HCl, 10 mM MgCl2 and 50 μM [γ-32P]ATP (specific activity, 500 c.p.m. pmol−1), plus 0.5 mM EGTA or 0.5 mM CaCl2, 60 μg ml−1 phosphatidylserine and 3 μg ml−1 diolein (pH 7.4, 30°C, 10 min). Reactions were started by addition of [γ-32P]ATP and stopped by spotting a 40 μl aliquot of the reaction mixture onto Whatman P-81 phosphocellulose filters (4 cm2), which were rapidly soaked in 75 mM H3PO4. The filters were washed (×3) in the same solution (20 min each), dried, and assayed for radioactivity in scintillation mixture. PKC activity was calculated as the difference between 32P incorporated into the GS substrate peptide in the presence of CaCl2-phosphatidylserine- diolein vs. EGTA. PKC activity was determined in cells exposed for 2 min to 24 h to high D-glucose (25 mM) in the absence or presence of PMA (100 nM, 30 min), calphostin C (100 nM, 30 min) or PD-98059 (10 μM, 30 min to 24 h). Results were expressed as picomoles 32P per milligram of protein per minute (pmol (mg protein)−1 min−1).

Conversion of L-[3H]arginine into L-[3H]citrulline

Confluent endothelial cell monolayers in 24-well plates cultured in M199 containing 5 or 25 mM D-glucose (24 h) were incubated with 100 μM L-[3H]arginine (4 μCi ml−1, 30 min, 37°C) in the absence or presence of 100 μM L-NAME, 10 μM histamine (last 5 min of a 30 min incubation period), PMA (100 nM, 30 min), calphostin C (100 nM, 30 min) or PD-98059 (10 μM, 30 min). Aliquots of 100 g of the cation ion-exchange resin Dowex50W (50X8-200) in its protonated form were converted into the sodium ion form by incubation with 200 ml 1 n NaOH. After calibration of the Dowex column, 200 μl of endothelial cells digested in 95 % formic acid (∼5 × 105 cells) were passed through the column and eluates of H2O and NaOH were collected (Contreras et al. 1997; Sobrevia et al. 1998). The amount of L-[3H]citrulline produced after 30 min incubation with L-[3H]arginine was determined in the H2O eluate and expressed as disintegrations per minute per 106 cells per 30 min (d.p.m. (106 cells)−1 (30 min)−1).

Immunoblotting

Confluent second passage endothelial cells in 24-well plates were exposed for 24 h to culture medium without serum and containing either 5 or 25 mM D-glucose. Monolayers were washed twice with Krebs solution (37°C) and then incubated in M199 containing 5 or 25 mM D-glucose in the absence or presence of PD-98059 (10 μM, 30 min to 24 h) or L-NAME (100 μM, 30 min to 24 h). Cell monolayers were also exposed to 100 μM SNAP (30 min). The reaction was stopped by washing monolayers with ice-cold PBS containing 200 μM sodium orthovanadate. Cells were lysed in buffer containing 63.5 mM Tris-HCl (pH 6.8), 10 % (v/v) glycerol, 2 % (w/v) sodium dodecyl sulphate (SDS), 1 mM sodium orthovanadate, 1 mM 4-(2-aminoethyl)benzenesulphonyl fluoride (AEBSF), 50 μg ml−1 leupeptin and 5 % (v/v) 2-mercaptoethanol. Protein cell lysates (20-30 μg) were separated by 10 % polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon-P (0.45 μm pore size polyvinylidene difluoride (PVDF) membranes), which were blocked for 3 h in bovine serum albumin (BSA; 3 % in Tris-buffered saline-Tween (TBST) containing 50 mM Tris-HCl, 150 mM NaCl, 0.02 % (v/v) Tween 20, pH 7.4) and probed with the primary (rabbit) polyclonal antibody anti-phosphorylated p44-p42mapk or anti-phosphorylated p42mapk, or with anti-non-phosphorylated-phosphorylated p42mapk (diluted 1/1000 in 0.2 % BSA-TBST). Membranes were then washed (×6) in TBST and incubated for 1 h in TBST-0.2 % BSA containing horseradish peroxidase-conjugated goat anti-rabbit antibody (1/1000). Protein bands were detected using enhanced chemiluminescence (ECL) detection reagents.

Materials

Newborn and fetal calf serum, NBMPR and all other reagents were purchased from Sigma. Collagenase Type II from Clostridium histolyticum was from Boehringer Mannheim (Germany) and Bradford protein reagent was from BioRad Laboratories (Herts, UK). [2,8,5′-3H]Adenosine (60 Ci mmol−1) and D-[1-14C]-mannitol (49.3 mCi mmol−1) were from NEN (Dreieich, Germany). Receptor agonists and antagonists were obtained from RBI (UK). Antiphosphotyrosine and MAPK antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Statistics

Values are expressed as means ± s.e.m., where n indicates the number of different umbilical vein endothelial cell cultures with three to six replicate measurements per experiment. Statistical analyses were carried out on raw data using the Peritz F multiple means comparison test. Student’s t test was applied for unpaired data and P < 0.05 was considered statistically significant.

