DIABETES-INDUCED ACTIVATION OF SYSTEM Y(+) AND NITRIC-OXIDE SYNTHASE IN HUMAN ENDOTHELIAL-CELLS - ASSOCIATION WITH MEMBRANE HYPERPOLARIZATION

1. The activity of the hnman endothelial cell L-arginine transporter (system y(+)) has been correlated with cGMP production (index of nitric oxide) and prostacyclin (PGI(2)) 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 V-max for saturable L-arginine transport (4.6 +/- 0.13 vs. 9.9 +/- 0.5 pmol (mu g 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[H-3]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 [H-3]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 mu M), an inhibitor of nitric oxide synthase, abolished basal cGMP accumulation in nondiabetic and diabetic cells. 7. Histamine (10 mu M) 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 PGI(2) release was decreased markedly in diabetic cells. 9. Our findings demonstrate tl-lat 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 PGI(2) production.