These experiments are designed to provide a bridge between investigations on the cellular mechanisms which may modulate the permeability of endothelial barriers in culture, and investigations in intact microvascular beds.
The specific aims of the project are to test four hypotheses: The first hypothesis states that increased calcium ion influx into endothelial cells is a necessary condition to increase microvessel permeability to water and macromolecules. The second hypothesis states that membrane potential of the endothelial cell is a regulator of calcium influx. The third hypothesis states that the physical and chemical environment of the microvessel modifies changes in permeability and calcium influx associated with exposure to an inflammatory stimulus, and the fourth hypothesis states that vesicle exchange and specific cellular uptake processes are more important mechanisms for the transport of macromolecules than solvent drag when microvessels are in the lowest permeability state. The investigators have developed methods to measure changes in cytoplasmic calcium concentration, [Ca++]i, and the membrane potential of endothelial cells forming the walls of individually perfused microvessels. They have found that in microvessels exposed to calcium ionophores, processes which depolarize the endothelial cell membrane reduce calcium influx and decrease microvessel permeability while processes which hyperpolarize the membrane increase calcium influx and permeability. Preliminary experiments suggest similar mechanism may regulate other high permeability states. The investigators have also developed perfusion conditions where the solvent drag contribution to macromolecule flux across the microvessel wall is negligible. They are therefore able to investigate cellular mechanisms which may contribute to macromolecular exchange. The four hypotheses are a logical extension of investigations carried out in the current funding period. To test the hypotheses, the investigators will use microperfusion methods to measure [Ca++]i, calcium influx, and microvessel permeability under identical experimental conditions. Permeability will be increased using receptor dependent and receptor independent changes in permeability in venular microvessel of frog and hamster mesentery. The investigations provide a direct experimental route to further understanding of the processes which increase and decrease capillary permeability under conditions which lead to edema and loss of organ function.
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