RESULTS

Modulation of adenosine transport by elevated D-glucose

As we have previously described (Sobrevia et al. 1994), adenosine transport was saturable and fitted best by a single Michaelis-Menten equation, inhibited by > 95 % by NBMPR, and unaffected by removal of extracellular Na+. Adenosine transport was reduced after incubation of cells with 25 mM D-glucose. After 24 h, half-maximal inhibition occurred with 10 ± 2 mM D-glucose (Fig. 1A). Half-maximal inhibition of adenosine transport by 25 mM D-glucose was detected at 6 ± 1 h, with maximal inhibition of transport rates achieved within 8-12 h and sustained over 24 h (Fig. 1B).

Figure 1. Time course and concentration-dependent effect of D-glucose on adenosine transport in human umbilical vein endothelial cells.

Figure 1

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A, NBMPR-sensitive adenosine transport (10 μM, 22 °C, 20 s) was determined in cells incubated for 24 h in M199 containing serum and D-glucose (5-25 mM). The D-glucose concentration producing half-maximal inhibition of adenosine transport was 10 ± 2 mM. B, endothelial cells cultured in M199 were exposed for the specified periods of time to 5 mM D-glucose (○), 25 mM D-glucose (•) or 5 mM D-glucose + 20 mM D-mannitol (□), and 10 μM adenosine transport was determined as described in A. The D-glucose-induced time-dependent, half-maximal inhibition was at 6 ± 1 h. Values denote means ± s.e.m. of 6 different cell cultures. *P < 0.05 vs. the corresponding controls (5 mM D-glucose or 5 mM D-glucose + 20 mM D-mannitol).

Exposure of cells to 25 mM D-glucose was associated with a reduction in the Vmax for saturable adenosine transport, with negligible changes in Km (Table 1). The reduction in adenosine transport capacity induced by D-glucose was specific for this hexose, since D-fructose or 2-deoxy-D-glucose had no effect on the kinetics of adenosine transport (Table 1). The reduction of Vmax for adenosine transport was not due to osmotic effects of D-glucose, since 20 mM D-mannitol plus 5 mM D-glucose had no such effect.

Table 1.

Effect of elevated d-glucose on the kinetics of adenosine transport in human umbilical vein endothelial cells

Conditions Km (μm) Vmax (pmol (106 cells)−1s−1)
d-Glucose (5 mm) 83 ± 30 617 ± 72
d-Glucose (25 mm) 71 ± 38 344 ± 58*
d-Fructose (25 mm) 67 ± 14 638 ± 66
2-Deoxy-D-glucose (25 mm) 82 ± 22 578 ± 58
d-Mannitol (20 mm) 89 ± 12 498 ± 61
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Saturable kinetics of adenosine transport (0.015-500 μm) were determined in cells indubabed for 24 h in M 199 containing serum and either 5 or 25 mm d-glucose. Experiments using 25 mm d-fructose, 25 mm 2-deoxy-d-glucose or 20 mm d-mannitol were performed in cells cultured in the presence of 5 mm d-glucose. Values are means ± s.e.m., n = 3-4 different cell cultures.

*

P < 0.05 vs. values in 5 mm d-glucose.

Effects of D-glucose on [3H]NBMPR binding

The concentration dependence of [3H]NBMPR binding to confluent endothelial cell monolayers exposed to 5 or 25 mM D-glucose in a single experiment is shown in Fig. 2. [3H]NBMPR binding kinetics exhibited a 2-fold reduction (P < 0.05) in the maximal number of [3H]NBMPR binding sites (Bmax) in cells cultured in 25 mM D-glucose (Bmax, 1.4 ± 0.2 pmol (106 cells)−1; n = 4) compared with cells exposed to 5 mM D-glucose (Bmax, 2.5 ± 0.2 pmol (106 cells)−1; n = 4). However, the affinity of [3H]NBMPR binding was unaffected by elevated D-glucose (apparent Kd, 0.16 ± 0.04 and 0.19 ± 0.07 nM for 5 and 25 mM D-glucose, respectively). To assess the effects of D-glucose on the specificity of adenosine transport in HUVECs, we examined the inhibitory effects of nucleosides and nucleobases on NBMPR-sensitive adenosine transport (Table 2). Nucleosides inhibited adenosine transport with similar efficacy in endothelial cells exposed to either 5 or 25 mM D-glucose, with the order of potency: formycin B > guanosine > uridine. The nucleobases, adenine, hypoxanthine and guanine, at final concentrations up to 8 mM, did not inhibit adenosine transport. The facilitated-diffusion nucleoside transport inhibitor dilazep markedly inhibited adenosine transport in endothelial cells exposed to both 5 and 25 mM D-glucose (Table 2). These results indicate that adenosine transport in cells incubated with 25 mM D-glucose occurs via the same route as that in cells incubated with 5 mM D-glucose, but is reduced because of reduced transporter numbers.

