Hormone-induced Ca2+ signals regulate secretion in many types of cells, including hepatocytes. Ca2+ signals begin as Ca2+ waves in nearly all cell types, and in hepatocytes such waves may play a role in regulation of secretion. The goal of this project is to define the mechanism of Ca2+ wave propagation in hepatocytes, and to determine whether Ca2+ waves play a regulatory role in canalicular contraction, a Ca2+-mediated event important for bile secretion. The following hypothesis will be tested: Ca2+ waves are initiated by sequential release of Ca2+, first from inositol trisphosphate (IP3)-sensitive Ca2+ stores in the apical region of hepatocytes, then from Ca2+-sensitive Ca2+ stores in the basolateral region. These Ca2+ waves then spread from cell to cell across gap junctions, in a fashion that depends on the type(s) of gap junctions expressed. The apical Ca2+ increase in each cell stimulates canalicular contraction, so that intercellular spread of a Ca2+ wave leads to sequential contraction of the hepatocyte canaliculi, which in turn permits peristaltic propulsion of bile. To test this hypothesis, the specific aims of this project are: 1. To determine whether distinct IP3-sensitive and IP3-insensitive Ca2+ pools can be mobilized in the apical and basolateral region of hepatocytes, respectively, Subcellular Ca2+ signals will be elicited by microinjection of specific agonists and antagonists into isolated rat hepatocytes within couplets and triplets, and Ca2+ signals will be detected in these cells using confocal microscopy. 2. To determine whether gap junctions consisting of connexin26, 32, or 43, each of which are expressed in epithelial cells within liver, modulate the spread of intercellular Ca2+ waves in distinct fashions. Ca2+ increases in individual SKHep1 hepatoma cells will be induced by microinjection of specific second messengers, and intercellular coupling will be measured by cell-to-cell spread of the Ca2+ waves. These cells normally do not express gap junctions but have been transfected with each of these three types of gap junction proteins, and features of the intercellular Ca2+ waves in each of these three SKHep1 cell lines will be quantified and compared. 3. To determine whether canalicular contractions in hepatocyte couplets require an apical increase in Ca2+, require an apical increase in Ca2+, and whether such apical Ca2+ increases must propagate from cell to cell to permit sequential contraction of neighboring canaliculi in hepatocyte triplets. Apical and basolateral Ca2+ increases each will be elicited selectively by microinjection of specific agonists and antagonists into hepatocyte couplets and triplets, and subcellular Ca2+ and canalicular contractions will be measured simultaneously using confocal microscopy and optical planimetry. By defining the mechanisms of Ca2+ wave propagation in this fashion, this project may clarify the importance of intra- and intercellular Ca2+ waves for regulating bile secretion. This work is of potential significance for understanding secretion in other organs consisting of polarized epithelia as well, including the kidney, exocrine pancreas and gastrointestinal tract, since similar polarized Ca2+ waves occur in the cells comprising those tissues. This research also may reveal how signals within communicating epithelial cells integrate to permit an organized, organ- level response to hormonal stimulation, a question of fundamental importance in cell physiology.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
First Independent Research Support & Transition (FIRST) Awards (R29)
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General Medicine A Subcommittee 2 (GMA)
Program Officer
Serrano, Jose
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Yale University
Internal Medicine/Medicine
Schools of Medicine
New Haven
United States
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Weerachayaphorn, Jittima; Amaya, Maria Jimena; Spirli, Carlo et al. (2015) Nuclear Factor, Erythroid 2-Like 2 Regulates Expression of Type 3 Inositol 1,4,5-Trisphosphate Receptor and Calcium Signaling in Cholangiocytes. Gastroenterology 149:211-222.e10
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Reed, Anamika M; Husain, Sohail Z; Thrower, Edwin et al. (2011) Low extracellular pH induces damage in the pancreatic acinar cell by enhancing calcium signaling. J Biol Chem 286:1919-26

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