Calcium and Protein Kinase C Mediate High-Glucose-Induced Inhibition of Inducible Nitric Oxide Synthase in Vascular Smooth Muscle Cells

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Abstract
Abnormal vascular smooth muscle (VSMC) proliferation is a key feature in diabetes-associated atherosclerotic disease. Since nitric oxide inhibits VSMC tone, migration, adhesion, and proliferation, we examined the effects of high glucose on IL-1β-induced NO release from VSMCs in culture. Confluent smooth muscle cells, preincubated with either 5 mmol/L (mM) or 20 mmol/L (mM) glucose for 48 hours, were stimulated with IL-1β. Nitrite was measured in the culture medium after 24 hours. IL-1β-induced a 15-fold increase in NO production in normal glucose medium. Glucose (10 to 30 mmol/L (mM)) significantly reduced the response to IL-1β. High glucose (20 mmol/L (mM) inhibited IL-1β-evoked NO production by approximately 50%. IL-1β-stimulated [3H] citrulline-forming activity of the nitric oxide synthase (NOS) was also significantly lower in high-glucose-exposed cells, and this was reflected in diminished cellular levels of NOS protein. To assess the role of protein kinase C (PKC), membrane PKC activity was measured, and glucose (20 mmol/L (mM)) significantly increased it. Immunoblotting of the membranes revealed a glucose-induced increase in the PKC βII isoform. 1,2-Dioctanoyl-glycerol, a PKC activator, mimicked the high-glucose effect on IL-1β-induced NO release, while staurosporine, a PKC inhibitor, reversed it. The role of calcium in the glucose-mediated inhibition of cytokine-induced NO release was determined by treatment with BAPTA, an intracellular chelator of calcium. BAPTA partially reversed the inhibitory effects of glucose. Increasing intracellular calcium by A23187, an ionophore or thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPase, significantly decreased IL-1β-induced NO release and NOS expression. These results indicate that glucose-induced inhibition of IL-1β-stimulated NO release and NOS expression may be mediated by PKC activation and increased intracellular calcium.