Figure 2. Effect of D-glucose on the concentration dependence of [3H]NBMPR binding to human umbilical vein endothelial cells.

Figure 2

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Cells cultured for 24 h in M199 containing serum and either 5 mM D-glucose (○) or 25 mM D-glucose (•) were washed and then preincubated in Krebs solution for 15 min in the absence or presence of 10 μM NBMPR. The same cell monolayers were then incubated with [3H]NBMPR for 30 min at 22 °C in Krebs solution. Specific cell-associated radioactivity, defined as the difference between total binding and binding in the presence of 10 μM NBMPR, is plotted against the final free concentration of [3H]NBMPR. The individual data points of one representative experiment of 4 are shown.

Table 2.

Effects of nucleosides and nucleobases on adenosine transport in human umbilical vein endothelial cells

Ki(μm)
5 mm d-glucose 25 mm d-glucose
Formycin B 49 ± 13 57 ± 12
Guanosine 106 ± 32 79 ± 29
Uridine 247 ± 24 278 ± 76
Dilazep 0.003 ± 0.001 0.004 ± 0.001
Adenine No inhibition No inhibition
Hypoxanthine No inhibition No inhibition
Guanine No inhibition No inhibition
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The apparent inhibition constants (Ki) were calculated using the expression: Ki= IC50/(1 +[Ado]/Km), where Km is the apparent Km value for adenosine tranport, [Ado] is the adenosine concentration (10 μm) and IC50 is the half-maximal inhibitory concentration of the inhibitors. Maximal concentrations for potential inhibitors were 2.5 mm for adenine and guanine, and 8 mM for hypoxanthine. Values are means ± s.e.m., n = 4-6 different cell cultures.

Involvement of protein kinase C in D-glucose-induced reduction of adenosine transport

Treatment of cells for 30 min with PMA, an activator of PKC, significantly reduced adenosine transport (Fig. 3), while the inactive analogue 4α-PDD had no effect (not shown). Calphostin C, an inhibitor that acts at the diacylglycerol (DAG)- or phorbol ester-binding site of PKC (Kobayashi et al. 1989), significantly inhibited the effects of PMA treatment and had no effect on basal rates of adenosine transport (Fig. 3).

Figure 3. Involvement of protein kinase C in D-glucose-induced inhibition of adenosine transport in human umbilical vein endothelial cells.

Figure 3

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Cells were pretreated for 24 h in M199 containing 5 or 25 mM D-glucose and then exposed to phorbol 12-myristate 13-acetate (PMA) and/or calphostin C for 30 min before NBMPR-sensitive adenosine transport (10 μM, 20 s, 22 °C) was measured. *P < 0.05 vs. values in 5 mM D-glucose. Values denote means ± s.e.m. of 3-4 different cell cultures.

Figure 3 also shows that inhibition of adenosine transport by elevated D-glucose was abolished when cells were incubated with calphostin C, suggesting that the effect of D-glucose on adenosine transport depends on the activity of PKC. Exposure of endothelial cells to PMA for the last 30 min of a 24 h incubation period with 25 mM D-glucose did not further potentiate the inhibition of adenosine transport induced by D-glucose.

To determine directly whether elevated D-glucose alters PKC activity, cytosol and membrane fractions were prepared from monolayers of endothelial cells. Table 3 shows that elevated D-glucose increased by 5.5-fold the PKC activity in the membrane fraction, an effect that was inhibited completely by calphostin C. Endothelial PKC activity in the membrane fraction was increased by PMA, but not by 4α-PDD. PMA activation of PKC was abolished by co-incubation of cells with calphostin C.

Table 3.

