Triangulation Mapping of Oxidative Bursts Released by Single Fibroblasts by Amperometry at Microelectrodes

It has been previously established that a lesion created by a microcapillary in the membrane of a single aerobic cell (from skin or immune origin) was sufficient to induce a local membrane depolarization and the ensuing release of oxidative bursts. Their kinetic and quantitative features reveal the activity of cell constitutive enzymes, namely, NADPH oxidases and NO synthases, prone to produce rapidly reactive oxygen and reactive nitrogen species. Until now, the spatial resolution provided by microelectrodes has been exploited in this context to characterize the chemical composition of oxidative bursts at several cell types with high collection efficiency. In the present work, spatial features of the oxidative bursts from single human fibroblasts were investigated using a step-by-step geometrical mapping approach. The spatial locations of cell active zones and of the extent of the activated area, when a cell membrane was stressed by a microcapillary's tip of 1-microm radius, have been addressed. On cells of large dimensions such as fibroblasts, ROS and RNS emission originated from a disk surface of the membrane limited to approximately 15-microm radius around the approximately 1-microm hole created by the microcapillary. This experimental result was rationalized through a simple physicochemical model designed to portray the extent of the membrane activated area due to ion concentration variations resulting from the pinhole channel created across the cell membrane. This is consistent with the fact that the activation of constitutive enzymatic complexes (NOX and NOS) is hypothesized to be a consequence of local variations of ion concentrations such as K(+), Na(+) or possibly Ca(2+). Our results showed that the calculated area near the cell membrane where the ion concentration gradients are significant was equivalent to the area of species release measured experimentally.