Rody San Martín

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

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.

Equilibrative Nucleoside Transporter 1 Expression Is Downregulated by Hypoxia in Human Umbilical Vein Endothelium

Reduced oxygen level (hypoxia) induces endothelial dysfunction and release of the endogenous nucleoside adenosine. Human umbilical vein endothelium (HUVEC) function in an environment with 3% to 5% O2 and exhibit efficient adenosine membrane transport via human equilibrative nucleoside transporters 1 (hENT1). We studied whether adenosine transport and hENT1 expression are altered by hypoxia in HUVEC. Hypoxia (0 to 24 hours, 2% and 1% O2) reduced maximal hENT1-adenosine transport velocity (Vmax) and maximal nitrobenzylthionosine (NBMPR, a high-affinity hENT1 protein ligand) binding, but increased extracellular adenosine concentration. Hypoxia also reduced hENT1 protein and mRNA levels, effects unaltered by N&ohgr;-nitro-l-arginine methyl ester (l-NAME, nitric oxide synthase [NOS] inhibitor) or PD-98059 (inhibitor of mitogen-activated protein kinase kinase 1 and 2 [MEK1/2]). Hypoxia reduced endothelial NOS (eNOS) activity and eNOS phosphorylation at Ser1177, but increased eNOS protein level. Hypoxia increased (1 to 3 hours), but reduced (24 hours) p42/44mapk phosphorylation. Thus, hypoxia-increased extracellular adenosine may result from reduced hENT1-adenosine transport in HUVEC. Hypoxia effect seems not to involve NO, but p42/44mapk may be required for the relatively rapid effect (1 to 3 hours) of hypoxia. These results could be important in diseases where the fetus is exposed to intrauterine environments poor in oxygen, such as intrauterine growth restriction, or where adenosine transport is altered, such as gestational diabetes.

Nitric oxide reduces adenosine transporter ENT1 gene (SLC29A1) promoter activity in human fetal endothelium from gestational diabetes.

Human umbilical vein endothelial cells (HUVEC) from gestational diabetes exhibit reduced adenosine uptake and increased nitric oxide (NO) synthesis. Adenosine transport via human equilibrative nucleoside transporters 1 (hENT1) is reduced by NO by unknown mechanisms in HUVEC. We examined whether gestational diabetes‐reduced adenosine transport results from lower hENT1 gene (SLC29A1) expression. HUVEC from gestational diabetes exhibit reduced SLC29A1 promoter activity when transfected with pGL3‐hENT1−2154 compared with pGL3‐hENT1−1114 constructs, an effect blocked by NG‐nitro‐L‐arginine methyl ester (L‐NAME, NOS inhibitor), but unaltered by S‐nitroso‐N‐acetyl‐L,D‐penicillamine (SNAP, NO donor). In cells from gestational diabetes transfected with pGL3‐hENT1−2154, L‐NAME increased, but SNAP did not alter promoter activity and hENT1 expression. However, in cells from normal pregnancies L‐NAME increased, but SNAP reduced promoter activity and hENT1 expression. Adenovirus‐silenced eNOS expression increased hENT1 expression and activity in cells from normal or gestational diabetic pregnancies. Thus, reduced adenosine transport may result from downregulation of SLC29A1 expression by NO in HUVEC from gestational diabetes. These findings explain the accumulation of extracellular adenosine detected in cultures of HUVEC from gestational diabetes. In addition, fetal endothelial dysfunction could be involved in the abnormal fetal development and growth seen in gestational diabetes. J. Cell. Physiol. 208: 451–460, 2006.

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 NG‐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. J. Cell. Physiol. 209: 826–835, 2006.

5'-ectonucleotidase mediates multiple-drug resistance in glioblastoma multiforme cells.