Effects of d-glucose and PMA or PKC activity in the cytosol and membrane fractions from human umbilical vein endothelial cells

5 mm d-glucose 25 mm d-glucose
Conditions Cytosol Membrane Cytosol Membrane
Control 123 ± 13 27 ± 13* 55 ± 12* 147 ± 20‡
PMA (100 nm) 61 ± 5* 189 ± 11‡ 49 ± 12* 149 ± 12‡
Calphostin C(100 nm) 107 ± 23 17 ± 6 131 ± 25§ 43 ± 16§
PMA + calphostin C 146 ± 29† 17 ± 12† 150 ± 32§ 55 ± 11§
4α-PDD (100 nm) 117 ± 22† 32 ± 12† 39 ± 19* 169 ± 27‡
l-NAME (100 μm) 145 ± 25† 19 ± 7* 27 ± 2* 187 ± 25‡
l-NAME + PMA 24 ± 9* 170 ± 22‡ 35 ± 31* 145 ± 10‡
SNAP (100 μm) 107 ± 38 27 ± 12* 47 ± 14* 127 ± 12‡
SNAP + PMA 55 ± 15* 191 ± 27‡ 36 ± 9* 173 ± 13‡
SNAP + l-NAME 143 ± 8* 16 ± 34* 41 ± 12* 182 ± 10‡
PD-98059 (10 μm) 116 ± 8† 17 ± 14† 15 ± 12* 147 ± 13‡
PD-98059 + PMA 17 ± 32* 197 ± 17‡ 37 ± 12* 269 ± 64‡
PD-98059 + l-NAME 117 ± 27† 32 ± 11† 59 ± 13* 178 ± 41‡
PD-98059 + l-NAME+SNAP 129 ± 17† 22 ± 10† 39 ± 14* 161 ± 32‡
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Cytosolic and membrane fractions of HUVECs were prepared from endothelial cell monolyaers cultured for 24 h in M199 containing 5 or 25 mm d-glucose (as described in Methods). The activity of PKC was estimated as the difference between 32P information from [γ−32P]ATP into a synthetic glycogen synthase PKC substrate peptide in the presence of CACl2-phosphatidylserine-diolein vs. EGTA (Castro et al. 1998). Experiments were performed in endothelial cells exposed to M199 (Control) or M199 contaning different molecules for 30 min at the indicated concentrations. PMA, phorbol 12-myristate 13-acetate; 4α-PDD, 4α-phorbol 12, 13-didecanoate; l-NAME, NG-nitro-L-arginine methyl ester; SNAP, S-nitroso-N-acetyl-l,d-penicillamine. Values are in pmol (mg protein)−1 min−1; means +/- s.e.m., n = 3-4 different cell cultures.

*

P < 0.05 vs. control values for cytosol fraction in 5 mm d-glucose

†

P < 0.04 vs. values in PMA-treated cells

‡

P < 0.05 vs. control values for membrane fraction in 5 mm d-glucose

§

P < 0.05 vs. corresponding control values in 25 mM D-glucose.

Involvement of MAPK in D-glucose-induced reduction of adenosine transport

To establish whether inhibition of adenosine transport by elevated D-glucose involves activation of MAPK and tyrosine kinase pathways, endothelial cells were exposed to the MEK-1 inhibitor PD-98059 or the general tyrosine kinase inhibitor genistein. The pronounced inhibition of adenosine transport induced by 25 mM D-glucose was prevented when cells were exposed simultaneously to elevated D-glucose and PD-98059 (Fig. 4A). Similarly, the inhibitory effects of D-glucose on adenosine transport were prevented by co-treatment of cells with genistein (Fig. 4B), but not by co-treatment with daidzein, a less active analogue of genistein (data not shown).

Figure 4. Inhibition of the effect of D-glucose on adenosine transport by PD-98059 or genistein in human umbilical vein endothelial cells.

Figure 4

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NBMPR-sensitive adenosine transport (10 μM, 20 s, 22 °C) was measured in cell monolayers incubated in Krebs solution. Cells were initially preincubated for 24 h in M199 containing serum and either 5 mM D-glucose (□) or 25 mM D-glucose (▪), in the absence or presence of 10 μM PD-98059 (A) or 10 μM genistein (B). Values denote means ± s.e.m. of 6 different cell cultures. *P < 0.05 vs. all other values.

Endothelial cell monolayers were exposed to elevated D-glucose and phosphorylated forms of p42-44mapk were detected by immunoblotting. Phosphorylation of p42-44mapk was increased in endothelial cells exposed to 25 mM D-glucose for 12 and 24 h (Fig. 5A; lanes B and C, respectively). When endothelial cells were exposed simultaneously to elevated D-glucose (25 mM for 24 h) and the MEK-1 inhibitor PD-98059, phosphorylation of p42-44mapk was significantly attenuated. When endothelial cells were incubated for shorter periods of time (0.5-8 h) with 25 mM D-glucose, phosphorylation of p42-44mapk was significantly increased, with a maximum being reached after about 2 h but remaining elevated for 24 h (Fig. 5B). Simultaneous incubation of endothelial cells with PD-98059 did not alter basal PKC activity in cells cultured in 5 mM D-glucose or the stimulatory effect of elevated D-glucose on the PKC activity in cytosol or membrane fractions (see Table 3). In contrast, PMA-induced phosphorylation of p42-44mapk was blocked by calphostin C (Fig. 5C). These results suggest that inhibition of adenosine transport by elevated D-glucose requires PKC activation followed by activation of the p42-44mapk pathway.

