Testosterona inhibe la actividad de la aldosterona sintasa silvestre y quimérica in vitro

Background: Familial hyperaldosteronism type I is caused by the generation of a chimeric aldosterone synthase enzyme (ASCE) which is regulated by ACTH instead of angiotensin II. We have reported that in vitro, the wild-type (ASWT) and chimeric aldosterone synthase (ASCE) enzymes are inhibited by progesterone and estradiol does not affect their activity. Aim: To explore the direct action of testosterone on ASWT and ASCE enzymes. Material and Methods: HEK-293 cells were transiently transfected with vectors containing the full ASWT or ASCE cDNAs. The effect of testosterone on AS enzyme activities was evaluated incubating HEK-cells transfected with enzyme vectors and adding deoxycorticosterone (DOC) alone or DOC plus increasing doses of testosterone. Aldosterone production was measured by HPLC-MS/MS. Docking of testosterone within the active sites of both enzymes was performed by modelling in silico. Results: In this system, testosterone inhibited ASWT (90% inhibition at five pM, 50% inhibitory concentration (IC50) =1.690 pM) with higher efficacy andpotency than ASCE (80% inhibition at five pM, IC50=3.176 pM). Molecular modelling studies showed different orientation of testosterone in ASWT and ASCE crystal structures. Conclusions: The inhibitory effect of testosterone on ASWT or ASCE enzymes is a novel non-genomic testosterone action, suggesting that further clinical studies are needed to assess the role of testosterone in the screening and diagnosis of primary aldosteronism.

Sodium Intake Is associated With Endothelial Damage Biomarkers and Metabolic Dysregulation.

BACKGROUND: Mounting evidence has associated high sodium (HS) intake with hypertension, cardiovascular disease, and stroke. We investigated whether HS intake modulates the parameters of endothelial damage, inflammation, and oxidative stress. METHODS: We used a cross-sectional study design including 223 Chilean subjects (6.9-65.0 years old). We measured aldosterone, renin activity, cortisol, cortisone, adiponectin, leptin, hsCRP, interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), plasminogen activator inhibitor type 1 (PAI-1), metalloproteinase (MMP)-9 and MMP-2 activity, and malondialdehyde. Sodium and creatinine were measured in 24-hour urine samples. The subjects were divided by sodium intake, high sodium (HS): ≥150 mEq/day, n = 118, and adequate sodium (AS): <150 mEq/day, n = 105. RESULTS: We observed a positive correlation between urinary sodium excretion and blood pressure (r = 0.1669, P = 0.0124 for systolic and r = 0.2416, P = 0.0003 for diastolic), glycemia (r = 0.2660, P < 0.0001), and triglycerides (r = 0.1604, P = 0.0175) and a highly significant correlation between sodium excretion and PAI-1 (r = 0.2701, P < 0.0001). An inverse correlation was observed between urinary sodium and HDL-cholesterol (r = -0.2093, P = 0.0018) and adiponectin (r = -0.2679, P < 0.0001). In a linear regression model, urinary sodium excretion remained significantly associated with PAI-1 values even after adjusting for age, gender, and BMI. The HS group had higher blood pressure, glycemia, HOMA-IR, atherogenic index of plasma, and PAI-1 values than the group with AS intake. CONCLUSIONS: HS intake is associated with endothelial damage (high PAI-1) and metabolic dysregulation. On the other hand, inflammation and oxidative stress parameters are not modified by sodium intake.

Estudio e identificación de microRNA en exosomas urinarios en hipertensión arterial mineralocorticoide / Study and identification of microRNA in urinary exosomes in mineralocorticoid arterial hypertension

About 15% of the essential hypertensive patients would have a low activity of the 11ßHSD2 enzyme, which inactivates cortisol (F) to cortisone (E). Gene expression can be negatively regulated by miRNA. Urinary exosomes and their specific content (miRNA/proteins) represent a valuable tool as a biomarker for the diagnosis and prognosis of the disease. Aim: To evaluate the expression of miRNA specific for 11ßHSD2 in samples of urinary exosomes and to determine its association with biochemical variables associated with mineralocorticoid metabolism. Subjects and Methods: Cross-sectional study in subjects between 10-60 years. They were classified into subjects with high F/E (> p75) and low cortisone (< p25) and control subjects. The urinary exosomes were isolated with the Invitrogen kit. Bioinformatic analysis was performed with Mir Walk to identify specific miRNAs of HSD11B2. The expression of miRNA was evaluated by qRT PCR. The comparisons were made with the Mann-Whitney test. Results: 7.1% of the subjects are suggestive of a partial deficiency of 11ßHSD2 (NC-AME). The expression of miR-488 was higher in NC-AME than in controls (5839 ± 1719 vs 3,437 ± 2,581; p = 0.01). We found positive associations between mir-615 and ARP; miR-488 and the sodium/potassium ratio; miR-1205 with age and urinary sodium excretion; miR-494 with age, activity MMP9 and NGAL. Conclusion: We identified high expression of miR488 in NC-AME subjects and associations of miRNAs with biochemical variables associated with mineralocorticoid metabolism. Thus, exosomes and their miRNA content could be potential regulators and biomarkers of 11ßHSD2 activity.

