Exchange of ionic species and water with the environment is a normal physiological function of living cells that lead to fluctuations of cell volume, activation of volume-sensitive membrane CT channels and water fluxes and to recovery of cell volume. PI 3-kinases, tyrosine kinases are simultaneously activated, but their cellular effectors and mechanisms of Cl-channel regulation are elusive, Our long-term goal is to identify intracellular signal transduction sensitive Cl-channels and cell volume adaptation. The hypotheses are: 91) activation of phosphatidylinositol 3- kinases (PI3Ks) and protein kinases C (PKCs) is crucial for rapid regulation of Cl- channels and adequate early responses to cell volume perturbation; and (2) that PI3K-mediated activation of effector kinase cascades, i.e. MAP kinase (p38 MAPK), PAK and ROCK, triggers reorganization of the actin cytoskeleton, which then modulates activation of ClC-2 and ClC-3 channels and elicits late responses to hypotonic swelling or hypertonic shrinkage of pulmonary artery smooth muscle cells (PASMCs).
Four specific aims will test the hypothesis:
Aim 1 : Activation of PI 3-kinases and PKCs mediate channel conductance and volume adaptations; identify PKC enzymes that regulate Cl-channel current; show activation of other PI3K-dependent pathways such as MAPKs, PAKs, ROCKs, and Akt/PKB.
Aim 2 : Cell volume-regulated activation of p38 MAPKs and phosphorylation of putative target proteins leading to actin remodeling. We will study the role of p38 MAPK - >Hsp27 pathway in remodeling of actin structures and Cl-channels.
Aim 3 : Actin cytoskeleton rearrangement mediated by PAKs and ROCKs and role in activation of volume regulatory events. We will: study PAK- and ROCK-mediated phosphorylation of MLC and effects on actin assembly and Cl-channel activity; analyze activation-dependent translocation of LIMK, association of LIMK association and regulation of Cl-channels.
Aim 4 : Expression and cellular functions of the SGK; study activation, intracellular localization and potential SGK targets; and test role of SGK in phosphorylation and function of Cl- channels. This project will generate novel information on the intracellular pathways involved in regulation of the native volume-sensitive Cl-channels and help better understand the volume adaptation machinery of smooth muscle cells.
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