Glioblastoma multiforme (GBM) cells are characterised by their extreme chemoresistance. The activity of multiple‐drug resistance (MDR) transporters that extrude antitumor drugs from cells plays the most important role in this phenomenon. To date, the mechanism controlling the expression and activity of MDR transporters is poorly understood. Activity of the enzyme ecto‐5′‐nucleotidase (CD73) in tumor cells, which hydrolyses AMP to adenosine, has been linked to immunosuppression and prometastatic effects in breast cancer and to the proliferation of glioma cells. In this study, we identify a high expression of CD73 in surgically resected samples of human GBM. In primary cultures of GBM, inhibition of CD73 activity or knocking down its expression by siRNA reversed the MDR phenotype and cell viability was decreased up to 60% on exposure to the antitumoral drug vincristine. This GBM chemosensitization was caused by a decrease in the expression and activity of the multiple drug associated protein 1 (Mrp1), the most important transporter conferring multiple drug resistance in these cells. Using pharmacological modulators, we have recognized the adenosine A3 receptor subtype in mediation of the chemoresistant phenotype in these cells. In conclusion, we have determined that the activity of CD73 to trigger adenosine signaling sustains chemoresistant phenotype in GBM cells. J. Cell. Physiol. 228: 602–608, 2013.

Adenosine A 2B receptor-mediated VEGF induction promotes diabetic glomerulopathy

Diabetic nephropathy ranks as the most devastating kidney disease worldwide. It characterizes in the early onset by glomerular hypertrophy, hyperfiltration and mesangial expansion. Experimental models show that overproduction of vascular endothelial growth factor (VEGF) is a pathogenic condition for podocytopathy; however the mechanisms that regulate this growth factor induction are not clearly identified. We determined that the adenosine A2B receptor (A2BAR) mediates VEGF overproduction in ex vivo glomeruli exposed to high glucose concentration, requiring PKCα and Erk1/2 activation. The glomerular content of A2BAR was concomitantly increased with VEGF at early stages of renal disease in streptozotocin-induced diabetic rats. Further, in vivo administration of an antagonist of A2BAR in diabetic rats blocked the glomerular overexpression of VEGF, mesangial cells activation and proteinuria. In addition, we also determined that the accumulation of extracellular adenosine occurs in glomeruli of diabetic rats. Correspondingly, raised urinary adenosine levels were found in diabetic rats. In conclusion, we evidenced that adenosine signaling at the onset of diabetic kidney disease is a pathogenic event that promotes VEGF induction.

Combined Use of Anticancer Drugs and an Inhibitor of Multiple Drug Resistance-Associated Protein-1 Increases Sensitivity and Decreases Survival of Glioblastoma Multiforme Cells In Vitro

Glioblastoma multiforme (GBM) is a brain tumour characterised by a remarkably high chemoresistance and infiltrating capability. To date, chemotherapy with temozolomide has contributed only poorly to improved survival rates in patients. One of the most important mechanisms of chemoresistance comes about through the activity of certain proteins from the ATP-binding cassette superfamily that extrudes antitumour drugs, or their metabolites, from cells. We identify an increased expression of the multiple drug resistance-associated protein 1 (Mrp1) in glioblastoma multiforme biopsies and in T98G and G44 cell lines. The activity of this transporter was also confirmed by measuring the extrusion of the fluorescent substrate CFDA. The sensitivity of GBM cells was low upon exposure to temozolomide, vincristine and etoposide, with decreases in cell viability of below 20% seen at therapeutic concentrations of these drugs. However, combined exposure to vincristine or etoposide with an inhibitor of Mrp1 efficiently decreased cell viability by up to 80%. We conclude that chemosensitization of cells with inhibitors of Mrp1 activity might be an efficient tool for the treatment of human GBM.