Cortisol/cortisone ratio and matrix metalloproteinase-9 activity are associated with pediatric primary hypertension.

OBJECTIVE: To identify novel biomarkers associated with pediatric primary hypertension. METHODS: We recruited 350 participants (4-16 years). Anthropometric parameters and aldosterone, plasma renin activity, cortisol, cortisone, Homeostasis Model Assessment Insulin Resistance (HOMA-IR), high-sensitivity C-reactive protein, adiponectin, IL-6, plasminogen activator inhibitor type 1 levels and matrix metalloproteinase-9 and matrix metalloproteinase-2 (MMP-9 and MMP-2) activities were measured. Genomic DNA was isolated. Patients with altered glucose metabolism, severe obesity [BMI-SD score (BMI-SDS) > 2.5], renovascular disease, primary aldosteronism and apparent mineralocorticoid excess syndrome were excluded. RESULTS: In selected participants (n = 320), SBP was positively correlated with BMI-SDS (r = 0.382, P < 0.001), HOMA-IR (r = 0.211, P < 0.001), MMP-9 activity (r = 0.215, P < 0.001) and the cortisol/cortisone ratio (r = 0.231, P < 0.001). DBP showed similar correlations with these variables. No correlation was observed with aldosterone or plasma renin activity. Participants were categorized as hypertensive (n = 59) or nonhypertensive (n = 261). In the univariate analysis, hypertensive patients had higher BMI-SDS (P < 0.001), HOMA-IR (P < 0.001), high-sensitivity C-reactive protein (P < 0.001), MMP-9 activity (P < 0.001), plasminogen activator inhibitor type 1 (P < 0.001) and cortisol/cortisone ratio (P < 0.001) than nonhypertensive patients. Multiple regression analysis showed that the variables that remained associated with hypertension were higher BMI-SDS [odds ratio (OR) = 3.74; 95% confidence interval (CI) = 1.84-7.58], a higher cortisol/cortisone ratio (OR = 3.92; 95% CI = 1.98-7.71) and increased MMP-9 activity (OR = 4.23; 95% CI = 2.15-8.32). CONCLUSION: We report that MMP-9 activity and the cortisol/cortisone ratio were higher in pediatric primary hypertensive patients, and these associations were independent of the effect of obesity. The potential role of these novel biomarkers in predicting hypertension risk and blood pressure regulation warrants further investigation.

Expression Pattern of Scavenger Receptors and Amyloid-ß Phagocytosis of Astrocytes and Microglia in Culture are Modified by Acidosis: Implications for Alzheimer's Disease.

The pathological hallmarks of Alzheimer's disease (AD) are amyloid-ß (Aß) plaques, neurofibrillary tangles, and glia activation. The pathology also includes vascular amyloidosis and cerebrovascular disease. Vascular compromise can result in hypoperfusion, local tissue hypoxia, and acidosis. Activated microglia and astrocytes can phagocytose Aß through membrane receptors that include scavenger receptors. Changes in glial cells induced by extracellular acidosis could play a role in the development of AD. Here, we assess whether extracellular acidosis changes glial cell properties relevant for Aß clearance capacity. Incubation of glial cells on acidified culture medium (pH 6.9 or 6.5) for 24-48 h resulted in decreased cell diameter, with thinner branches in astrocytes, slight reduction in cell body size in microglia, a transient decrease in astrocyte adhesion to substrates, and a persistent decrease in microglia adhesion compared with control media (pH 7.4). Astrocyte Aß phagocytosis decreased at pH 6.9 and 6.5, whereas microglia phagocytosis only transiently decreased in acidified media. Scavenger receptors class B member I (SR-BI) increased and scavenger receptors-macrophage receptors with collagenous structures (SR-MARCO) decreased in astrocytes cultured at pH 6.5. In contrast, in microglia exposed to pH 6.5, expression of SR-BI and SR-MARCO increased and fatty acid translocase (CD-36) decreased. In conclusion, the acidic environment changed the adhesiveness and morphology of both microglia and astrocytes, but only astrocytes showed a persistent decrease in Aß clearance activity. Expression of scavenger receptors was affected differentially in microglia and astrocytes by acidosis. These changes in scavenger receptor patterns can affect the activation of glia and their contribution to neurodegeneration.

