The objective of this research is to understand the mechanisms and pathways by which macromolecules move across the capillary wall in skeletal muscle. The approach is to measure fundamental membrane parameters descriptive of the geometry of the transport pathways. These parameters, the solvent drag and osmotic reflection coefficients (sigma f and sigma d), will be measured.for a series of proteins under 1) normal permeability conditions, 2) increased.permeability due to the application of inflammatory mediators and 3) cold-induced inflammation. The measurement techniques are new, previous ones being inappropriate and inaccurate for use in this tissue. The Integral-Mass Balance (IMB) method determines sigma f from measurement of the accumulated losses of water and macromolecule(s) from the circulation induced by a period of fluid filtration. Only hematocrit and macromolecule concentration measurements are required. The IMB method is particularly suited to measure sigma f for two or more molecules simultaneously and to measure the rapid changes in permeability which can occur in inflammation. The Isogravimetric-Osmotic (IO) method determines sigma d from the ratio of the change in the isogravimetric capillary pressure (Pci) to the imposed change in the macromolecule osmotic pressure in an individual experiment. Pci is measured by a new and rapid stop-flow method. The experiments will be performed in the isolated, perfused hindlimb preparation which has been used to develop these measurement methods. The proteins used will be ribonuclease-A, ovalbumin, albumin, gamma-immunoglobulin-G (IgG) and gamma-immunoglobulin-M (IgM) which span a size range of about 1.5.12 nm in radius. The studies proposed will measure 1) sigma f for pairs of proteins in individual experiments. 2) sigma d for certain proteins, 3) both sigma f and sigma d for a protein in individual experiments to test the equality of these coefficients, 4) the time course of sigma f changes due to inflammatory mediators and 5) the effect of cold- induced tissue trauma on these parameters. The body of data developed will be new. It will serve as a basis for hypotheses of fluid transport mechanisms and pathway structure in skeletal muscle. This information is vitally important to the understanding of normal transport processes and of the altered ones seen in edema, tissue trauma and shock.
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