David E. Cochrane - US grants

Mast cells can be stimulated to secrete histamine by a variety of non-immunological agents. These secretagogues are believed to act independently of the IgE molecule and differ in this and several other significant ways from immunologic agents such as antigen or anti-IgE serum. Evidence accumulated over the past few years convincingly suggests an active role for several of these non-immunologic agents in the physiological regulation of mast cell function and raises the distinct possibility of new, as yet unknown, roles for the mast cell. The mechanisms by which these non-immunologic secretagogues activate and control mast cell secretion are only partially understood. It is the objective of this research proposal to study these mechanisms. For this, the characteristics of the Ca-entry mechanism activated by stimulation with non-immunologic secretagogues will be determined using the process of inactivation (i.e., the decay in Ca permeability). Initial experiments will characterize Ca movements in the absence of secretion using 45Ca efflux from cells loaded with 45Ca by stimulation. The specificity of the inactivation process, the effect of membrane potential, the cellular changes that may precede and/or accompany inactivation, recovery from inactivation, and whether non-immunologic and innumologic secretagogues activate a common Ca-"channel" mechanism will be determined. Secondly, the binding characteristics for non-immunologic secretagogues, the possibility of a common binding site, and whether "down regulation" of binding occurs during the inactivation process will be determined. Thirdly, the characteristics of the non-immunologic peptide secretagogues, neurotensin, bradykinin, and substance P. will be directly determined and compared to the more fully studied, synthetic secretagogue, compound 48/80. Fourth, non-immunologic agents are believed to act independently of the IgE molecule. This conjecture will be directly tested. Finally, the characteristics of the cellular Ca pool that is accessible to chelation and whether cellular Ca is released from a membrane fraction will be determined.

Mast cells are an important source of histamine (H) and are involved in several common pathological conditions including various urticarias, atopic eczema, and inflammation. Neurotensin (NT) has been shown to bind to specific receptor sites on the isolated mast cell surface, to elicit H release from isolated mast cells, to evaluate H levels in the blood when given intravenously, and to increase cutaneous vascular permeability. Preliminary experiments in our laboratories have demonstrated that very low concentrations of NT (Less than 10 to the -10M) can significantly increase H release from perfused slices of skin, that there is a means to form NT-related peptides in extracts of skin, and that these NT-related peptides can release H from isolated mast cells. It is the objective of this project to study in detail this NT-mast cell system in order to ascertain its possible physiological and/or pathological significance. Using perfused slices of skin and isolated peritoneal mast cells, the effect of NT on the release of H will be characterized in terms of ionic, metabolic, and pharmacologic requirements; the possible involvement of other effectors of H release in the response will be tested, whether additional mediators of inflammation accompany the H released by NT will be determined, and the cellular source of the released H will be confirmed. The chemical, immunochemical, and biological properties of the NT-related material that can be extracted from skin will be characterized. Finally, the effects of physiological and/or pathological stimuli to the skin on the level of these NT-related peptides and the level of H in the skin will be determined. Skin slices and mast cells will be isolated from adult rats. Histamine will be assayed fluorometrically and radioenzymatically. NT will be assayed by radioimmunoassay and by receptor binding-assay. NT-related material will be characterized by HPLC and biological activity assayed by ability to release H from skin slices and mast cells. Changes in blood flow and vascular permeability in vivo will be followed using radioactive microspheres and the escape of I125 albumin, respectively. The experiments proposed in this application will provide new and significant information concerning the biological role of NT and the relationship between this peptide and the mast cell in health as well as in disease states.