S. Rojas

Insulin Reverses D-Glucose–Increased Nitric Oxide and Reactive Oxygen Species Generation in Human Umbilical Vein Endothelial Cells

Vascular tone is controlled by the L-arginine/nitric oxide (NO) pathway, and NO bioavailability is strongly affected by hyperglycaemia-induced oxidative stress. Insulin leads to high expression and activity of human cationic amino acid transporter 1 (hCAT-1), NO synthesis and vasodilation; thus, a protective role of insulin on high D-glucose–alterations in endothelial function is likely. Vascular reactivity to U46619 (thromboxane A2 mimetic) and calcitonin gene related peptide (CGRP) was measured in KCl preconstricted human umbilical vein rings (wire myography) incubated in normal (5 mmol/L) or high (25 mmol/L) D-glucose. hCAT-1, endothelial NO synthase (eNOS), 42 and 44 kDa mitogen-activated protein kinases (p42/44mapk), protein kinase B/Akt (Akt) expression and activity were determined by western blotting and qRT-PCR, tetrahydrobiopterin (BH4) level was determined by HPLC, and L-arginine transport (0–1000 μmol/L) was measured in response to 5–25 mmol/L D-glucose (0–36 hours) in passage 2 human umbilical vein endothelial cells (HUVECs). Assays were in the absence or presence of insulin and/or apocynin (nicotinamide adenine dinucleotide phosphate-oxidase [NADPH oxidase] inhibitor), tempol or Mn(III)TMPyP (SOD mimetics). High D-glucose increased hCAT-1 expression and activity, which was biphasic (peaks: 6 and 24 hours of incubation). High D-glucose–increased maximal transport velocity was blocked by insulin and correlated with lower hCAT-1 expression and SLC7A1 gene promoter activity. High D-glucose–increased transport parallels higher reactive oxygen species (ROS) and superoxide anion (O2 •–) generation, and increased U46619-contraction and reduced CGRP-dilation of vein rings. Insulin and apocynin attenuate ROS and O2 •– generation, and restored vascular reactivity to U46619 and CGRP. Insulin, but not apocynin or tempol reversed high D-glucose–increased NO synthesis; however, tempol and Mn(III)TMPyP reversed the high D-glucose–reduced BH4 level. Insulin and tempol blocked the high D-glucose–increased p42/44mapk phosphorylation. Vascular dysfunction caused by high D-glucose is likely attenuated by insulin through the L-arginine/NO and O2 •–/NADPH oxidase pathways. These findings are of interest for better understanding vascular dysfunction in states of foetal insulin resistance and hyperglycaemia.

Hyperglycaemia Inhibits Thymidine Incorporation and Cell Growth via Protein Kinase C, Mitogen‐Activated Protein Kinases and Nitric Oxide in Human Umbilical Vein Endothelium

An elevated extracellular concentration of D‐glucose (i.e. hyperglycaemia) inhibits cell proliferation and incorporation of the endogenous nucleoside thymidine into DNA in human umbilical vein endothelial cells (HUVECs). Cells in their log‐phase of growth (3.7 ± 0.3 days, n = 27) incubated for 30 min with 25 mM D‐glucose, but not with equimolar concentrations of L‐glucose or D‐mannitol, exhibited reduced [3H]thymidine incorporation and cell growth rate, with no change in cell viability (> 98%), total DNA, protein content or cell volume. Incubation with D‐glucose activated protein kinase C (PKC), endothelial NO synthase (eNOS), p42 and p44 mitogen‐activated protein kinases (p42/44mapk), but inhibited superoxide dismutase (SOD). Incubation with D‐glucose also increased cGMP and cAMP levels. The effect of D‐glucose was blocked by the PKC inhibitor calphostin C, the MAP kinase kinase 1/2 (MEK1/2) inhibitor PD‐98059, the eNOS inhibitor L‐NAME, the protein kinase G (PKG) inhibitor KT‐5823 and the protein kinase A (PKA) inhibitor KT‐5720. In the presence of 5 mM D‐glucose, [3H]thymidine incorporation and cell growth were reduced by the PKC activator phorbol 12‐myristate 13‐acetate (PMA), the NO donor S‐nitroso‐N‐acetyl‐L,D‐penicillamine (SNAP), dibutyryl cGMP, dibutyryl cAMP and the Ca2+ ionophore A‐23187. The effect of A‐23187 was blocked by calphostin C and PD‐98059. D‐Glucose‐dependent inhibition of thymidine incorporation and cell proliferation is associated with increased PKC, eNOS, and MEK1/2, but decreased SOD activity, and higher intracellular levels of cGMP, cAMP and Ca2+ in HUVECs. These are cellular mechanisms which may reduce endothelial cell growth in pathological conditions such as in diabetes mellitus or hyperglycaemia.