The Expression of RAC1 and Mineralocorticoid Pathway-Dependent Genes are Associated With Different Responses to Salt Intake.

BACKGROUND: Rac1 upregulation has been implicated in salt-sensitive hypertension as a modulator of mineralocorticoid receptor (MR) activity. Rac1 could affect the expression of oxidative stress markers, such as hemoxigenase-1 (HO-1) or nuclear factor-B (NF-κB), and the expression of neutrophil gelatinase-associated lipocalin (NGAL), a cytokine upregulated upon MR activation. AIM: We evaluated RAC1 expression in relation of high salt intake and association with MR, NGAL, HO-1, and NF-κB expression, mineralo- and glucocorticoids levels, and inflammatory parameters. SUBJECTS AND METHODS: We studied 147 adult subjects. A food survey identified the dietary sodium (Na) intake. RAC1 expression was considered high or low according to the value found in normotensive subjects with low salt intake. We determined the gene expression of RAC1, MR, NGAL, HO-1, NF-κB, and 18S, isolated from peripheral leukocytes. We measured aldosterone, cortisol, sodium, potassium excretion, metalloproteinase (MMP9 y MMP2), and C-reactive protein. RESULTS: We identified 126 subjects with high Na-intake, 18 subjects had high, and 108 low-RAC1 expression. The subjects with high-RAC1 expression showed a significant increase in MR (P = 0.0002), NGAL (P < 0.0001) HO-1 (P = 0.0004), and NF-κB (P < 0.0001) gene expression. We demonstrated an association between RAC1 expression and MR (R sp 0.64; P < 0.0001), NGAL (R sp 0.48; P < 0.0001), HO-1 (R sp 0.53; P < 0.0001), and NF-κB (R sp0.52; P < 0.0001). We did not identify any association between RAC1 and clinical or biochemical variables. CONCLUSIONS: RAC1 expression was associated with an increase in MR, NGAL, NF-κB, and HO-1 expression, suggesting that RAC1 could be a mediator of cardiovascular damage induced by sodium, and may also useful to identify subjects with different responses to salt intake.

LC-MS/MS Method for the Simultaneous Determination of Free Urinary Steroids.

Cortisol homeostasis is implicated in hypertension and metabolic syndrome. Two enzymes modulate cortisol availability; 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) preferentially converts inactive cortisone to cortisol, whereas 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) converts cortisol to cortisone. In contrast, 5α and 5ß reductases inactivate cortisol by conversion to its tetrahydrometabolites: tetrahydrocortisol, allo-tetrahydrocortisol and tetrahydrocortisone. A subtle local increase in cortisol can be detected by measuring 24-h urine metabolites, LC-MS/MS being the reference method. The 11ß-HSD2 activity is assessed based on the cortisol/cortisone ratio, and the 11ß-HSD1 activity on the (tetrahydrocortisol + allo-tetrahydrocortisol)/tetrahydrocortisone ratio. To better understand hypertension and/or metabolic syndrome pathogenesis a method for simultaneous determination of cortisol, cortisone, tetrahydrocortisol, allo-tetrahydrocortisol and tetrahydrocortisone was developed and validated in an LC coupled with the new detector AB Sciex QTrap® 4500 tandem mass spectrometer. The steroids were extracted from 1 mL urine, using cortisol-D4 as internal standard. The quantification range was 0.1-120 ng/mL for cortisol and cortisone, and 1-120 ng/mL for tetrahydrometabolites, with >89 % recovery for all analytes. The coefficient of variation and accuracy was <10 %, and 85-105 %, respectively. Our LC-MS/MS method is accurate and reproducible in accordance with Food and Drug Administration guidelines, showing good sensitivity and recovery. This method allows the assessment of 11ß-HSD2 and 11ß-HSD1 activities in a single analytical run providing an innovative tool to explain etiology of misclassified essential hypertension and/or metabolic syndrome.

Polymorphisms in the RAC1 gene are associated with hypertension risk factors in a Chilean pediatric population.

