Importance of large conductance calcium-activated potassium channels (BKCa) in interleukin-1b-induced adhesion of monocytes to endothelial cells

K.M. Burgazli, C.J. Venker, M. Mericliler, N. Atmaca, M. Parahuleva, A. Erdogan

Department of Internal Medicine and Angiology, Wuppertal Research and Medical Center, Wuppertal, Germany. burgazlk@uni-koeln.de

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• Angiology

OBJECTIVE: The present study investigated the role of the large conductance calcium-activated potassium channels (BKCa) in interleukin-1b (IL-1b) induced inflammation.

MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVECs) were isolated and cultured. Endothelial cell membrane potential measurements were accomplished using the fluorescent dye DiBAC4(3). The role of BKCa was assessed using iberiotoxin, a highly selective BKCa inhibitor. Changes in the calcium intracellular calcium were investigated using Fura-2-AM imaging. Fluorescent dyes DCF-AM and DAF-AM were further used in order to measure the formation of reactive oxygen species (ROS) and nitric oxide (NO) synthesis, respectively. Endothelial cell adhesion tests were conducted with BCECF-AM adhesion assay and tritium thymidine uptake using human monocytic cells (U937). Expression of cellular adhesion molecules (ICAM-1, VCAM-1) was determined by flow cytometer.

RESULTS: Interleukin-1b induced a BKCa dependent hyperpolarization of HUVECs. This was followed by an increase in the intracellular calcium concentration. Furthermore, IL-1b significantly increased the synthesis of NO and ROS. The increase of intracellular calcium, radicals and NO resulted in a BKCa dependent adhesion of monocytes to HUVECs. Endothelial cells treated with IL-1b expressed both ICAM-1 and VCAM-1 in significantly higher amounts as when compared to controls. It was further shown that the cellular adhesion molecules ICAM-1 and VCAM-1 were responsible for the BKCa-dependent increase in cellular adhesion. Additionally, inhibition of the NADPH oxidase with DPI led to a significant downregulation of IL-1b-induced expression of ICAM and VCAM, as well as inhibition of eNOS by L-NMMA, and intracellular calcium by BAPTA.

CONCLUSIONS: Activation of the endothelial BKCa plays an important role in the IL-1b-induced monocyte adhesion to endothelial cells.

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