This is a resubmission of a small grant proposal in which the applicant, Dr. Kenneth Izutsu, proposes to apply a new methodology to the study of the regulation of, and the mechanisms underlying, the exocytosis of secretory granules, using patch clamp instrumentation capable of measuring whole-cell capacitances. Because exocytosis of secretory granules adds membrane to the surface membrane area of the cell, and because whole-cell capacitance is directly proportional to the surface membrane area, this technique can be used effectively as a measure of exocytosis of secretory granules. These studies, similar to previous studies in other cell types, are to be performed in rat submandibular gland acinar cells in primary culture. These cells have the advantage of not being a transformed cell line, of having numerous secretory granules, and of being fairly well characterized from a biochemical point of view. Preliminary data show that procedures have been successfully developed for the preparation of dispersed, individual acinar cells, and that routine recordings can be made from these cells in the whole-cell patch clamp mode. In response to the critique of the first submission, more details are provided concerning the methods to be used to correlate secretion of granule contents with cell membrane capacitance changes. Specifically, it is proposed to use cell immunoblots to measure secretion of specific proteins from individual cells. Dr. Bertil Hille will serve as the consultant on installing and using the cell capacitance hardware and software as well as on performing the cell immunoblot assay, and Dr. Richardo Martinez, who collaborated on the development of the acinar cell preparation and has performed many biochemical studies on submandibular acinar cells, will serve as a consultant on the protein secretion aspects of the studies.
The specific aims i n the revised application are:
Specific Aim l: To obtain and install the equipment necessary to make cell capacitance measurements using the whole-cell patch clamp technique.
Specific Aim 2 : To test whether cell capacitance changes are correlated with secretion of secretory granule contents in single rat submandibular gland acinar cells in primary culture. This will primarily be tested using norepinephrine stimulation.
Specific Aim 3 : To test whether endocytosis of secretory granule membranes as measured by cell capacitance changes, is inhibited in the absence of external Ca2+.
Specific Aim 4 : To test whether ion channels in the secretory granule membranes contribute to the cell conductance changes observed during granule exocytosis. The applicant explains that the first of these aims involves installing the equipment, the second involves the major experimental work, and the third and fourth aims involve additional studies using the methodology developed and used in the first two aims to further validate the system. The applicant suggests that, if successful, this technique should open a new approach to the study of the regulation of the exocytosis of secretory granules.
