Cytosolic Ca2+ (Cai2+) regulates a wide range of cell functions, from secretion to metabolism to cell growth and death. It is unknown how Ca2+ simultaneously controls such diverse activities in an individual cell, although Cai2+ waves and other types of Ca2+ gradients may be responsible by allowing distinct Cai2+ signals to occur in different subcellular regions. The parent grant for this FIRCA proposal tests the hypothesis that Cai2+ + waves in hepatocytes result from sequential release of distinct subcellular Ca2+ pools, and that localized, subcellular increases in Cai2+ canregulate specific hepatocyte functions. The current proposal would extend the hypothesis of the parent grant in an important way, by testing whether subcellular and intercellular Cai2+ waves regulate secretion in pancreatic acinar cells, a well-established model of polarized epithelium.
The specific aims of this project are: 1. To determine the mechanism by which Cai2+ + waves spread across individual acinar cells. We will test whether Cai2+ can be increased selectively in either the apical or basolateral region. Subcellular Cai2+ signals will be elicited by microinjection of caged second messengers, followed by localized (apical or basolateral) release of these agonists using flash photolysis. Also, we will inject agents to specifically enhance or antagonize the effects of these second messengers to determine which of these messenger molecules affect Cai2+ waves. Individual acinar cells within intact acini will be used, and Cai2+ signals will be detected by confocal microscopy. 2. To determine the mechanism by which Cai2+ waves spread from cell to cell in the intact pancreatic acinus. We will test which second messengers must cross gap junctions in order to coordinate the spread of Cai2+ wavesfrom cell to cell. Increases in Cai2+ in individual acinar cells will be induced by microinjection of caged second messengers followed by their release through flash photolysis in the presence or absence of gap junction blockers. The resulting Cai2+ waves will be measured throughout the entire acinus using confocal microscopy. 3. To determine the relationship between subcellular and intercellular Cai2+ increases and a Ca2+-mediated event, exocytosis. We will testif apical exocytosis requires only that Cai2+ increases in the apical region, and whether exocytosis is potentiated if such apical Cai2+ increases are coordinated among neighboring cells. Localized apical or basolateral Cai2+ signals will be elicited in acinar cells by flash photolysis. Cai + and exocytosis will be detected simultaneously using confocal microscopy.
| Guatimosim, Silvia; Amaya, Maria Jimena; Guerra, Mateus T et al. (2008) Nuclear Ca2+ regulates cardiomyocyte function. Cell Calcium 44:230-42 |
| Hernandez, Erick; Leite, M Fatima; Guerra, Mateus T et al. (2007) The spatial distribution of inositol 1,4,5-trisphosphate receptor isoforms shapes Ca2+ waves. J Biol Chem 282:10057-67 |
| Husain, Sohail Z; Grant, Wayne M; Gorelick, Fred S et al. (2007) Caerulein-induced intracellular pancreatic zymogen activation is dependent on calcineurin. Am J Physiol Gastrointest Liver Physiol 292:G1594-9 |
| Correa, Paulo Renato A V; Kruglov, Emma A; Thompson, Mayerson et al. (2007) Succinate is a paracrine signal for liver damage. J Hepatol 47:262-9 |
