A method is presented whereby it is possible to measure the cytoplasmic concentration of ionized calcium (Ca-i) and rates of hormone secretion from the same population of cells. Ca-i is measured with the photoprotein aequorin which is loaded into the cells by 1 of 2 methods: hypoosmotic shock or scrape loading. I propose to determine the method to incorporate the most aequorin while producing the least damage to the cells with this method. I propose to measure the magnitude and time course of the changes in Ca-i and rates of hormone secretion evoked by a number of secretagogues, to determine how these changes are influenced by cAMP changes in inositol triphosphate metabolism, and the hormones estradiol and triidothyronine. Preliminary results suggest that quin-2 may buffer the effects of the secretagogues on Ca-i and hormone so I propose to determine if the incorporation of quin-2 affects the effect of secretagogues on Ca-i of aequorin loaded cells. Finally I propose to use aequorin to test the effects of inisotol triiphosphate on the release of calcium from intracellular stores of permeabilized cells and determine how the release may be altered by cAMP and calmodulin.
| Pershadsingh, H A; Lee, L Y; Snowdowne, K W (1989) Evidence for a sodium/calcium exchanger and voltage-dependent calcium channels in adipocytes. FEBS Lett 244:89-92 |