Altered expression and localization of insulin receptor in proximal tubule cells from human and rat diabetic kidney

Diabetes is the major cause of end stage renal disease, and tubular alterations are now considered to participate in the development and progression of diabetic nephropathy (DN). Here, we report for the first time that expression of the insulin receptor (IR) in human kidney is altered during diabetes. We detected a strong expression in proximal and distal tubules from human renal cortex, and a significant reduction in type 2 diabetic patients. Moreover, isolated proximal tubules from type 1 diabetic rat kidney showed a similar response, supporting its use as an excellent model for in vitro study of human DN. IR protein down‐regulation was paralleled in proximal and distal tubules from diabetic rats, but prominent in proximal tubules from diabetic patients. A target of renal insulin signaling, the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), showed increased expression and activity, and localization in compartments near the apical membrane of proximal tubules, which was correlated with activation of the GSK3β kinase in this specific renal structure in the diabetic condition. Thus, expression of IR protein in proximal tubules from type 1 and type 2 diabetic kidney indicates that this is a common regulatory mechanism which is altered in DN, triggering enhanced gluconeogenesis regardless the etiology of the disease. J. Cell. Biochem. 114: 639–649, 2013.

Transcription factor Pit-1 expression is modulated upon seasonal acclimatization of eurythermal ectotherms: Identification of two Pit-1 genes in the carp

A second Pit‐1 gene in carp (Cyprinus carpio), including the complete structural gene and 1.1 kb of promoter region, was identified and completely sequenced. The exon‐intron structure was determined, and reverse transcription‐polymerase chain reaction (RT‐PCR) experiments suggest that only one Pit‐1 splice variant is present in carp pituitary. The effect of seasonal acclimatization on the extent of Pit‐1 gene expression was studied in summer‐ and winter‐acclimatized carp. Quantitative RT‐PCR analysis revealed a clear increase of Pit‐1 mRNA in the pituitaries from summer‐acclimatized carp compared with the winter‐adapted fish. In situ hybridization of pituitary gland sections with riboprobes representing the complete 5′‐transactivating region of carp Pit‐1 depicted a significantly higher Pit‐1 mRNA level in the rostral pars distalis of the summer‐acclimatized fish where prolactin is expressed in a manner that resembles the seasonal increase observed in the proximal pars distalis and the pars intermedia. The cell‐ and temporal‐specific transcription of Pit‐1 supports its role in the molecular mechanisms that underly the acclimatization process undergone by eurythermal fish as a result of the physical effects of seasonal changes on their habitat. J. Cell. Biochem. 75:598–609, 1999.

D-glucose stimulation of L-arginine transport and nitric oxide synthesis results from activation of mitogen-activated protein kinases p42/44 and Smad2 requiring functional type II TGF-β receptors in human umbilical vein endothelium

Elevated extracellular D‐glucose increases transforming growth factor β1 (TGF‐β1) release from human umbilical vein endothelium (HUVEC). TGF‐β1, via TGF‐β receptors I (TβRI) and TβRII, activates Smad2 and mitogen‐activated protein kinases p44 and p42 (p42/44mapk). We studied whether D‐glucose‐stimulation of L‐arginine transport and nitric oxide synthesis involves TGF‐β1 in primary cultures of HUVEC. TGF‐β1 release was higher (∼1.6‐fold) in 25 mM (high) compared with 5 mM (normal) D‐glucose. TGF‐β1 increases L‐arginine transport (half maximal effect ∼1.6 ng/ml) in normal D‐glucose, but did not alter high D‐glucose‐increased L‐arginine transport. TGF‐β1 and high D‐glucose increased hCAT‐1 mRNA expression (∼8‐fold) and maximal transport velocity (Vmax), L‐[3H]citrulline formation from L‐[3H]arginine (index of NO synthesis) and endothelial NO synthase (eNOS) protein abundance, but did not alter eNOS phosphorylation. TGF‐β1 and high D‐glucose increased p42/44mapk and Smad2 phosphorylation, an effect blocked by PD‐98059 (MEK1/2 inhibitor). However, TGF‐β1 and high D‐glucose were ineffective in cells expressing a truncated, negative dominant TβRII. High D‐glucose increases L‐arginine transport and eNOS expression following TβRII activation by TGF‐β1 involving p42/44mapk and Smad2 in HUVEC. Thus, TGF‐β1 could play a crucial role under conditions of hyperglycemia, such as gestational diabetes mellitus, which is associated with fetal endothelial dysfunction. J. Cell. Physiol. 212:626–632, 2007.