Modulation of adenosine transport by insulin in human umbilical artery smooth muscle cells from normal or gestational diabetic pregnancies

Abstract

Adenosine transport was measured in human cultured umbilical artery smooth muscle cells, isolated from non‐diabetic or gestational diabetic pregnancies, under basal conditions and after pretreatment in vitro with insulin. Adenosine transport in non‐diabetic smooth muscle cells was significantly increased by insulin (half‐maximal stimulation at 0.33 ± 0.02 nm, 8 h) and characterized by a higher maximal rate (Vmax) for nitrobenzylthioinosine (NBMPR)‐sensitive (es) saturable nucleoside transport (17 ± 5 vs. 52 ± 12 pmol (μg protein)−1 min−1, control vs. insulin, respectively) and maximal binding sites (Bmax) for [3H]NBMPR (0.66 ± 0.07 vs. 1.1 ± 0.1 fmol (μg protein)−1, control vs. insulin, respectively), with no significant changes in Michaelis‐Menten (Km) and dissociation (Kd) constants. In contrast, in smooth muscle cells from diabetic pregnancies, where the values of Vmax for adenosine transport (59 ± 4 pmol (μg protein)−1 min−1) and Bmax for [3H]NBMPR binding (1.62 ± 0.16 fmol (μg protein)−1) were significantly elevated by comparison with non‐diabetic cells, insulin treatment (1 nm, 8 h) reduced the Vmax for adenosine transport and Bmax for [3H]NBMPR binding to levels detected in non‐diabetic cells. In non‐diabetic cells, the stimulatory effect of insulin on adenosine transport was mimicked by dibutyryl cGMP (100 nm) and reduced by inhibitors of phosphatidylinositol 3‐kinase (10 nm wortmannin), nitric oxide synthase (100 μmNG‐nitro‐l‐arginine methyl ester, l‐NAME) or protein synthesis (1 μm cycloheximide), whereas inhibition of adenylyl cyclase (100 μm SQ‐22536) had no effect. Wortmannin or SQ‐22536, but not l‐NAME or cycloheximide, attenuated the inhibitory action of insulin on the diabetes‐induced stimulation of adenosine transport. Protein levels of inducible NO synthase (iNOS) were similar in non‐diabetic and diabetic cells, but were increased by insulin (1 nm, 8 h) only in non‐diabetic smooth muscle cells. Our results suggest that adenosine transport via the es nucleoside transporter is modulated differentially by insulin in either cell type. Insulin increased adenosine transport in non‐diabetic cells via NO and cGMP, but inhibited the diabetes‐elevated adenosine transport via activation of adenylyl cyclase, suggesting that the biological actions of adenosine may be altered under conditions of sustained hyperglycaemia in uncontrolled diabetes.

Intrauterine Growth Retardation Is Associated With Reduced Activity and Expression of the Cationic Amino Acid Transport Systems y + /hCAT-1 and y + /hCAT-2B and Lower Activity of Nitric Oxide Synthase in Human Umbilical Vein Endothelial Cells

“…8,21,22 Membranes were washed (ϫ6) in Tris buffer saline Tween (TBST, 50 mmol/L Tris/HCl, 150 mmol/L NaCl, 0.02% v/v Tween 20, pH 7.4), and incubated (1 hour) in TBST/0.2% BSA containing horseradish peroxidase-conjugated goat anti-rabbit antibody. Proteins were detected using enhanced chemiluminescence (ECL) detection reagents and quantitated by densitometry using an Ultrascan XL enhanced laser densitometer (LKB Instruments).…”

Inhibition of Nitrobenzylthioinosine-Sensitive Adenosine Transport by Elevated d -Glucose Involves Activation of P 2Y2 Purinoceptors in Human Umbilical Vein Endothelial Cells

“…Tracer uptake was terminated by rinsing the monolayers (3 times) with 200 L ice-cold Krebs solution containing 10 mol/L NBMPR, and cell radioactivity was determined by liquid scintillation counting. 6,8 Adenosine transport was also determined in cells exposed to the P 2Y antagonists reactive blue 2 (RB2, 0.1 to 100 nmol/L, 5 minutes or 24 hours), pyridoxal phosphate-6-azophenyl-2Ј,4Ј-disulfonic acid (PPADS, 0.1 to 100 nmol/L, 5 minutes or 24 hours), 20,21 or the G␣ s protein inhibitor 8-(3-benzamido-4-methylbenzamido)-naphthalene-1,3,4-trisulfonic acid (suramin, 100 mol/L, 15 minutes or 24 hours). 22 Cells were then exposed to ATP (0.1 to 100 mol/L, 2 minutes), which is a nucleotide hydrolyzed by ectonucleotidases in human endothelium, 5 ATP-␥-S (0.1 to 100 mol/L, 2 minutes or 24 hours), which is a nonhydrolyzable analogue of ATP, 23 ADP (0.1 to 100 mol/L, 2 minutes), UTP (0.1 to 100 mol/L, 2 minutes), or ␣,␤-methylene ATP dilithium (␣,␤-MeATP, 0.1 to 100 mol/L, 2 minutes), which is a nonselective P 2X purinoceptor agonist, in the absence or presence of RB2, PPADS, or suramin.…”

“…[1][2][3][4] Plasma and tissue levels of adenosine are regulated by an efficient membrane transport mediated by the Na ϩ -independent, nitrobenzylthioinosine (NBMPR)-sensitive equilibrative nucleoside transporter (system es or ENT1) 3,4 in human vascular endothelium 5,6 and smooth muscle. 7,8 Human ENT1 (hENT1) expression in Raji cells (a human B-lymphocyte cell line) is dependent on NO levels and the activity of protein kinase C (PKC). 9 Incubation of human umbilical vein endothelial cells (HUVECs) with 25 mmol/L D-glucose for 24 hours has been reported to reduce the NBMPR-sensitive adenosine transport associated with increased protein levels and the activity of endothelial NO synthase, intracellular Ca 2ϩ , PKC, and mitogen-activated protein kinases p42/p44 mapk .…”

The Effect of Insulin on Expression Level of Nucleoside Transporters in Diabetic Rats

“…Cell-specific changes in NBMPR-sensitive transport of adenosine in cultured human diabetic cells have been also reported. It was demonstrated that in endothelial cells isolated from human diabetic umbilical vein, NBMPR-sensitive adenosine transport was reduced (Sobrevia et al, 1994), whereas in smooth muscle cells isolated from diabetic human umbilical artery, adenosine transport was significantly elevated (Aguayo et al, 2001). …”