BACKGROUND: The GTPase Rac1 has been implicated in hypertension as a modulator of mineralocorticoid receptor activity. Our aim is to investigate the frequency of polymorphisms rs10951982 (intron 1, G>A) and rs836478 (intron 3, T>C) in the RAC1 gene and perform association studies with clinical and biochemical parameters in a Chilean pediatric cohort. METHODS: Two hundred two normotensive (NT) subjects (aged 4-16 years) were divided into 2 groups: NT subjects with hypertensive parents (NH; n = 103) and NT subjects with NT parents (NN; n = 99). We measured markers of inflammation (high-sensitivity C-reactive protein, interleukin 6 (IL-6), interleukin 8, and tumor necrosis factor α), endothelial damage (Plasminogen activator inhibitor-1 metalloproteinase-9, and metalloproteinase-2), and oxidative stress (malondialdehyde). Data were expressed as median and interquartile range (IQR). RESULTS: We found differences in polymorphism rs836478 (intron 3, C>T) in both genotypic (χ(2) = 15.2, 2 df; P = 0.0005) and allelic (X(2)=5.5, 1 df; P = 0.01) frequencies in NH vs. NN subjects. NH subjects with a TT genotype showed increase MMP9 expression (median = 2.3, IQR - 1.6-3.2; vs. median = 1.6, IQR = 1.6-2.3 AU; P = 0.01) and lower IL-6 expression (median = 8.8, IQR = 7.0-11.8; vs. median = 12.1, IQR = 8.2-14.7 pg/ml; P = 0.02) compared with subjects with TC/CC genotype. No difference in the allelic frequency distribution was seen in the polymorphism rs10951982 (NH vs. NN: χ(2)=0.2, 1 df; P = 0.6). For this SNP, NN subjects with GA/AA genotype showed decreased diastolic BP indexes compared with subjects with native GG genotype (median = 1.08, IQR = 1.0-1.2; vs. median = 0.99, IQR = 0.94-1.1; P = 0.02). CONCLUSIONS: We report the frequency of polymorphisms rs836478 and rs10951982 of the RAC1 gene in a Spanish-Amerindian cohort. The polymorphism rs836478 was associated with an increased expression in markers of inflammation and endothelial damage (MMP9 and IL-6) in pediatric subjects with a hypertensive genetic background.

Identification of novel 11ß-HSD1 inhibitors by combined ligand- and structure-based virtual screening.

11 beta-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) converts cortisone to cortisol in a NADPH dependent manner. Overexpression of 11ß-HSD1 in key metabolic tissues is related to the development of type 2 diabetes, obesity, hypertension and metabolic syndrome. Using crystal structures of human 11ß-HSD1 in complex with inhibitors as source of structural information, a combined ligand and structure-based virtual screening approach was implemented to identify novel 11ß-HSD1 inhibitors. A selected group of compounds was identified in silico and further evaluated in cell-based assays for cytotoxicity and 11ß-HSD1 mediated cortisol production inhibitory capacity. The expression of 11ß-HSD1 and 11ß-HSD2 in human LS14 adipocytes was assessed during differentiation. Biological evaluation of 39 compounds in adipocytes and steroids quantification by HPLC-MS/MS identify 4 compounds that exhibit 11ß-HSD1 mediated cortisol production inhibitory activity with potencies in the micromolar range. Two compounds showed to be selective for the 11ß-HSD1 reductase activity and over 11ß-HSD2 isoform, and thus represent novel leads for the development of more active derivatives with higher efficacies targeting intracellular cortisol levels in type 2 diabetes and metabolic syndrome.

Different effects of progesterone and estradiol on chimeric and wild type aldosterone synthase in vitro.