Transport and metabolism of adenosine in the perfused human placenta

Uptake and metabolism of adenosine by human placenta were studied using the single-circulation paired-tracer technique. When isolated cotyledons were perfused through the fetal (basal) circulation at mean pressures of 36 +/- 3.3 mmHg and mean flow rates of 6.6 +/- 0.3 ml/min the maximal [3H]adenosine uptake was 51.3 +/- 3.9 per cent. The uptake was not changed when the vascular resistance was pharmacologically increased. Adenosine uptake was significantly inhibited by adenosine, inosine and nitrobenzylthioinosine (NBMPR), but was unaffected by hypoxanthine. The kinetic analysis of adenosine transport showed it to be a saturable and, Na(+)-independent process, with a Km of 60.8 microM and a Jmax of 0.148 mumol/min. Thin layer chromatographic analysis showed that about 65 per cent of [3H]adenosine was metabolized (10-30 sec) in a single passage through the fetoplacental circulation. [3H]hypoxanthine and [3H]adenine were the major products recovered in the venous perfusate. In the presence of NBMPR the fractional recovery of [3H]adenine and [3H]phosphorylated derivatives was reduced while that of [3H]hypoxanthine was increased. These overall results show that the uptake of adenosine is a Na(+)-independent, NBMPR-sensitive, carrier-mediated process, which appears to be specific for nucleosides, and suggests that metabolization of adenosine proceeds both intra- and extracellularly.

Effects of ethanol ingestion on amino acid uptake in the dog liver in vivo.

The effect of ethanol ingestion on the uptake of labeled amino acids was studied in the in situ autoperfused dog liver. Ethanol was administered orally, as a 15% water solution, in a dose of 4 g/kg body weight/day as the only source of water for 2 days. Amino acid uptake was measured in anesthetized dogs by means of the single-passage, multiple-tracer dilution technique. In control animals, hepatic uptake of 14C-glycine, 3H-alpha-aminoisobutyric acid (3H-AIB) and 3H-L-leucine were 50, 15 and 66%, respectively. In the ethanol-treated dogs, glycine and AIB uptake was reduced by 70 and 63%, respectively. L-leucine uptake was reduced by only 23%. The plasma concentration of the naturally occurring amino acids was significantly increased after ethanol treatment, probably due to a reduced influx into hepatocytes. Simultaneous measurements of hemodynamic parameters showed a significant increase in the portal vein pressure of ethanol-treated animals, whereas the portal vein blood flow and hepatic extracellular volume were unaffected.

L-arginine transport at the fetal side of human placenta: effect of aspirin in pregnancy.

l‐Arginine transport by the fetal side of human placenta was investigated through the characterization of l‐[3H]arginine uptake in isolated perfused cotyledon. Competitive inhibition experiments suggest the presence of at least two transport systems: a Na+‐independent, pH‐insensitive system inhibitable by cationic amino acids, similar to system y+, and a Na+‐dependent system which recognizes both cationic and neutral amino acids only in the presence of Na+, i.e. a Bo,+‐like system. The kinetic analysis of l‐arginine uptake in the presence of Na+ revealed that the process is mediated by saturable components: a high‐affinity system (Km= 167 ± 18.0 μM; Vmax= 0.174 ± 0.012 μmol min−1) and a low‐affinity carrier (Km= 980 ± 112 μM; Vmax= 1.60 ± 0.12 μmol min−1). In the absence of Na+, l‐arginine uptake was fitted by one model with a Michaelis‐Menten constant of 200 ± 24.8 μM. These results suggest that the high‐affinity component corresponds to the Na+‐independent system y+, whilst the low‐affinity system may represent the activity of the Na+‐dependent Bo,+ transporter. Kinetic studies in placentae taken from aspirin‐treated pregnancies showed that l‐arginine is transported with a significantly higher affinity (Km= 42.5 ± 5.7 μM), but with a lower capacity (Vmax= 0.064 ± 0.003 μmol min−1) than in the non‐treated group. The latter finding suggests that aspirin would facilitate the uptake of the NO precursor only at very low arginine concentrations.

