Trauma provokes the release of a large number of bioactive mediators, such as lipids, hormones and cytokines (Project I-A). These ligands elicit immediate changes in function and metabolism, but also modify the cellular response(s) to subsequent stimuli (both destructively or constructively). The mechanism(s) of these adapted responses (termed priming or preconditioning, respectively) remain the focus of our investigations (with Projects II, III, IV, VII, and IX). Deservedly, ligand activated signaling by second messengers, ion-flux and protein phosphorylation continue to receive extensive study. But ligand binding also triggers receptor internalization. Using transformed models we (and others) now accept that activated receptors for several classes of bioactive mediators (such as catechols, lipids and TNFa) are internalized by clathrin coated vesicles. Clathrin coated endosomes are transported about the cell on microtubular tracks. Surprisingly, recent studies indicate that receptor internalization is necessary to initiate a novel phase of signaling. Signals emanating from internalized G-protein coupled receptors (GPCR) affect transcriptionally directed protein kinase cascades (including PKC and the MAPK modules) causing sustained changes in cell responsiveness (with Project V). However it is unclear how previous signaling events influence the interactions between internalized receptors, the endocytotic machinery and the microtubular cytoskeleton, in mammalian, especially human cells. Moreover it is unclear whether pro-inflammatory cells such as macrophages respond similarly to vulnerable targets such as endothelial or smooth muscle cells (with Project VI). We hypothesize that circulating mediators (adrenergic, lysolipid and cytokines ) (with Projects V, VII and IX) released after trauma augment internalization for membranes, receptors and subsequent signal traffic. We initially focus on ligands selected for i. pertinence to trauma, ii. mediation by GPCR, and iii, activation of PKC and MAPK modules. We will examine how these test ligands influence a. constitutive membrane/receptor trafficking and b. internalization dependent signaling in a set of myeloid and non myeloid cells from humans and rodents. These studies will form the basis for evaluating whether currently accessible therapeutic strategies can be directed against clathrin coated endocytosis and microtubule based trafficking.
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