During the past few years, studies of the initiation of PMN responses have revealed much about stimulus-response coupling, particularly with respect to the role of intracellular calcium. However, many questions remain to be answered and several new lines of investigation have been opened. This application proposes five lines of research, four of which are largely dependent upon permeabilized cell systems developed by the applicant, and which will permit a far better understanding of the critical early events in stimulus-response coupling. 1) We propose to further refine the permeabilized cell system which permit the investigator to freely manipulate the intracellular milieu. 2) In view of the critical role played by the mobilization of membrane-bound calcium in the earliest events in stimulus-response coupling, we will further characterize the chlortetracycline fluorescence response. 3) The role of intracellular free calcium will be investigated using permeabilized PMN, which will permit manipulation of these calcium levels with chelating buffers. 4) The roles of other putative messengers, such as lipids and inositol triphosphate, will be explored using the """"""""leaky"""""""" PMN. 5) Finally, we will use intramolecular pyrene eximer formation to address the problem of measuring rapid changes in membrane microviscosity. Examination of PMN from patients with Chediak-Higashi syndrome, which have defects directly relevant to stimulus-secretion coupling, will complement these studies. These lines of investigation should prove fruitful in revealing the underlying mechanisms of stimulus-response coupling in PMN. These studies will have relevance to a basic understanding of cellular responses to external stimuli.