BACKGROUND: Familial hyperaldosteronism type I (FH-I) is caused by the unequal recombination between the 11beta-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) genes, resulting in the generation of a CYP11B1/B2 chimeric gene and abnormal adrenal aldosterone production. Affected patients usually show severe hypertension and an elevated frequency of stroke at a young age. Aldosterone levels rise during pregnancy, yet in pregnant women with FH-1, their hypertensive condition either remains unchanged or may even improve. The purpose of this study was to investigate in vitro whether female sex steroids modulate the activity of chimeric (ASCE) or wild type (ASWT) aldosterone synthase enzymes. METHODS: We designed an in vitro assay using HEK-293 cell line transiently transfected with vectors containing the full ASCE or ASWT cDNAs. Progesterone or estradiol effects on AS enzyme activities were evaluated in transfected cells incubated with deoxycorticosterone (DOC) alone or DOC plus increasing doses of these steroids. RESULTS: In our in vitro model, both enzymes showed similar apparent kinetic parameters (Km = 1.191 microM and Vmax = 27.08 microM/24 h for ASCE and Km = 1.163 microM and Vmax = 36.98 microM/24 h for ASWT; p = ns, Mann-Whitney test). Progesterone inhibited aldosterone production by ASCE- and ASWT-transfected cells, while estradiol demonstrated no effect. Progesterone acted as a competitive inhibitor for both enzymes. Molecular modelling studies and binding affinity estimations indicate that progesterone might bind to the substrate site in both ASCE and ASWT, supporting the idea that this steroid could regulate these enzymatic activities and contribute to the decay of aldosterone synthase activity in chimeric gene-positive patients. CONCLUSIONS: Our results show an inhibitory action of progesterone in the aldosterone synthesis by chimeric or wild type aldosterone synthase enzymes. This is a novel regulatory mechanism of progesterone action, which could be involved in protecting pregnant women with FH-1 against hypertension. In vitro, both enzymes showed comparable kinetic parameters, but ASWT was more strongly inhibited than ASCE. This study implicates a new role for progesterone in the regulation of aldosterone levels that could contribute, along with other factors, to the maintenance of an adequate aldosterone-progesterone balance in pregnancy.

Differences in acute lung response to elastase instillation in two rodent species may determine differences in severity of emphysema development.

Elastase intratracheal instillation induces early emphysema in rodents. However, Syrian Golden hamsters develop more severe emphysema than Sprague-Dawley rats. We have reported species differences in oxidant/antioxidant balance modulating antiprotease function early after instillation. We now hypothesize that other components of the initial lung response to elastase might also be species-dependent. Sprague-Dawley rats and Syrian Golden hamsters received a single dose of pancreatic elastase (0.55 U/100 g body wt) to study acute lung injury biomarkers. Using serum, lung, and bronchoalveolar lavage fluid (BALF) samples, we evaluated changes in alveolar-capillary permeability, alpha 1-antitrypsin (α(1)-AT) concentration and activity, glutathione content, and proinflammatory cytokines. Rats showed a large increase in alveolar-capillary permeability and few hemorrhagic changes, whereas hamsters exhibited large hemorrhagic changes (P < 0.01) and mild transendothelial passage of proteins. Western blots showed a 30-fold increase in BALF α(1)-AT concentration in rats and only a 7-fold increase in hamsters (P < 0.001), with [α(1)-AT-elastase] complexes only in rats, suggesting differences in antiprotease function. This was confirmed by the α(1)-AT bioassay showing 20-fold increase in α(1)-AT activity in rats and only twofold increase in hamsters (P < 0.001). In rats, results were preceded by a 3-, 60-, and 20-fold increase in IL-6, IL-1ß, and TNF-α respectively (P < 0.001). In hamsters, only IL-1ß and TNF-α showed mild increases. All parameters studied were back to baseline by 4 days. In conclusion, several components of the initial lung response showed species differences. Cytokine release pattern and functional inhibition of α(1)-AT were the most significant components differing among species and could account for differences in susceptibility to elastase.

Differences in lung glutathione metabolism may account for rodent susceptibility in elastase-induced emphysema development.

Syrian Golden hamsters develop more severe emphysema than Sprague-Dawley rats after intratracheal instillation of the same dose of elastase/body weight. Although species variations in antielastase defenses may largely explain these results, other variables, such as differences in lung antioxidants, cannot be overlooked since oxidative stress modulates antiprotease activity. We propose that elastase instillation might affect lung glutathione (GSH) metabolism differently in these species. Our aim was to study in hamsters and rats, lung glutathione metabolism at different times, from the stage of diffuse alveolar damage to advanced emphysema. We measured total and oxidized glutathione content as well as activity and expression of enzymes related to GSH synthesis and redox cycling: gamma-glutamylcysteine synthetase, glutathione peroxidase, and glutathione reductase. Whereas rats showed no significant changes in these measurements, hamsters showed significant derangement in GSH metabolism early after elastase instillation: 25% fall in total GSH (P < 0.05) with no increase in oxidized glutathione associated with reduced enzyme activities 24 h after elastase [60% for gamma-glutamylcysteine synthetase (P < 0.01), 30% for glutathione peroxidase (P < 0.01), and 75% for glutathione reductase (P < 0.001)]. GSH homeostasis was restored at the end of the first week, involving transient increased expression of these enzymes. We conclude that elastase induces significant alterations in GSH metabolism of hamster lungs and no overall change in rat lungs. Although differences in disease severity may account for our findings, the hamster becomes vulnerable to functional inhibition of alpha(1)-antitrypsin by oxidants and thus, even more susceptible to injury than it would be, considering only its low alpha(1)-antitrypsin level.

