title: Hyperglycaemia inhibits thymidine incorporation and cell growth via protein kinase C, mitogen-activated protein kinases and nitric oxide in human umbilical vein endothelium.
abstract: An elevated extracellular concentration of D-glucose (i.e. hyperglycaemia) inhibits cell proliferation and incorporation of the endogenous nucleoside thymidine into DNA in human umbilical vein endothelial cells (HUVECs). Cells in their log-phase of growth (3.7 +/- 0.3 days, n = 27) incubated for 30 min with 25 mM D-glucose, but not with equimolar concentrations of L-glucose or D-mannitol, exhibited reduced [3H]thymidine incorporation and cell growth rate, with no change in cell viability (> 98 %), total DNA, protein content or cell volume. Incubation with D-glucose activated protein kinase C (PKC), endothelial NO synthase (eNOS), p42 and p44 mitogen-activated protein kinases (p42/44(mapk)), but inhibited superoxide dismutase (SOD). Incubation with D-glucose also increased cGMP and cAMP levels. The effect of D-glucose was blocked by the PKC inhibitor calphostin C, the MAP kinase kinase 1/2 (MEK1/2) inhibitor PD-98059, the eNOS inhibitor L-NAME, the protein kinase G (PKG) inhibitor KT-5823 and the protein kinase A (PKA) inhibitor KT-5720. In the presence of 5 mM D-glucose, [3H]thymidine incorporation and cell growth were reduced by the PKC activator phorbol 12-myristate 13-acetate (PMA), the NO donor S-nitroso-N-acetyl-L,D-penicillamine (SNAP), dibutyryl cGMP, dibutyryl cAMP and the Ca2+ ionophore A-23187. The effect of A-23187 was blocked by calphostin C and PD-98059. D-Glucose-dependent inhibition of thymidine incorporation and cell proliferation is associated with increased PKC, eNOS, and MEK1/2, but decreased SOD activity, and higher intracellular levels of cGMP, cAMP and Ca2+ in HUVECs. These are cellular mechanisms which may reduce endothelial cell growth in pathological conditions such as in diabetes mellitus or hyperglycaemia.
authors.affiliation: Cellular and Molecular Physiology Laboratory, Department of Physiology, Faculty of Biological Sciences, University of Concepción, PO Box 160-C, Concepción, Chile.
authors.forename: Susana
authors.initials: S
authors.lastname: Rojas
authors.forename: Romina
authors.initials: R
authors.lastname: Rojas
authors.forename: Liliana
authors.initials: L
authors.lastname: Lamperti
authors.forename: Paola
authors.initials: P
authors.lastname: Casanello
authors.forename: Luis
authors.initials: L
authors.lastname: Sobrevia
mesh_terms: D001786:Blood Glucose / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism / Q000502:physiology; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D004789:Enzyme Activation; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors*; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D052250:Nitric Oxide Synthase Type III; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors; D013936:Thymidine / Q000378:metabolism; D014471:Umbilical Veins / Q000378:metabolism
chemical_list: D001786:Blood Glucose; D009569:Nitric Oxide; D000242:Cyclic AMP; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D013482:Superoxide Dismutase; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D006152:Cyclic GMP; D005947:Glucose; D002118:Calcium; D013936:Thymidine