MECHANISMS INVOLVED IN LYMPHATIC CONTRACTILE ACTIVITY

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My long-term career goals are to advance the level of understanding about the roles and mechanisms of an important but poorly understood organ system, the lymphatic system. My immediate career objectives are to: establish a reputation for independent, quality scientific studies; to obtain tenure; to train graduate students and postdoctoral fellows; and to contribute to the advancement of the scientific societies, study section and editorial boards. Receipt of this award will help advance my research program by providing incentives to the college to free up both time and money for the PI to focus on scientific research. The lymphatic system is a specialized part of the circulation that is intimately involved in the maintenance of normal body fluid homeostasis. It is thought to be one of the principal safety factors against gross edema formation. The lymphatic system is also involved in nutrient absorption, lymphocyte circulation and antigen presentation. These tasks are accomplished via lymph circulation. Fluid is actively pumped through the lymphatic system by the spontaneous generation of lymphatic contractions. The general objectives of these studies will be to elucidate the mechanisms involved in the generation and modulation of spontaneous lymphatic contractions. The following specific aims will be addressed: 1) Determine the role of the endothelium, smooth muscle cells and their interactions in the pressure sensitivity of the lymphatic pump; 2) Determine the role of the endothelium, smooth muscle cells and their interactions in the flow sensitivity of the lymphatic pump; 3) Evaluate the regulation of lymphatic intracellular calcium during spontaneous contractions; 4) Evaluate the alterations in lymphatic intracellular calcium during pressure- and flow-induced changes in the lymphatic pump; and 5) Evaluate the site of lymphatic pacemaker activity. These studies will be conducted in isolated, pressurized collecting lymphatics from the mesentery of the rat small intestine. The lymphatics will be dissected out of the tissue and cannulated. The pressure and flow through the vessel will be carefully controlled by varying the inlet and outlet pressures. The role of the endothelium and endothelial-smooth muscle interactions will be investigated using techniques to inhibit and/or remove the lymphatic endothelium. Lymphatic contractions will be monitored using videomicroscopic techniques. The lymphatic diameter will be continuously measured throughout the experiments. From the lymphatic diameter tracings, functional indices of the lymph pump will be evaluated. Intracellular levels of calcium in the lymphatics during spontaneous contractions will be monitored using the fluorescent calcium-sensitive dye Fura-2. The cellular mechanisms involved in the regulation of intracellular calcium will be investigated. The influence of pressure and flow on lymphatic calcium regulation will also be evaluated. Evaluation of membrane potential within the cells of the lymphatic vessels will be performed using the potentiometric fluorescent dye Di-8-ANEPPS. Investigation of the pacemaker activity within the lymphatic will be performed using fluorescent imaging techniques. The results of these investigations should provide considerable insight into the mechanisms involved in the generation and alteration of spontaneous lymphatic contractions and will be the basis for continued studies of the role of the lymphatic system in fluid and macromolecular homeostasis, fat absorption, lymphocyte circulation, and antigen presentation.