HIGH GLUCOSE‐INDUCED HUMAN UMBILICAL VEIN ENDOTHELIAL CELL HYPERPERMEABILITY IS DEPENDENT ON PROTEIN KINASE C ACTIVATION AND INDEPENDENT OF THE Ca2+–NITRIC OXIDE SIGNALLING PATHWAY

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Abstract
1. Endothelial barrier dysfunction plays a pivotal role in the pathogenesis of diabetic vascular complications. The precise molecular mechanisms by which hyperglycaemia causes the increased permeability in endothelial cells are not yet well understood. In the present study, we investigated whether high concentrations of glucose induce endothelial permeability through the activation of protein kinase C (PKC) and/or the calcium-nitric oxide (NO) signalling pathway in human umbilical vein endothelial cells (HUVEC). 2. Endothelial permeability was measured by albumin diffusion across endothelial monolayers under the stimuli of high glucose (HG; 20 mmol/L), 100 nmol/L phorbol-myristate-acetate (PMA) or 100 nmol/L histamine. The intracellular calcium concentration ([Ca2+]i) was detected in HUVEC using the fluorescent probe fura-2 AM. The effects of PKC inhibitors (LY379196 and hypocrellin A) and the NO synthase (NOS) inhibitor NG-monomethyl-L-arginine (L-NMMA) on endothelial permeability and [Ca2+]i were determined. 3. High glucose and PMA increased endothelial permeability associated with decreased [Ca2+]i, whereas histamine triggered significant increases in endothelial permeability, accompanied by increases in [Ca2+]i in HUVEC. Hypocrellin A (HA) and LY379196 reversed both HG- and histamine-induced endothelial permeability. The NOS inhibitor L-NMMA only abolished histamine- and not HG-induced endothelial permeability. Neither LY379196, HA nor L-NMMA had any significant effects on alterations in [Ca2+]i caused by HG and histamine. 4. These results indicate that increased endothelial permeability in HUVEC induced by HG is dependent on PKC activity and is independent of the [Ca2+]i-NO pathway. Increased endothelial permeability due to other inflammatory factors, such as histamine, may also be mediated by the PKC pathway. Thus, PKC inhibitors would be a potential therapeutic approach to endothelial dysfunction induced by hyperglycaemia, as well as other inflammatory factors, in diabetes.