The effects of hormones such as catecholamines and vasopressin on hepatic metabolism are mediated by alterations in the concentration of cytosolic free Ca2+ ([Ca2+]c), largely as a result of Ca2+ mobilization from intracellular stores by the second messenger inositol 1,4,5- trisophosphate (IP3). The [Ca2+]c responses to these hormones are organized in the form of [Ca2+]c oscillations and waves whose frequency is controlled by agonist dose. Frequency-modulated [Ca2+]c oscillations may serve a number of signaling functions, including improved fidelity, sensitivity and targeted regulation of specific processes. [Ca2+}c waves are also likely to play a role in propagating [Ca2+]c signals to distal parts of the cell, in signaling between cells and in establishing polarized functional responses. It is clear that the principal element of the [Ca2+]c oscillation mechanism is the IP3 receptor Ca2+ channel (IP3R), which is sensitive to positive and negative feedback regulation by Ca2+ and IP3. However, the evidence also indicates that other Ca2+ signaling components may be involved in establishing the spatial and temporal organization of [Ca2+]c signaling, either by modulating the Ca2+ feedback regulation of the IP3R or by superimposing additional Ca2+ - feedback loops on the essential IP3R oscillator. We propose to investigate how these additional inputs interact with IP3-dependent [Ca2+]c oscillations to enrich the frequency range and spatial pattern of oscillatory [CA2+]c waves. Our studies are designed to address the following questions: 1. Does Ca2+ - feedback on IP3 metabolism (phospholipase C or IP3 3-kinase) play a role in generating [Ca2+]c oscillations and waves? 2. What is the role of the ryanodine receptor in Ca2+ release and regenerative Ca2+ wave propagation? 3. What are the consequences of the close coupling between IP3-dependent Ca2+ release and mitochondrial Ca2+ uptake for the temporal and spatial organization of [Ca2+]c signals? 4. What defines the polarized initiation site for [Ca2+]c waves in hepatocytes; subcellular heterogeneity in IP3R function or spatially localized interactions of the IP3R with other Ca2+ signaling components? These studies will make use of biochemical, molecular and imaging techniques for studies of Ca2+ signaling in intact hepatocytes, together with a recently developed permeabilized cell system in which we can reconstitute IP3-dependent Ca2+ oscillations and waves.
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