Diabetes-induced activation of system y+ and nitric oxide synthase in human endothelial cells: association with membrane hyperpolarization.
1. The activity of the human endothelial cell L-arginine transporter (system y+) has been correlated with cGMP production (index of nitric oxide) and prostacyclin (PGI2) release in umbilical vein endothelial cells cultured from normal or gestational diabetic pregnancies. 2. In non-diabetic and diabetic cells, transport of L-arginine was Na+ and pH independent, inhibited by other cationic L-arginine analogues and unaffected by neutral amino acids. 3. Diabetes was associated with an increased Vmax for saturable L-arginine transport (4.6 +/- 0.13 vs. 9.9 +/- 0.5 pmol (microgram protein)-1 min-1, P < 0.01), but had no effect on initial rates of transport for L-serine, L-citrulline, L-leucine or 2-deoxyglucose. 4. In non-diabetic and diabetic cells, elevated K+ resulted in a concentration-dependent inhibition in the initial rates of transport for L-arginine and the membrane potential-sensitive probe tetra[3H]phenylphosphonium (TPP+). 5. When resting membrane potential was measured using the whole-cell patch voltage clamp technique, diabetic cells were hyperpolarized (-78 +/- 0.3 mV) compared with non-diabetic cells (-70 +/- 0.04 mV, P < 0.04). Accumulation of [3H]TPP+ was also increased in diabetic compared with non-diabetic cells. 6. Basal intracellular cGMP levels were elevated 2.5-fold in diabetic cells, and L-NAME (100 microM), an inhibitor of nitric oxide synthase, abolished basal cGMP accumulation in non-diabetic and diabetic cells. 7. Histamine (10 microM) had no effect on L-arginine transport but evoked significant increases in cGMP in non-diabetic and diabetic cells, which were completely inhibited by L-NAME but unaffected by superoxide dismutase. 8. Basal and histamine-stimulated PGI2 release was decreased markedly in diabetic cells. 9. Our findings demonstrate that gestational diabetes is associated with phenotypic changes in fetal endothelial cells, which result in a membrane hyperpolarization, activation of the human endothelial cell L-arginine transporter (system y+), elevation of basal nitric oxide synthesis and decreased PGI2 production.
Sobrevia
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L
Vascular Biology Research Centre, King's College London, UK.
Cesare
P
P
Yudilevich
D L
DL
Mann
G E
GE
D001120:Arginine / Q000378:metabolism*; D002478:Cells, Cultured; D003920:Diabetes Mellitus / Q000378:metabolism* / Q000503:physiopathology; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000201:enzymology*; D011464:Epoprostenol / Q000378:metabolism; D005260:Female; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D011247:Pregnancy
D001120:Arginine; D011464:Epoprostenol; D019001:Nitric Oxide Synthase