Figure 5. Mitogen-activated protein kinase phosphorylation in human umbilical vein endothelial cells after stimulation with D-glucose.

Figure 5

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A, immunoblot of phosphorylated p42-p44mapk as described in Methods. Endothelial cells were deprived of serum for 24 h, washed and incubated in M199 containing 5 mM (lane A) or 25 mM D-glucose for 12 h (lane B), or 24 h (lane C), or 25 mM D-glucose and 10 μM PD-98059 (24 h, lane D). B, details as in A, but showing the time course of the effect of treatment with 25 mM D-glucose. C, effect of PMA (100 nM, 30 min) on phosphorylation of p42-p44mapk and its inhibition by co-incubation with either calphostin C or PD-98059. The blots shown in A-C are representative of similar blots of 3 different cell cultures.

Involvement of NO in the inhibition of adenosine transport by elevated D-glucose

We have previously reported that elevated D-glucose increases the activity and protein levels of eNOS in HUVECs, with similar time and concentration dependencies to those for the effect of D-glucose on adenosine transport (Sobrevia et al. 1996; Mann et al. 1998). In the present study, we therefore examined whether NO is involved in the regulation of adenosine transport by D-glucose. Adenosine transport in endothelial cells cultured in either 5 or 25 mM D-glucose was significantly increased following treatment of cells with L-NAME (Fig. 6A). The NO donor SNAP inhibited adenosine transport in endothelial cells cultured in 5 mM D-glucose but had no effect in cells cultured for 24 h in 25 mM D-glucose (Fig. 6A). The effect of SNAP on adenosine transport in cells cultured in 5 mM D-glucose was blocked by PD-98059. In parallel experiments, D-glucose-induced phosphorylation of p42mapk was abolished by co-incubation of endothelial cells with L-NAME (Fig. 6B).

Figure 6. Effect of nitric oxide on adenosine transport and mitogen-activated protein kinase phosphorylation in human umbilical vein endothelial cells after stimulation with D-glucose.

Figure 6

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A, cells were cultured for 24 h in M199 containing 5 mM (□) or 25 mM (▪) D-glucose. Cells were then incubated for 30 min in Krebs solution (with 5 or 25 mM D-glucose) in the absence (Control) or presence of NG-nitro-L-arginine methyl ester (L-NAME, 30 min), S-nitroso-N-acetyl-l,D-penicillamine (SNAP, 30 min) or SNAP + PD-98059 (24 h), and NBMPR-sensitive adenosine transport (10 μM, 20 s, 22 °C) was then determined. Values denote means ± s.e.m. of n = 6 different cell cultures. *P < 0.05 vs. control value in 5 mM D-glucose; **P < 0.05 vs. corresponding control and SNAP values. B, immunoblot of non-phosphorylated (p42) and phosphorylated (p42∼P) p42mapk as described in Methods. Endothelial cells were deprived of serum for 24 h, washed and incubated in M199 containing 5 or 25 mM D-glucose for 24 h in the absence or presence of L-NAME. C, cell monolayers were washed and incubated in Krebs solution for 30 min in the absence or presence of SNAP, PD-98059 or SNAP + PD-98059 before detection of phosphorylated p42-p44mapk as described in Methods. D, details as in C, but cells were incubated with PMA alone or with increasing concentrations of L-NAME. The blots shown in B-D are representative of similar blots obtained from 3 different cell cultures.

Further evidence for the involvement of NO as an inducer of p42-44mapk phosphorylation is shown in Fig. 6C and D. Phosphorylation of p42-44mapk was induced by incubation of endothelial cells with SNAP for 30 min, an effect that was substantially inhibited by PD-98059, while PMA-induced phosphorylation of p42-44mapk was blocked by L-NAME.

Table 4 illustrates the effects of inhibitors or activators of PKC and a MEK-1 inhibitor on D-glucose-induced activation of eNOS, assayed by formation of L-[3H]citrulline from L-[3H]arginine. Cells cultured in 25 mM D-glucose exhibited a 2-fold higher formation of L-[3H]citrulline compared with cells cultured in 5 mM D-glucose. When endothelial cells were exposed to PMA, L-[3H]citrulline formation was increased in cells cultured in 5 mM D-glucose, but cells in 25 mM D-glucose were unaffected by this phorbol ester. The effect of PMA on L-[3H]citrulline formation in 5 mM D-glucose was inhibited by calphostin C or L-NAME. In endothelial cells cultured in 25 mM D-glucose, calphostin-C and L-NAME also inhibited L-[3H]citrulline formation. Table 4 also shows that, in endothelial cells exposed to the MEK-1 inhibitor PD-98059, basal formation of L-[3H]citrulline was unaffected in both cell culture conditions. Furthermore, PMA-induced formation of L-[3H]citrulline was unaffected by incubation of endothelial cells with PD-98059, suggesting that formation of L-[3H]citrulline from L-[3H]arginine in HUVECs may not be dependent on the activity of MEK-1.

