We seek to elucidate the initial steps in, and the membrane receptor responsible for, granulocyte stimulation by specific stimuli. We shall concentrate first on two of the latter, opsonized particles and antigen-antibody complexes. We plan to analyze the response of normal human granulocytes as well as that from patients with granulocyte disorders such as chronic granulomatous disease. Our approaches are outgrowths of the techniques we designed for elucidation of the stimulus-response mechanism of another secretory cell, the human platelet, techniques which may be generally applicable to the study of secretory cells. In pursuit of this goal we are preparing fully biologically active photoreactive derivatives of the C3b fragment of complement and of the Fc portion of a specific IgG. We will then use these derivatives, allow them to bind to granulocytes in the dark for a very short time, photoactivate covalent bond formation between the derivatives and their membrane receptors, and then solubilize the membranes, isolating and identifying the receptor-stimulus complex by standard means. The same derivatives can be used to help elucidate the mechanism whereby binding to the receptor is transmitted as a signal (by selective and covalent blocking of these receptors), and to correlate this mechanism with the rapid changes in membrane potential and in intragranulocyte pH and (Ca++), (by using specific probes or indicators) and the stimulation of oxygen metabolite formation. We shall also investigate the involvement of the granulocyte's contractile system in the stimulus-response (by using an indicating actin). The effect of perturbants such as specific cation blockers (amiloride) or membrane active agents (chlorpromazine) on the parameters described above and on the accessibility of receptors shall also be probed. These studies should help us to clarify the mechanism, to understand the cause of defects (congenital or induced) in, and to suggest amelioration for defects in granulocyte stimulation.
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