We will study the linked transport and metabolic mechanisms that export the products of the biochemical reactions providing energy for skeletal muscle contraction, namely H+ and lactate, mainly in fast glycolytic fibers, and CO2, mainly in slow oxidative fibers. Our hypotheses are: (1) the rapid production of H+ by glycolysis would reduce cytosolic pH to dangerous levels and damage muscle fibers if the H+ were not as rapidly removed by forming CO2, which diffuses Out of the cell to the blood, a process accelerated by the highly active isozyme carbonic anhydrase (CA II). (2). Additional H+ is carried out of the muscle cells very much more slowly by transporters, some with lactate in a possible co-transporter and some in exchange with Na+. (3) The high concentration of the low activity isozyme CA III in slow oxidative muscle fibers facilitates the diffusion of metabolic CO2 into the capillary blood. (4) CA V, the mitochondrial isozyme, provides HCO3 to condense with pyruvate, that has been produced from lactate by LDH, to produce oxaloacetate which either initiates gluconeogenesis or enters the Krebs Cycle and is oxidized, in either case removing some lactate. Experimentally we will: (1) in anesthetized rabbits during stimulated muscle contraction measure pH by 32P-NMR and selectively inhibit CA II, in order to demonstrate that this retards the bicarbonate buffering of H+ in fast glycolytic fibers, and inhibit CA III to show that this interferes with CO2 excretion and lowers cytosolic pH. (2) In cultured skeletal muscle cells in suspension measure lactate and HCO3- transport, CA activity and effect of the enzyme's inhibition on substrate metabolism to support hypotheses 2 and 3. (3) In muscle mitochondria measure the effect of CA V inhibition on gluconeogenesis and oxidative metabolism from pyruvate, supporting hypothesis 4. (4) Determine the facilitation of CO2 diffusion in a thin electrolyte film containing slow oxidative cytosol or CA III, in support of Hypothesis 3.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Research Project (R01)
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Metabolism Study Section (MET)
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Lymn, Richard W
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University of Pennsylvania
Schools of Medicine
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Liu, M; Walter, G A; Pathare, N C et al. (2007) A quantitative study of bioenergetics in skeletal muscle lacking carbonic anhydrase III using 31P magnetic resonance spectroscopy. Proc Natl Acad Sci U S A 104:371-6
Wroblewski, Krzysztof; Spalthoff, Simon; Zimmerman, Un-Jin et al. (2005) The role of carbonic anhydrase in the recovery of skeletal muscle from anoxia. J Appl Physiol 99:488-98
Lahiri, Sukhamay; Forster 2nd, Robert E (2003) CO2/H(+) sensing: peripheral and central chemoreception. Int J Biochem Cell Biol 35:1413-35