The medullary collecting duet (MCD) is the final arbiter of renal excretion of sodium chloride, potassium, urea, and hydrogen ions. Studies of whole medulla have shown that ion transport is coupled to both aerobic and anaerobic glycolysis but little else is known concerning the metabolism of either H+ secreting outer MCD cells (OMCD) or Na+ absorbing inner MCD (IMCD) cells. Recently we have isolated both OMCD and IMCD cells from the rabbit in quantities sufficient for routine biochemical analysis. These cells share morphologic, biochemical, and transport properties of MCD cells in situ. In addition, we have shown MCD cells have high rates of anaerobic glycolysis, that they depend exclusively on exogenous substrate for aerobic metabolism, and that this rate of energy generation is tightly coupled to transport. The proposed studies will examine in detail glucose metabolism in IMCD and OMCD cells and how metabolism is coupled to transport. Initially, experiments will measure the rates of glucose uptake, aerobic and anerobic glycolysis, as well as intracellular ATP/ADP levels, and ion transport activity as a function of external glucose concentration. This will establish the significance of variations in extracellular glucose concentration on collecting duct function and will provide new insights into the interrelationships of glucose uptake, metabolism, and ion transport in MCD cells. Secondly, we will measure steady-state glucose utilization, lactate generation, and oxygen consumption under conditions of stimulated and inhibited transport in MCD cells. These studies will determine the portion of cellular energy generation which supports transport under a variety of conditions, and will reveal how the cells augment and diminish energy production to meet changing demands of ion transport. Finally, we will measure incorporation of radiolabel from glucose carbons into selected intermediates and products of metabolism and analyze the data using a computer modeling technique, thus quantifying substrate flux through each component step of the cellular metabolic pathways under different ion transport conditions. This sophisticated analytical approach will be novel for renal research and will enable us to identify the sites and mechanisms of control of metabolism. MCD cell suspensions represent an ideal system for studying the coupling of transport and intermediary metabolism and the techniques proposed have all been used successfully by the authors in cell suspension studies.
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