investigator's application): Large and often repetitive increases of cytosolic [Ca2+] ([Ca2+]c) mediate the effects of various hormones that act through the second messenger inositol 1,4,5-triphosphate (IP3) in many cell types. Elevated [Ca2+]c results in activation of Ca2+-sensitive components that participate in a wide range of cellular functions. Full activation of a particular target would be evoked by prolonged Ca2+ increases, however, simultaneous full activation of various targets in the cells by permanently high [Ca2+] could lead to cell injury. The physiological range of stimulation appears to be restricted to the transient [Ca2+]c increases and in many cases the intensity of hormonal stimulation is converted into the frequency of brief and uniform [Ca2+]c increases (frequency modulation). It is well established that the bursting phase of Ca2+ mobilization occurs when IP3 opens Ca2+ release channels on the endoplasmic reticulum Ca2+ store using positive feed back effects exerted by the released Ca2+. However, little is known about the mechanism of the falling phase of Ca2+ transients. The present proposal is directed towards elucidating the mechanisms that are responsible for the deactivation of [Ca2+]c transients using hepatocytes and endocrine cells of the adrenals as a model system. The investigators suggest, that IP3-induced (time dependent) inactivation of the IP3 receptor (IP3R) is an important factor in the falling phase of [Ca2+]c transients. It is hypothesized that inactivation of the IP3R is particularly large if IP3R is activated by IP3 in Ca2+-sensitized state that may occur at modest increases of IP3 evoked by physiological levels of hormones. The investigators also suggest that Ca2+ released by IP3 is transiently accumulated in mitochondria, allowing mitochondria to contribute to the decay of [Ca2+]c transients and subsequently to recharge IP3 sensitive Ca2+ stores. The investigators propose that mitochondria are not only an important target of the cytosolic Ca2+ signal, but also represent a Ca2+ store that controls the ability of the endoplasmic reticulum Ca2+ store to generate [Ca2+]c transients. A major component of these studies will rely on imaging approaches to measure dynamics of Ca2+ movements within both intact and permeabilized cells, including some novel techniques that permit studies of IP3 action at the level of the Ca2+ stores. Imaging methods will be used in combination with electrophysiology and molecular biology.
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