Detection of NO at Small Carbon Electrodes as a Means to Assess the Inhibition Efficiency in its Release from Foetal Pig Islet-Like Cell Clusters

Abstract

As a xenotransplant, foetal pig islet-like cell clusters (ICCs) are tissues with a potential to reverse hyperglycaemia in Type I diabetes. Following the exposure of ICCs to some pro-inflammatory cytokine factors, the amino acid L-arginine is catalytically cleaved into NO. A cascade of events is then triggered by the NO, leading to tissue apoptosis. In our laboratory, N-monomethyl-L-arginine (an antagonist of NO formation) and heat shock treatment (an inhibitor of NO synthetic enzyme) have been used to prevent pathological injuries in cytokine-induced ICCs. Chronoamperometry at Nafion-coated carbon cylinder electrodes (<5 µm tip diameter) adjacent to cytokine-induced ICCs was employed in this study to periodically monitor NO release and diffusion in ICCs, so that the inhibition efficiency of the respective treatments could be evaluated. Using cytokine-induced ICCs with L-arginine, the electrode recorded a current increase, arising from the oxidation of released NO, over a period of 30 min, followed by a current plateau that persisted for 3 h. In contrast, no corresponding current increase was observed in control experiments involving non-cytokine-induced ICCs with L-arginine, demonstrating the reliability of the direct detection of NO at the carbon electrodes. When the cells were subjected to N-monomethyl-L-arginine or mild heat shock pre-treatment, a decrease in the oxidation current of released NO was observed, demonstrating their inhibitive role in down-regulating NO generation and release.