Table 4.

l-[3H]Citrulline formation from L-[3H]arginine in human umbilical vein endothelial cells

Conditions 5 mm d-glucose 25 mm d-glucose
Control 4102 ± 200 8200 ± 1201*
l-NAME (100 μm) 1233 ± 250* 1455 ± 750‡
Ca2+ free Krebs solution 873 ± 311* 1622 ± 189‡
PMA (100 nm) 6884 ± 244* 7226 ± 466*
Calphostin C (100 nm) 3677 ± 234† 3266 ± 255†‡
PMA + calphostin C 3215 ± 677† 5077 ± 239†‡
PMA + l-NAME 679 ± 245*† 1177 ± 245†‡
PD-98059 (10 μm) 3704 ± 367† 8922 ± 1066*
PD-98059 + l-NAME 702 ± 105*† 477 ± 215†‡
PD-98059 + PMA 7759 ± 401* 8059 ± 944†‡
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Formation of l-[3H]citrulline from l-[3H]arginine was determined in endothelial cell monolayers cultured for 24 h in M199 containing 5 or 25 mm d-glucose as described in Methods (see Sobrevia et al. 1998). Endothelial cells were exposed to normal or Ca2+ -free Krebs solution containing L-arginine (100 μm) and l-[3H]arginine (4μCi ml−1, 37°C) for 30 min in the absece or presence of other molecules at the indicated concentrations. Values are in d.p.m. (106 cells)−1 (30 min)−1; means ± s.e.m., n = 3-8 different cell cultures.

*

P < 0.05 vs. control values in 5 mm d-glucose

†

P < 0.05 vs. values in cells cultured in 5 mm d-glucose and incubated with PMA

‡

P < 0.05 vs. control values in 25 mm d-glucose.

DISCUSSION

This study has established that NBMPR-sensitive (es) adenosine transport in human fetal vein endothelial cells is inhibited in a concentration- and time-dependent manner by elevated D-glucose. Inhibition of adenosine transport by D-glucose apparently involves PKC, NO and MAPK pathways. To our knowledge, these findings provide the first evidence that adenosine transport is modulated by extracellular D-glucose through protein kinase-dependent pathways in human fetal endothelial cells, implying that biological actions of adenosine can be altered under conditions of sustained hyperglycaemia.

Effect of D-glucose on adenosine transport

Hyperglycaemia induces alterations in endothelial function, such as inhibition of protein turnover, activation of L-arginine transport, and enhanced expression and activity of eNOS (Suschek et al. 1994; Sobrevia et al. 1996; Cosentino et al. 1997; Mann et al. 1998). In this study, we have shown that inhibition of adenosine transport in HUVECs exposed to elevated D-glucose is characterised by a reduction in the maximal velocity of transport (Vmax), with no detectable changes in the intrinsic properties of a single population of transporters. In HUVECs, elevated D-glucose has previously been shown to stimulate the Vmax for L-arginine and L-lysine transport, and the time course (8-12 h) and half-maximal inhibition (K½ = 10 ± 2 mM) of adenosine transport are similar to those in previous studies reporting D-glucose-induced stimulation of L-arginine transport and NO synthesis in HUVECs (Sobrevia et al. 1996) and inhibition of Na+,K+-ATPase activity in rabbit aorta (Gupta et al. 1992). Thus, the effects of D-glucose in human endothelial cells are not restricted solely to nucleoside transporters, as hyperglycaemia also alters the activity of other membrane transport proteins. In parallel studies we have amplified es (hENT1), but not ei (hENT2, insensitive to nanomolar concentrations of NBMPR), transporter mRNAs (see Baldwin et al. 1999) by reverse transcription polymerase chain reaction in HUVECs (data not shown). This observation supports our earlier kinetic studies (Sobrevia et al. 1994) and suggests the presence only of es-like nucleoside transporters in this cell type. Consistent with this, [3H]NBMPR was found to bind to a single population of high affinity sites in HUVECs cultured in low or high D-glucose, but with a decreased availability of es transporters in endothelial cells treated with 25 mM D-glucose.

