The goal of this research is to determine the cellular and molecular basis of intracellular calcium signalling mechanisms operating within cultured smooth muscle cells. Cell biological and biochemical approaches will be applied to analyze the molecular basis of two recently identified mechanisms controlling the release and transfer of calcium within cells activated by inositol 1,4,5-triphosphate (InsP3) and guanosine 5'- triphosphate (GTP), respectively. The project has three specific objectives: 1. Identification of the nature and organization of intracellular calcium pools. Studies will assess the function of calcium regulatory organelles in clonal and primary smooth muscle cell cultures, identify at the subcellular level those organelles responsive to InsP3 and GTP, and use electron microscopy and electron probe microanalysis to localize intracellular calcium pools. 2. Analysis of the mechanisms of intracellular calcium transport activated by InsP3 and GTP. Measurements will be made on the nature and specificity of ion fluxes activated by InsP3 and GTP, and on the physical and biochemical changes in the properties of intracellular membranes which correlate with GTP- activated calcium translocation. 3. Molecular characterization of the calcium translocation mechanisms. Studies will attempt to isolate and identify proteins involved in InsP3 and GTP-activated calcium translocation and to determine immunochemically the existence of known calcium-regulatory proteins within discrete membrane subfractions from cells. These studies represent a comprehensive approach to understanding the cellular location and molecular basis of the two calcium regulatory mechanisms, and will assess the validity of a model based on prior work which predicts that GTP induces the translocation of calcium between distinct calcium pumping organelles, including that responsive to InsP3. Changes in intracellular free calcium are involved in the signal transduction pathways for a large number of important cellular messages such as hormones and electrical excitation. The regulation of intracellular calcium by cellular organelles is thus critical for the function of all cells. With prior NSF support Dr. Gill has discovered that in subcellular fractions two calcium pumping activities can be distinguished (in addition to mitochondria), presumably corresponding to different organelles. The molecule, GTP, causes calcium to shift from one of these calcium-pumping organelle to another. These results reveal a new level of complexity of cellular calcium regulation and signal transduction. Continuation of support for this research will permit further analysis of this important finding and permit more detailed characterization of the mechanisms involved in regulation of intracellular calcium.
