Cytosolic Ca2+ (Cai2+) regulates a wide range of cell functions, from secretion to metabolism to cell death. It is unknown how Cai2+ simultaneously controls such diverse activities in an individual cell, although Cai2+ waves and other types of Cai2+ gradients may be responsible by allowing distinct Cai2+ signals to occur in different subcellular regions. Physiologic roles have been identified for Cai2+ waves in some cell types, but neither the mechanism of Cai2+ wave propagation nor its physiologic significance is established in hepatocytes. The hypothesis of this project is that Cai2+ waves in hepatocytes result from sequential release of distinct subcellular Ca2+ pools, and that localized, subcellular increases in Cai2+ can regulate specific hepatocyte functions. To test this hypothesis, the specific aims of this project are: 1. To determine the mechanism by which Cai2+ waves spread across individual hepatocytes. We will test whether there are distinct types of inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ stores in the apical region and the basolateral region. Subcellular Cai2+ signals will be elicited by microinjection of caged InsP3, Ca2+, or both, followed by localized (apical or basolateral) release of these agonists using two-photon flash photolysis. Also, we will inject agents to specifically antagonize the effects of InsP3, or Ca2+ to determine which of these messenger molecules affect Cai2+ waves. Isolated rat hepatocyte triplets will be used, and Cai2+ signals will be detected by confocal line scanning microscopy. 2. To determine the mechanism by which Cai2+ waves spread from cell to cell in liver. We will test whether gap junctions consisting of connexin26, 32, or 43, each of which are expressed in liver, modulate the cell-to-cell spread of Cai2+ waves in distinct fashions. Increases in Cai2+ in individual SKHep1 hepatoma cells will be induced by microinjection of caged second messengers, followed by their release through flash photolysis. The resulting Cai2+ waves will be measured and compared using confocal video microscopy. This cell line normally does not express gap junctions but has been transfected with these three different types of gap junction proteins. 3. To determine the relationship between subcellular Cai2+ increases and a Ca2+- mediated event, canalicular contraction. We will test if canalicular contraction requires only that Cai2+ increases in the apical region, and whether such apical Cai2+ increases must spread from cell to cell to permit sequential contraction of neighboring canaliculi. Localized apical or basolateral Cai2+ signals will be elicited in isolated rat hepatocyte couplets and triplets by two-photon flash photolysis. Cai2+ and canalicular contractions will be detected simultaneously using confocal microscopy and optical planimetry.
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