Involvement of PKC in the inhibition of adenosine transport by elevated D-glucose

Hyperglycaemia has been shown to increase the activity of PKC in several cell types (Lee et al. 1989; King et al. 1996; Koya & King, 1998; reviewed in Poston & Taylor, 1995; Sobrevia & Mann, 1997). Moreover, activation of PKC is involved in the modulation of membrane transport of metabolic substrates, such as adenosine (Sen et al. 1993; Mun et al. 1998), uridine (Lee et al. 1991; Soler et al. 1998), L-arginine (Racké et al. 1998), L-taurine (Han et al. 1996), D-glucose (Davidson et al. 1991) and inositol (Guzmán & Crews, 1992). In our experiments, incubation of endothelial cells with the PKC activator PMA reduced initial rates of adenosine transport, an effect blocked by calphostin C, an inhibitor of DAG- and phorbol ester-sensitive PKC isozymes (Kobayashi et al. 1989). Parallel experiments demonstrated that PMA increased the activity of PKC in the membrane fraction of endothelial cells.

Inhibition of adenosine transport in human endothelial cells caused by PMA was similar to the findings reported for adenosine transport in chromaffin cells (Delicado et al. 1991), undifferentiated neuroblastoma cells (Sen et al. 1999), and T84 intestinal epithelial cells (Mun et al. 1998) and for uridine transport in human B lymphocytes (Soler et al. 1998). However, our results in HUVECs contrast with our previous findings in bovine adrenal medulla endothelial cells (BAMECs), where activation of PKC with PMA did not alter adenosine transport (Sen et al. 1996). As micro- and macrovascular endothelial cells exhibit structural and functional differences (see King et al. 1983), a lack of regulation of adenosine transport in BAMECs compared with HUVECs may be due to differences in the source of endothelial cells (micro vs. macrovessel), species (bovine vs. human) and/or adult vs. fetal endothelium.

The PKC isoforms α, ε and ζ (Ross & Joyner, 1997) and β1 (Deisher et al. 1993) have been reported to be the most prominent isoforms in human umbilical vein endothelial cells. Since the PKCα and PKCε isozymes are phorbol ester sensitive and inhibited by calphostin C (Kobayashi et al. 1989), it is likely that these isoforms are involved in the inhibition of adenosine transport induced by PMA treatment.

Incubation of HUVECs with high D-glucose has been shown to induce the activities of PKCα and ε (Morigi et al. 1998). Our results confirm that elevated D-glucose increased the activity of PKC in the membrane fraction, but decreased its activity in the cytosol fraction of endothelial cells, an effect that was blocked by calphostin C. Thus, D-glucose-sensitive PKCα and ε isoforms may mediate the inhibitory actions of elevated D-glucose on adenosine transport.

Involvement of MAPK in D-glucose-mediated inhibition of adenosine transport

In the present study, phosphorylation of p42-44mapk was induced by incubation of human endothelium with elevated D-glucose. When endothelial cells were exposed to PD-98059 (an inhibitor of MEK-1) or genistein (a general tyrosine kinase inhibitor), D-glucose-induced inhibition of adenosine transport was abolished. These results suggest that the phosphorylation of p42-p44mapk induced by elevated D-glucose is involved in the inhibition of adenosine transport. The ability of elevated D-glucose to activate p42-p44mapk is consistent with previous reports in mesangial cells (Hayama et al. 1997; Haneda et al. 1997), isolated pancreatic islets (Burns et al. 1997), the murine β-pancreatic cell line MIN6 (Benes et al. 1998), the glucose-sensitive insulinoma cell line INS-1 (Frödin et al. 1995; Khoo & Cobb, 1997) and rat aorta smooth muscle cells (Natarajan et al. 1999).

The present study also shows that the PKC activity induced by D-glucose or PMA was unaltered when endothelial cells were co-incubated with the MEK-1 inhibitor PD-98059, suggesting that the increase in PKC activity does not depend on the activation of the MEK-1 pathway in HUVECs. Phosphorylation of p42-p44mapk was induced in HUVECs challenged with PMA, confirming a previous report in this cell type (May et al. 1996). PMA-induced phosphorylation of p42-44mapk was blocked by calphostin C and PD-98059. PMA has been shown to induce p42-44mapk phosphorylation via activation of PKCα and ζ in rat mesangial cells (Haneda et al. 1997). Therefore, activation of these PKC isoforms by elevated D-glucose could result in a downstream regulation of MEK-1 in human endothelium.