High D-glucose reduces SLC29A1 promoter activity and adenosine transport involving specific protein 1 in human umbilical vein endothelium.

High D-glucose reduces human equilibrative nucleoside transporter 1 (hENT1)-mediated adenosine uptake involving endothelial nitric oxide synthase (eNOS), mitogen-activated protein (MAP) kinase kinases 1 and 2/MAP kinases p42/44 (MEK/ERKs), and protein kinase C (PKC) activation in human umbilical vein endothelium (HUVEC). Since NO represses SLC29A1 gene (hENT1) promoter activity we studied whether D-glucose-reduced hENT1-adenosine transport results from lower SLC29A1 expression in HUVEC primary cultures. HUVEC incubation (24 h) with high D-glucose (25 mM) reduced hENT1-adenosine transport and pGL3-hENT1(-1114) construct SLC29A1 reporter activity compared with normal D-glucose (5 mM). High D-glucose also reduced pGL3-hENT1(-1114) reporter activity compared with cells transfected with pGL3-hENT1(-795) construct. N(G)-nitro-L-arginine methyl ester (L-NAME, NOS inhibitor), PD-98059 (MEK1/2 inhibitor), and/or calphostin C (PKC inhibitor) blocked D-glucose effects. Insulin (1 nM) and phorbol 12-myristate 13-acetate (PMA, 100 nM, PKC activator), but not 4alpha-phorbol 12,13-didecanoate (4alphaPDD, 100 nM, PMA less active analogue) reduced hENT1-adenosine transport. L-NAME and PD-98059 blocked insulin effects. L-NAME, PD-98059, and calphostin C increased hENT1 expression without altering protein or mRNA stability. High D-glucose increased Sp1 transcription factor protein abundance and binding to SLC29A1 promoter, phenomena blocked by L-NAME, PD-98059, and calphostin C. Sp1 overexpression reduced SLC29A1 promoter activity in normal D-glucose, an effect reversed by L-NAME and further reduced by S-nitroso-N-acetyl-L,D-penicillamine (SNAP, NO donor) in high D-glucose. Thus, reduced hENT1-mediated adenosine transport in high D-glucose may result from increased Sp1 binding to SLC29A1 promoter down-regulating hENT1 expression. This phenomenon depends on eNOS, MEK/ERKs, and PKC activity, suggesting potential roles for these molecules in hyperglycemia-associated endothelial dysfunction.

Insulin restores glucose inhibition of adenosine transport by increasing the expression and activity of the equilibrative nucleoside transporter 2 in human umbilical vein endothelium.

L-Arginine transport and nitric oxide (NO) synthesis (L-arginine/NO pathway) are stimulated by insulin, adenosine or elevated extracellular D-glucose in human umbilical vein endothelial cells (HUVEC). Adenosine uptake via the human equilibrative nucleoside transporters 1 (hENT1) and 2 (hENT2) has been proposed as a mechanism regulating adenosine plasma concentration, and therefore its vascular effects in human umbilical veins. Thus, altered expression and/or activity of hENT1 or hENT2 could lead to abnormal physiological plasma adenosine level. We have characterized insulin effect on adenosine transport in HUVEC cultured in normal (5 mM) or high (25 mM) D-glucose. Insulin (1 nM) increased overall adenosine transport associated with higher hENT2-, but lower hENT1-mediated transport in normal D-glucose. Insulin increased hENT2 protein abundance in normal or high D-glucose, but reduced hENT1 protein abundance in normal D-glucose. Insulin did not alter the reduced hENT1 protein abundance, but blocked the reduced hENT1 and hENT2 mRNA expression induced by high D-glucose. Insulin effect on hENT1 mRNA expression in normal D-glucose was blocked by N(G)-nitro-L-arginine methyl ester (L-NAME, NO synthase inhibitor) and mimicked by S-nitroso-N-acetyl-L,D-penicillamine (SNAP, NO donor). L-NAME did not block insulin effect on hENT2 expression. In conclusion, insulin stimulation of overall adenosine transport results from increased hENT2 expression and activity via a NO-independent mechanism. These findings could be important in hyperglycemia-associated pathological pregnancies, such as gestational diabetes, where plasma adenosine removal by the endothelium is reduced, a condition that could alter the blood flow from the placenta to the fetus affecting fetus growth and development.