The long range objectives of this proposal are: (1) to determine the mechanism by which carbonic anhydrase (CA) regulates the partition of CO2 flux in mitochondria between the synthetic and export pathways; and (2) the mechanism by which the export flux of metabolic CO2 out of muscle is apportioned between simple diffusion of dissolved gas and facilitated transport. A prime fact in this investigation is that while pyruvate and Krebs cycle decarboxylation produce CO2 and not HCO3, the synthetic reactions, gluconeogenesis, ureagenesis and fatty acid synthesis all require HCO3- and not CO2. Concerning (1) we hypothesize that: (a) transport of HCO3- into the mitochondria + uncatalyzed hydration of CO2 is not rapid enough to maintain matrix [HCO3] in the face of its consumption by active synthetic reactions so CA is required to accelerate the formation of HCO3- ; and (b) that CO2 produced in mitochondria is preferentially channeled to provide HCO3- for the synthetic reactions and does not equilibrate with the CO2/HCO3- pool in the mitochondria and the cytosol. We will therefore measure the rate of transport of HCO3- into and the rate of production of HCO3- from CO2 in the mitochondria, both compared with the rate of consumption of HCO3-, in the mitochondria using rapid-mixing apparatus and isotopic exchange techniques. We will also measure the enrichment of 13C in urea produced in hepatocytes metabolizing 13C labelled pyruvate looking for channeling of the labelled carbon, and the influence of mitochondrial CA activity on it. Concerning (2) we hypothesize that muscle CA and myoglobin act together to increase the portion of exported CO2 that is facilitated, and that the flux of O2 into muscle synergized the facilitated flux of CO2, out of the muscle. We will measure the facilitation of CO2 transport through thin films of solution containing CA and myoglobin, and of muscle, and the CA activity in intact skeletal muscle cells and bundles. We will determine the importance of CA in the export of 13C labelled CO2 produced by metabolism of labelled pyruvate in perfused isolated muscle in situ. We expect these experiments will provide new and significant information about the gradients and regulation of the economy of CO2 and HCO3- in hepatocytes and in muscle.