Involvement of nitric oxide in D-glucose-mediated inhibition of adenosine transport

NO has been shown to inhibit L-arginine transport (Patel et al. 1996; Ogonowski et al. 2000) and to stimulate L-cystine uptake (Li et al. 1999) in endothelial cells. When adenosine transport was determined in endothelial cells incubated with the eNOS inhibitor L-NAME, transport was increased in HUVECs. In addition, adenosine transport was decreased in HUVECs treated with the NO donor SNAP, suggesting that NO downregulates NMBPR-sensitive adenosine transport in human endothelial cells. The finding that adenosine transport in endothelial cells cultured under hyperglycaemic conditions was not further inhibited by SNAP suggests that the D-glucose-stimulated NO levels are sufficient to induce a maximal inhibition of system es in this cell type. Aliquots of 100 μM SNAP can generate ∼200 nM NO in solution (Ogonowski et al. 2000), thus it is feasible that similar levels of NO are produced by endothelial cells cultured in high D-glucose. The possibility that elevated NO in endothelial cells in hyperglycaemia is inhibiting adenosine transport is further supported by the results showing that incubation of cells with L-NAME leads to an increased transport of adenosine. Thus, in hyperglycaemia NO levels seem to be directly involved in the modulation of es transport system activity. The modulatory action of NO on nucleoside transport has recently been demonstrated in human B lymphocytes, where NO increases uridine transport via systems N1 and N5 (concentrative, Na+-dependent transporters), but decreases the activity of es transporters (Soler et al. 2000). It is worth noting that our present results in HUVECs contrast with our report that NBMPR-sensitive adenosine transport in human umbilical artery smooth muscle cells is increased by NO and cGMP but downregulated by cAMP (Aguayo & Sobrevia, 2000). Thus, modulation of the es system by NO seems to be different in vascular endothelial and smooth muscle cells, which could influence extracellular adenosine concentrations.

D-Glucose stimulates the expression and activity of eNOS in human endothelial cells (Sobrevia et al. 1996; Cosentino et al. 1997; Mann et al. 1998). Consistent with these results, we found that HUVECs cultured in the presence of elevated D-glucose exhibit a higher L-NAME-inhibitable and Ca2+-dependent formation of L-[3H]citrulline from L-[3H]arginine compared with cells cultured in 5 mM D-glucose. It has also been been reported that PKCα or PKCε enhances transcription of the eNOS gene in HUVECs (H. Li et al. 1998). In our experiments, the elevated PKC activity in endothelial cells exposed to D-glucose was unaltered following inhibition of eNOS and/or MEK-1 or when cells were exposed to the NO donor SNAP, indicating that D-glucose stimulation of PKC activity is independent of NO levels or MAPK phosphorylation.

It has been reported that NO induces phosphorylation of p42-44mapk through a protein kinase G-mediated activation of Raf-1, a serine/threonine kinase, in coronary postcapillary vein endothelium (Parenti et al. 1998; Hood & Granger, 1998). In HUVECs, D-glucose-induced phosphorylation of p42mapk was significantly inhibited in cells preincubated with the NO synthase inhibitor L-NAME. Further studies of adenosine transport established that the inhibitory effect of SNAP-derived NO was blocked following inhibition of MEK-1. In addition, incubation of endothelial cells with SNAP led to PD-98059-inhibitable phosphorylation of p42-44mapk.

In summary, our findings in human fetal venous endothelial cells establish that elevated D-glucose inhibits adenosine transport through a mechanism involving a reduction in the number of NBMPR-sensitive nucleoside transporters. Based on our results, we propose that the effect of hyperglycaemia on adenosine transport involves activation of DAG/phorbol ester-sensitive PKC isozymes leading to an activation of eNOS, followed by phosphorylation of p42-p44mapk and inhibition of adenosine transport. These studies extend our previous results in human fetal endothelium isolated from diabetic patients, where the maximal transport rate and the number of adenosine transporters were reduced compared with endothelial cells from non-diabetic patients (Sobrevia et al. 1994). Thus, inhibition of adenosine transport and metabolism in diabetes could be associated with elevated extracellular D-glucose levels, leading to increased extracellular levels of adenosine, which in turn may activate A2a-purinoceptors and NO production (Sobrevia et al. 1997). Enhanced NO production may account for the increased blood flow in certain organs in the early stages of diabetes but serves as a failing counter-regulatory mechanism in established diabetes.

Acknowledgments

This study was supported by Fondo Nacional de Ciencia y Tecnología (FONDECYT 1971321, 1000354 and 7000354), Dirección de Investigación (9733871-ID)-Universidad de Concepción (Chile) and the Wellcome Trust (UK). V. P. Montecinos and C. Aguayo hold CONICYT and MECESUP (Chile) PhD fellowships, respectively. C. Flores holds a University of Concepción MSc fellowship. We thank Ms Susana Rojas for her expert technical assistance in cell culture, the midwives of Hospital Las Higueras-Talcahuano and Hospital Regional-Concepción labour ward for the generous supply of umbilical cords, and Miss Isabel Jara for excellent secretarial assistance.

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