The precise physiological mechanisms behind enhanced extraction and utilization of glutamine by kidneys during metabolic acidosis is uncertain. This aspect of metabolism is important, for urinary ammonia produced from the two nitrogens of glutamine plays a major role in systemic acid-balance, and decarboxylation of the carbon skeleton of glutamine provides fuel for many metabolic processes. It is accepted by many that renal ammonia production from glutamine by dogs is regulated, to some extent, through two pathways; via phosphate-dependent glutaminase (PDG) and glutamate dehydrogenase (GDH). In 1968, I began to develop the pyridine nucleotide hypothesis (PNH) which states that during acidosis, decreased metabolism of oxidizable substrates in the TCA cycle provides more NAD+ and/or less NADH to the GDH pathway, which would enhance glutamate deamination, lower intramitochondrial glutamate concentrations, and augment glutamine deamidation. Over the last 13 years, my findings strongly and consistently support this hypothesis. Recently, other laboratories have reported data which also strenthen the PNH. Glutamate deamination is a rate limiting step and other substrates compete with glutamate formed from glutamine for oxidation, probably because the supply of NAD+ is rate limiting. The majority of work was performed in vitro. In vitro work outlines the possible metabolic pathways and mechanisms involved in ammoniagenesis; however, the actual pathways and relative contributions to ammonia production can be defined only by studies on intact functioning kidneys. Accordingly, the above theory, derived mainly from in vitro work, should be examined in intact dogs in order to have physiological relevance. To date, I have established in intact dogs that the GDH pathway activity is enhanced in acidosis, and that increasing the activity of this pathway can significantly augment ammonia production from glutamine. In addition, by analyzing substrates of glutamine metabolism, acidotic adaptation is more compatible with the """"""""pull"""""""" rather than the """"""""push"""""""" hypothesis in vivo. The immediate goals now are to establish in intact dogs that enhanced glutamate deamination during acidosis is physiologically important for adaptation and to ascertain whether substrates compete for oxidation. In addition, I wish to localize the sites where blocks in TCA cycle activity occur. The overall objective of the present proposal is to prove or disprove the PNH in intact dogs.