Hypoxanthine uptake at the fetal side of human placenta proceeds through a nucleobase-preferring carrier and a non-saturable process

Uptake and metabolism of hypoxanthine by human placenta were studied using the single-circulation paired-tracer technique. In isolated cotyledons perfused through the fetal (basal) circulation, at mean pressures of 31.7 +/- 4.0 mmHg and mean flow rates maintained at 5.5 +/- 0.15 ml/min, the [3H]hypoxanthine uptake was 36 +/- 2.4 per cent (16.5 +/- 1.1 pmol/g wet weight). Hypoxanthine uptake was significantly inhibited by unlabelled (mM) hypoxanthine (0.5), adenine (0.5), guanine (0.5) and papaverine (15.0), but was unaffected by nitrobenzylthioinosine (0.01). Adenosine failed to inhibit hypoxanthine uptake. The kinetic analysis of hypoxanthine uptake showed it to be partially mediated by a saturable (apparent K(m) = 12.1 +/- 1.85 microns; Jmax = 7.1 +/- 0.52 nmol/min) and Na(+)-dependent mechanism. A greater fraction of hypoxanthine influx proceeded through a non-saturable process. Thin layer chromatographic analysis of venous perfusate after the intra-arterial injection of [3H]hypoxanthine showed a negligible degradation of nucleobase. These overall results show that hypoxanthine uptake at the fetal side of human placenta occurs by a saturable plus a non-saturable process. The carrier showed specificity for nucleobases and high affinity-low capacity for hypoxanthine. Since the fetal blood concentration of hypoxanthine is normally low, its uptake would be mediated by the high affinity transport system. Because the non-saturable mechanism can be operative at high concentrations of hypoxanthine, it may have primary importance to clear the nucleobase coming from the fetus during intrauterine hypoxia.

Insulin Induces Relaxation and Decreases Hydrogen Peroxide-Induced Vasoconstriction in Human Placental Vascular Bed in a Mechanism Mediated by Calcium-Activated Potassium Channels and L-Arginine/Nitric Oxide Pathways

HIGHLIGHTS Short-term incubation with insulin increases the L-arginine transport in HUVECs. Short-term incubation with insulin increases the NO synthesis in HUVECs. Insulin induces relaxation in human placental vascular bed. Insulin attenuates the constriction induced by hydrogen peroxide in human placenta. The relaxation induced by insulin is dependent on BKCa channels activity in human placenta. Insulin induces relaxation in umbilical veins, increasing the expression of human amino acid transporter 1 (hCAT-1) and nitric oxide synthesis (NO) in human umbilical vein endothelial cells (HUVECs). Short-term effects of insulin on vasculature have been reported in healthy subjects and cell cultures; however, its mechanisms remain unknown. The aim of this study was to characterize the effect of acute incubation with insulin on the regulation of vascular tone of placental vasculature. HUVECs and chorionic vein rings were isolated from normal pregnancies. The effect of insulin on NO synthesis, L-arginine transport, and hCAT-1 abundance was measured in HUVECs. Isometric tension induced by U46619 (thromboxane A2 analog) or hydrogen peroxide (H2O2) were measured in vessels previously incubated 30 min with insulin and/or the following pharmacological inhibitors: tetraethylammonium (KCa channels), iberiotoxin (BKCa channels), genistein (tyrosine kinases), and wortmannin (phosphatidylinositol 3-kinase). Insulin increases L-arginine transport and NO synthesis in HUVECs. In the placenta, this hormone caused relaxation of the chorionic vein, and reduced perfusion pressure in placental cotyledons. In vessels pre-incubated with insulin, the constriction evoked by H2O2 and U46619 was attenuated and the effect on H2O2-induced constriction was blocked with tetraethylammonium and iberiotoxin, but not with genistein, or wortmannin. Insulin rapidly dilates the placental vasculature through a mechanism involving activity of BKCa channels and L-arginine/NO pathway in endothelial cells. This phenomenon is related to quick increases of hCAT-1 abundance and higher capacity of endothelial cells to take up L-arginine and generate NO.