Proteins and lipids define the diffusional field of nitric oxide

Nitric oxide (NO) fluxes released from the surface of individual activated macrophages or cells localized in small aggregates were measured with a novel polarographic self-referencing microsensor. NO fluxes could be detected at distances from the cells of 100-500 microm. The initial flux and the distance from the cells at which NO could be detected were directly related to the number of cells in the immediate vicinity of the probe releasing NO. Thus, whereas NO fluxes of approximately 1 pmol. cm(-2). s(-1) were measured from individual macrophages, aggregates composed of groups of cells varying in number from 18 to 48 cells produced NO fluxes of between approximately 4 and 10 pmol. cm(-2). s(-1). NO fluxes required the presence of L-arginine. Signals were significantly reduced by the addition of hemoglobin and by N-nitro-L-arginine methyl ester. NO fluxes were greatest when the sensor was placed immediately adjacent to cell membranes and declined as the distance from the cell increased. The NO signal was markedly reduced in the presence of the protein albumin but not by either oxidized or reduced glutathione. A reduction in the NO signal was also noted after the addition of lipid micelles to the culture medium. These results demonstrate that NO can be detected at significant distances from the cell of origin. In addition, both proteins and lipids strongly influence the net movement of free NO from macrophages. This suggests that these tissue components play an important role in regulating the biological activity of NO.