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Hyperglycaemia Inhibits Thymidine Incorporation and Cell Growth via Protein Kinase C, Mitogen‐Activated Protein Kinases and Nitric Oxide in Human Umbilical Vein Endothelium
Author(s) -
Rojas Susana,
Rojas Romina,
Lamperti Liliana,
Casanello Paola,
Sobrevia Luis
Publication year - 2003
Publication title -
experimental physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.925
H-Index - 101
eISSN - 1469-445X
pISSN - 0958-0670
DOI - 10.1113/eph8802515
Subject(s) - umbilical vein , nitric oxide , kinase , mitogen activated protein kinase , protein kinase a , endothelium , microbiology and biotechnology , medicine , chemistry , endocrinology , biochemistry , biology , in vitro
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 [ 3 H]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, [ 3 H]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 Ca 2+ 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 Ca 2+ in HUVECs. These are cellular mechanisms which may reduce endothelial cell growth in pathological conditions such as in diabetes mellitus or hyperglycaemia.