The alveolar macrophage is the first line of host defense in the lung against inhaled foreign material. The long term goal of this laboratory has been the elucidation of the biochemical and biophysical steps, and their regulation; which culminates in the inflammatory response. Although there is an improving picture of the biochemical sequence in macrophage activation, the mechanism(s) by which this sequence is regulated is poorly understood. Once this goal is achieved then it will be easier to develop drugs to regulate the activity of these cells. The overall objective of this proposal is to gain a better understanding of the regulation of pHi in the alveolar macrophage during stimulation and in turn characterize the effects of pHi on macrophage activation. Although the alveolar macrophage is the central inflammatory cell in the lung it has been inadequately studied. Specific objectives will be to: 1) describe ion exchangers which regulate intracellular pH in the macrophage, 2) determine the role of these ion exchangers during macrophage activation, and 3) determine the influence of intracellular pH changes on the activation sequence of the macrophage. The hypothesis that will be tested is that, at a minimum, there is a SITS sensitive bicarbonate/chloride exchanger and an amiloride sensitive sodium/proton exchanger that are important in regulating pHi of alveolar macrophages. Additional exchangers may also be involved and will be examined. Furthermore, pHi changes following macrophage activation could partially contribute to the regulation of the inflammatory response. The model system that will be used is isolated alveolar macrophages from guinea pigs. In order to test these hypotheses, these studies will utilize isotopes, inhibitors, pH and ion specific electrodes and media modification. Intracellular pH will be measured using two independent procedures (weak acid distribution and fluorescent probes). The effects of altered intracellular pH on the intracellular signalling pathway (receptor sensitivity, phosphatidyl inositol turnover, calcium mobilization and protein kinase C activity) will be measured to determine what and where the regulatory effects of pHi have on this pathway.
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