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

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 endothelia l cells cultured from normal or gestational diabetic pregnancies. 2. I n 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 o r 2-deoxyglucose. 4. In non-diabetic and diabetic cells, elevated K+ r esulted in a concentration-dependent inhibition in the initial rates o f 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, diab etic cells were hyperpolarized (-78 +/- 0.3 mV) compared with non-diab etic 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 intr acellular cGMP levels were elevated 2.5-fold in diabetic cells, and L- NAME (100 mu M), an inhibitor of nitric oxide synthase, abolished basa l cGMP accumulation in nondiabetic and diabetic cells. 7. Histamine (1 0 mu M) had no effect on L-arginine transport but evoked significant i ncreases in cGMP in non-diabetic and diabetic cells, which were comple tely inhibited by L-NAME but unaffected by superoxide dismutase. 8. Ba sal and histamine-stimulated PGI(2) release was decreased markedly in diabetic cells. 9. Our findings demonstrate tl-lat gestational diabete s is associated with phenotypic changes in fetal. endothelial cells, w hich result in a membrane hyperpolarization, activation of the human e ndothelial cell L-arginine transporter (system y(+)), elevation of bas al nitric oxide synthesis and decreased PGI(2) production.