The overall objective of the studies described in the present application is to determine the underlying mechanisms that regulate Ca2+ signals in the pancreatic acinar cell. These mechanisms are responsible for agonist- induced Ca2+ influx into the cytoplasm from the extracellular pace and from intracellular stores as well as patterns of cytoplasmic [Ca2+] changes such as waves and oscillations. Because the pancreatic cell serves as a model system for cell Ca2] changes such as waves and pathophysiologic mechanisms in a variety of tissues.
The specific aims of the present application include the following. 1.) The characterization of the cyclic GMP regulated transport mechanism mediating agonist-stimulated Ca2+ influx across the plasma membrane. 2.) The determination of the mechanisms responsible for Ca2+ regulation of guanylyl cyclase and cyclic GMP formation. 3.) The ultrastructural localization of specific types of agonist-sensitive Ca2+ stores. 4.) The determination of the roles and mechanisms of cyclic ADP-ribose, inositol 1,4,5-trisphosphate and cyclic GMP on Ca2+ release and uptake in various compartments of the agonist-sensitive Ca2+ store.
Aims 1 and 2 will be achieved by the molecular cloning and expression of a cyclic GMP-gated Ca2+ channel and a Ca2+-binding recoverin-like protein from the pancreatic acinar cell. Studies to determine the functioning and physiologic roles of these proteins will include measurements of Ca2+ changes with both electrophysiologic techniques, fluorescent dyes; and 45Ca2+ movements and the use of antibodies generated to these proteins.
Aim 3 will be achieved using antibodies to specific components of the stores; fluorescence and electron microscopy; and three dimensional reconstruction techniques.
Aim 4 will be achieved by determining the role of the messengers on Ca2+ transport in various purified microsomal preparations representing subcompartments of the intracellular Ca2+ store. The combined findings of the proposed studies will lead to a model describing the processes involved in agonist-regulated Ca2+ influx into cytoplasm as well as underlying processes involved in the generation of spaciotemporal patterns of cytoplasmic [Ca2+] changes in the pancreatic acinar cell.
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