The experimental program will be directed toward an elucidation of the factors which regulate ammonia synthesis by the mammalian kidney. Studies utilizing isolated renal cortical mitochondria will be performed to define the interrelationships between bicarbonate ion and H+ in regulating glutamine and glutamate metabolism. These studies will be extended to the intact cell utilizing 15N-glutamine and 14C-glutamine to define further the regulatory steps and pathways. 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 Alpha-ketoglutarate on glutamine transport and deamidation. To provide further insight into the factors controlling the accelerated rate of glutamate dehydrogenase flux in acidosis, the role of the mitochondrial oxidation reduction potential and protein synthesis will be assessed. The control of Alpha-ketoglutarate dehydrogenase flux will be investigated in detail by assessment of the role of calcium and H+ on the enzyme in kidney and liver. As part of this evaluation, mitochondrial free calcium and mitochondrial calcium efflux will be measured as a function of external pH. Utilizing cell fractionation methods, analysis of metabolite and pH profiles in the cytosolic and mitochondrial compartments of isolated renal tubules will be performed to understand better alterations in renal metabolism in acidosis. Of particular interest will be a determination of the contemporaneous changes in the cytosolic and mitochondrial pH as a function of external pH changes to determine whether metabolic rates may be effected by the pH gradient across the plasma and, particularly, the inner mitochondrial membrane. These studies will provide further insight into the biochemical mechanisms underlying the control of renal ammoniagenesis.
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