The effects of hormones such as catecholamines and vasopressin on hepatic metabolism and secretion 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-trisphosphate (IP3). The [Ca2+]c signals elicited by these hormones are organized in the form of [Ca2+]c oscillations and waves, whose frequency is controlled by hormone dose. Frequency-modulated [Ca2+]c oscillations may serve a number of regulatory functions, including improved fidelity, sensitivity and targeted regulation of specific processes. While the role of IP3-receptors in this pathway is well established, it has recently become evident that other Ca2+ release and Ca2+-feedback components contribute to extend the temporal and spatial range of [Ca2+]c oscillations. One way in which this might be achieved is through interplay between IP3R and the other major class of intracellular Ca2+ release channels, the ryanodine receptors (RyRs). RyRs are typically activated by Ca2+ through a Ca2+-induced Ca2+ release (CICR) mechanism, but they can also be activated by another second messenger, cyclic-ADPribose (cADPR). Although the RyRs have been well-characterized in excitable tissues (primarily muscle and brain), relatively little is known about the identity and properties of RyRs in nonexcitable cells. We have recently identified and cloned a unique RyR isoform from rat hepatocytes (RyR1b), which is derived from the RyR1 gene, but with an alternative start site that gives rise to a protein of only 40% the size of full length muscle RyRs. We hypothesize that RyR1b has distinct functional properties that underlie its contribution to [Ca2+]c signaling in hepatocytes and other nonexcitable secretory cells, where it may be the principal RyR isoform. Specifically, we propose that RyR1b plays a key role in enhancing and sustaining ER Ca2+ release initiated by IP3R activation. We will examine the potential role of RyR1b in two specific aims:
In Aim 1 we will carry out heterologous expression studies to investigate the function and regulatory properties of RyR1b, including regulation by [Ca2+]c, cADPR and its interactions with IP3 and the IP3R.
In Aim 2 we will investigate the subcellular distribution and regulation of RyR1b in primary hepatocytes, and determine how it contributes to the temporal and spatial pattern of [Ca2+]c signaling during hormonal stimulation of these cells. RyR1b appears to be a major new addition to the superfamily of intracellular Ca2+ release channels, with distinct properties. It may have specific functions that are tuned to the signaling requirements of nonexcitable secretory epithelia and endocrine cells. Thus, the proposed work is innovative and has the potential for high impact, with significant biomedical health relevance.
The proposed work will investigate the role and regulation of a newly discovered calcium signaling protein (RyR1b) that appears to play a role in mediating the effects of hormones on liver function. It is also postulated to participate in signaling in other tissues, including the pancreas and cells of the digestive tract. Since derangements of signaling in these tissues are frequently associated with disease states (eg. hepatitis, cholestasis, pancreatitis), characterization of the properties of RyR1b has significant potential public health impact. Its unique molecular structure may yield a novel therapeutic target.
