The experimental program will be directed toward an elucidation of the factors which regulate ammonium synthesis by the mammalian kidney. Studies utilizing isolated cortical mitochondria from dog and rat kidneys will be performed to define the relationships between mitochondrial matrix free Ca2+, H+ and HC03 in regulating glutamine and glutamate metabolism. Kinetic studies are also planned defining the characteristics of glutamine transport across the inner mitochondrial membrane. As part of these studies, we will evaluate the role of transport and metabolism of glutamate and ketoglutarate on glutamine transport and deamidation. Particular emphasis will be directed toward the control of ketoglutarate dehydrogenase flux and its effect on glutamate deamidation and deamidation by renal cortical mitochondria. Mitochondrial free Ca2+ and mitochondrial calcium efflux will be measured as a function of external pH, sodium, magnesium, and phosphate. We will reevaluate the effects of HC03- on succinate dehydrogenase utilizing concentrations of HC03- thought to occur in the matrix space in situ. Utilizing cell fractionation methods, analysis of metabolite profiles in the cytosolic and mitochondrial compartments of isolated renal tubules will be performed, particularly as a function of altered medium pH and HC03 - content in order to understand better alterations in renal metabolism in acidosis. Studies will also be performed to assess the effect of transporters for Na/H and Ca2+ on the cytosolic content of these ionic species, the interrelationship of acidosis and hormonal stimulation of gluconeogenesis and ammoniagenesis, and the involvement of other second messengers. These studies will provide further insight into the biochemical mechanisms underlying the control of renal ammoniagenesis.
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