TRANSGENIC MODELS OF RESPIRATORY CONTROL

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DESCRIPTION: The primary compensation for arterial hypoxemia is hyperventilation which is mediated by the O2-sensitive cells in the carotid body. The carotid body O2 chemoreceptors play a critical role in the maintenance of O2-homeostasis. The mechanisms by which the O2-sensitive (type I) cells in the carotid body detect a reduction in O2 tension and transduce this signal into the appropriate cellular responses leading to hyperventilation remains, for the most part, unknown. It is known, however, that type I cells express an O2-sensitive K channel that is inhibited by reduced O2 tension which, in turn, causes membrane depolarization and an increase in intracellular free Ca2+. We have shown that hypoxia-induced expression of the gene that encodes tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of dopamine, requires an increase in cytosolic Ca2+ and activation of calmodulin (CaM) in type I cells and in PC12 cells, an O2-sensitive cell line. We also found that neutralization of CaM in catecholamine cells in transgenic mice prevents hyperventilation and increased carotid body activity during hypoxia. In the proposed studies we shall investigate the molecular basis by which the Ca2+/CaM signal transduction system regulates carotid body function in transgenic mice during hypoxia. We hypothesize that an increase in intracellular free Ca2+ and activation of Ca2+/CaM target enzymes (CaM-KI, CaM-KII and CaIN) are involved in this critical process. We further hypothesize that calcium pumps (SERCA2, SERCA3 and PMCA2) play a major role in regulating the level of cytosolic Ca2+ during hypoxia. This is an important function which couples the level of intracellular free Ca2+ with the prevailing hypoxic stimulus. The Specific Aims of the proposed research are: 1) Determine the role of CaM-activated kinases and phosphatases in mediating the cellular responses to hypoxia in wild-type and genetically modified PC12 cells, 2) Determine the role of CaM activated-kinases and phosphatases in mediating the carotid body and ventilatory response to hypoxia in transgenic mice in which these pathways have been neutralized by a novel genetic approach, and 3) Determine the role of the CaM-sensitive plasma membrane and the sarco(endo)plasmic Ca2+-ATPases in mediating the cellular response to hypoxia. Studies are performed in the O2-sensitive PC12 cells, type I cells, transgenic mice and gene knockout mice. Findings from the proposed research should provide much needed information concerning the cellular and molecular basis of O2 chemosensitivity.