RESUMEN
1. 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. 2. 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 (V(max)) for nitrobenzylthioinosine (NBMPR)-sensitive (es) saturable nucleoside transport (17 +/- 5 vs. 52 +/- 12 pmol (microg protein)(-1) min(-1), control vs. insulin, respectively) and maximal binding sites (B(max)) for [(3)H]NBMPR (0.66 +/- 0.07 vs. 1.1 +/- 0.1 fmol (microg protein)(-1), control vs. insulin, respectively), with no significant changes in Michaelis-Menten (K(m)) and dissociation (K(d)) constants. 3. In contrast, in smooth muscle cells from diabetic pregnancies, where the values of V(max) for adenosine transport (59 +/- 4 pmol (microg protein)(-1) min(-1)) and B(max) for [(3)H]NBMPR binding (1.62 +/- 0.16 fmol (microg protein)(-1)) were significantly elevated by comparison with non-diabetic cells, insulin treatment (1 nM, 8 h) reduced the V(max) for adenosine transport and B(max) for [(3)H]NBMPR binding to levels detected in non-diabetic cells. 4. 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 microM N (G)-nitro-L-arginine methyl ester, L-NAME) or protein synthesis (1 microM cycloheximide), whereas inhibition of adenylyl cyclase (100 microM SQ-22536) had no effect. 5. Wortmannin or SQ-22536, but not L-NAME or cycloheximide, attenuated the inhibitory action of insulin on the diabetes-induced stimulation of adenosine transport. 6. 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. 7. 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.
Asunto(s)
Adenosina/metabolismo , Diabetes Gestacional/metabolismo , Insulina/fisiología , Músculo Liso Vascular/metabolismo , Embarazo/metabolismo , Arterias Umbilicales/metabolismo , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Células Cultivadas , AMP Cíclico/fisiología , Femenino , Humanos , Insulina/farmacología , Músculo Liso Vascular/citología , Óxido Nítrico/fisiología , Valores de Referencia , Arterias Umbilicales/citologíaRESUMEN
The effects of elevated D-glucose on adenosine transport were investigated in human cultured umbilical vein endothelial cells isolated from normal pregnancies. 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. 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. 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 microM, an inhibitor of nitric oxide synthase (NOS)) or PD-98059 (10 microM, an inhibitor of mitogen-activated protein kinase kinase 1). 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. Adenosine transport was inhibited by S-nitroso-N-acetyl-l, d-penicillamine (SNAP; 100 microM, an NO donor) but was increased in cells incubated with L-NAME. The effect of SNAP on adenosine transport was abolished by PD-98059. 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 microM, 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. 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.
Asunto(s)
Adenosina/metabolismo , Endotelio Vascular/embriología , Endotelio Vascular/metabolismo , Glucosa/farmacología , Tioinosina/análogos & derivados , Adenosina/antagonistas & inhibidores , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Feto/fisiología , Flavonoides/farmacología , Humanos , Proteínas Quinasas Activadas por Mitógenos/fisiología , NG-Nitroarginina Metil Éster/farmacología , Naftalenos/farmacología , Óxido Nítrico/fisiología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo III , Penicilamina/análogos & derivados , Penicilamina/farmacología , Proteína Quinasa C/fisiología , S-Nitroso-N-Acetilpenicilamina , Acetato de Tetradecanoilforbol/farmacología , Tioinosina/metabolismoRESUMEN
The transport properties of the nucleobase hypoxanthine were examined in the human umbilical vein endothelial cell line ECV 304. Initial rates of hypoxanthine influx were independent of extracellular cations: replacement of Na+ with Li+, Rb+, N-methyl-D-glucamine or choline had no significant effect on hypoxanthine uptake by ECV 304 cells. Kinetic analysis demonstrated the presence of a single saturable system for the transport of hypoxanthine in ECV 304 cells with an apparent K(m) of 320 +/- 10 microM and a Vmax of 5.6 +/- 0.9 pmol/10(6) cells per s. Hypoxanthine uptake was inhibited by the nucleosides adenosine, uridine and thymidine (apparent Ki 41 +/- 6, 240 +/- 27 and 59 +/- 8 microM respectively) and the nucleoside transport inhibitors nitrobenzylthioinosine (NBMPR), dilazep and dipyridamole (apparent Ki 2.5 +/- 0.3, 11 +/- 3 and 0.16 +/- 0.006 microM respectively), whereas the nucleobases adenine, guanine and thymine had little effect (50% inhibition at > 1 mM). ECV 304 cells were also shown to transport adenosine via both the NBMPR-sensitive and -insensitive nucleoside carriers. Hypoxanthine specifically inhibited adenosine transport via the NBMPR-insensitive system in a competitive manner (apparent Ki 290 +/- 14 microM). These results indicate that hypoxanthine entry into ECV 304 endothelial cells is mediated by the NBMPR-insensitive nucleoside carrier present in these cells.