The Development of an Electro-Optical Technique to Measure Superoxide Release from Pulmonary Alveolar Macrophages Exposed To Coal Dusts

"A microscope-based spectrophotometer has been developed to measure superoxide release from single pulmonary alveolar macrophages (PAM) or other phagocytes in culture. Results indicate that PAM respond to stimulation by phorbol myristate acetate (PMA). It appears that some PAM do not release 02 upon stimulation by PMA; however, those that do release 0, show differences in: (I) the total amount of 0, released, (2) the rate of release of 02 and (3) the time delay for release of 02. The significance of measuring 02 release from single cells is two-fold. It allows the simultaneous study of 02 release and electrical potential changes across the membrane of PAM and it allows the study of differences in 02 release among cells. Thus, the function of Normal PAM and the dysfunction of PAM exposed to coal dusts can be more closely studied on single cells.Pulmonary alveolar macrophages (PAM), free cells found in the lungs, protect the lungs by removing inhaled foreign debris and bacteria. The macrophages do this by engulfing and then internalizing the foreign matter (phagocytosis). Stimulation of PAM causes the release of super-oxide radical (02) which has been shown to be the primary metabolite involved in the killing of bacteria by phagocytes (3,8,12). The super-oxide radical is highly reactive and is spontaneously or enzymatically converted to other antibacterial oxidants, including hydrogen peroxide (H202), singlette oxygen ( 02), hydroxyl radical toil'), and hypochlorous acid (HOCl) (2, 3,8,11,12).Exposure of PAM to dusts may lead to dysfunction of this cell-type and to a greater risk of lung damage or infection upon subsequent exposure to toxic dusts. To date, much of the research with this cell-type has used cytotoxicity (cell death) as a measure of dysfunction. Although this is an important end point, PAM dysfunction may occur at doses below those which lead to cell death. In addition, dysfunction has been measured using large numbers of cells. In order to obtain a clear understanding of the mechanisms involved in dysfunction, measurements should be made on single cells. As mentioned earlier, one of the major roles of this cell type is its anti-bacterial actions. Therefore, asessment of release of superoxide radical in single cells is important in determining the functional behavior of PAM before and after exposure to dusts. Since measurements of 02 production have not been made on single cells the purpose of this study is to develop the methodology needed to study release of 02 from single cells.Recent studies have suggested that a relationship exists between the release of 02 and changes in the electrical properties of phagocytes. The change in membrane potential is thought to be the initial step in superoxide production (6,9). Stimulants which cause 02 release also cause changes in the transmembrane potential of phagocytes (9) and in the permeability of the cell membrane to ions (6). For example, stimulants which cause the release of 0, from neutrophils have also been shown to increase the membrane transport of Na+ , K+ and Ca+2 and stimulation of macrophages leads to increases in Na and perhaps Ca permeability with a subsequent change in membrane potential. In some cases, phagocytic activity has been shown to depend upon the extracellular concentrations of these ions (6). Holian and Daniele (7) have shown evidence that the redistribution ofcellular Ca in alveolar macrophages is an initial step leading to 0, release. In order to provide direct evidence that 02- production is related to electrical changes across the plasma membrane of phagocytes, these two phenomena must be analyzed simultaneously on single cells. Ultimately, measurements of 0, release using the methodology developed in this study will be combined with electrophysiological tech¬niques to study both superoxide release and membrane potential changes simultaneiously on single PAM, and to examine the effects of coal and mineral dusts on their physiological fu"