CALCIUM AND PROTEIN-KINASE-C MEDIATE HIGH-GLUCOSE-INDUCED INHIBITION OF INDUCIBLE NITRIC-OXIDE SYNTHASE IN VASCULAR SMOOTH-MUSCLE CELLS

Abnormal vascular smooth muscle (VSMC) proliferation is a key feature in diabetes-associated atherosclerotic disease. Since nitric oxide inh ibits VSMC tone, migration, adhesion, and proliferation, we examined t he effects of high glucose on IL-1 beta-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 wi th IL-1 beta. Nitrite was measured in the culture medium after 24 hour s. IL-1 beta-induced a 15-fold increase in NO production in normal glu cose medium. Glucose (10 to 30 mmol/L (mM)) significantly reduced the response to IL-1 beta. High glucose (20 mmol/L (mM)) inhibited IL-1 be ta-evoked NO production by approximately 50%. IL-1 beta-stimulated [H- 3] citrulline-forming activity of the nitric oxide synthase (NOS) was also significantly lower in high-glucose-exposed cells, and this was r eflected in diminished cellular levels of NOS protein. To assess the r ole of protein kinase C (PKC), membrane PKC activity was measured, and glucose (20 mmol/L (mM)) significantly increased it. Immunoblotting o f the membranes revealed a glucose-induced increase in the PKC beta II isoform. 1,2-Dioctanoyl-glycerol, a PKC activator, mimicked the high- glucose effect on IL-1 beta-induced NO release, while staurosporine, a PKC inhibitor, reversed it. The role of calcium in the glucose-mediat ed inhibition of cytokine-induced NO release was determined by treatme nt with BAPTA, an intracellular chelator of calcium. BAPTA partially r eversed the inhibitory effects of glucose. Increasing intracellular ca lcium by A23187, an ionophore or thapsigargin, an inhibitor of endopla smic reticulum Ca2+-ATPase, significantly decreased IL-1 beta-induced NO release and NOS expression. These results indicate that glucose-ind uced inhibition of IL-1 beta-stimulated NO release and NOS expression may be mediated by PKC activation and increased intracellular calcium.