Mast cells and their homologues, the basophil and the mucosal mast cell, are the primary effector cell type in a range of inflammatory reactions, such as hayfever and asthma. When stimulated by cross-linking of surface bound immunoglobulin E, mast cells release performed histamine from membrane-enclosed, cytoplasmic granules. Despite the importance of the storage of histamine in the secretory granules, little is known at the molecular level about the intragranular state of histamine or the nature of the intragranular environment. This is due in part to the lack of tools with which intact cells can be examined at the molecular level. It is the aim of this research (1) to characterize the intragranular environment of mast cell secretory granules with respect to pH, water content and the mobility of intragranular histamine, (2) to identify the changes in granules with respect to pH and histamine mobility that occur prior to extrusion of the granule during exocytosis, and (3) to fully characterize the binding of histamine by granules in terms of affinity constants and binding sites. These objectives will be accomplished by using noninvasive 1H nuclear magnetic resonance (NMR) spectroscopy to examine histamine in intact mast cells and in histamine-granule and histamine-glycosaminoglycan model systems. The mobility of histamine in intact cells, in mast cells which have been arrested following stimulation, and in granules will be determined from relaxation times for histamine protons. Affinity constants for the binding of histamine by granules and glycosaminoglycans will be determined from chemical shift and intensity measurements. Intragranular pH will be determined by using added imidazole as an NMR pH indicator, while a small, neutral, granule permeable molecule, such as methanol, will be used as a reporter of water volume. It is anticipated that these studies will contribute to an increased comprehension of mast cell behavior which is essential for deliberate perturbation of the secretory process for therapeutic purposes.
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