Date of Award
Doctor of Philosophy (PhD)
Professor Chris M. Wood
Fish engage in intensive exercise, both naturally or through human intervention, resulting in an extensive series of physiological and biochemical disturbances. The first part of the study assessed a range of different sampling, processing, and analytical methods for determining selected metabolites in trout muscle. The goal was to provide a set of reliable methods to preserve the original metabolic state in muscle. The second part of this study employed these methods and used rainbow trout (Oncorhynchus mykiss) as an in vivo model to examine the integrated responses of white muscle and plasma (acid-base, electrolytes, fluid volume, and metabolic state) to severe exercise. The investigation also addressed the issues of transmembrane distribution and transport of lactate, metabolic protons, and ammonia. In the third and largest section, an isolated-perfusion tail-trunk preparation (in vitro) was developed to characterize mechanisms involved in the regulation of lactate, ammonia, CO₂, HCO₃ˉ, and proton movement across the muscle cell membrane in both resting and post-exercise muscle.
Together, these studies yielded an integrated picture of the responses to intensive activity in trout white muscle. Exhaustive exercise induced a massive depletion of muscle glycogen, the majority of which was converted to lactate. The retention of lactate, and good carbohydrate conservation, suggested that, in situ glycogenesis, not oxidation, was the major fate of lactate in exercised muscle. The post-exercise redox state of muscle remained oxidative, challenging the traditional concept of "anaerobic" lactate production. The unorthodox involvement of fatty acid oxidation in exercised muscle was indicated by the elevated acyl-carnitine and decreased free carnitine levels. Small amounts of lactate and metabolic acid (ΔHm⁺) were released slowly from exercised muscle into extracellular fluid, and the two fluxes were uncoupled. H⁺ flux reacted to both pH and electrochemical gradients. Lactate distribution did not reach its electrochemical equilibrium, and changes in lactate efflux were more sensitive to the lactic anion (Lac) concentration gradient than to the lactic acid (HLac) gradient, suggesting the possible involvement of electro-neutral carrier-mediated lactate transport. For the first time, it was clearly demonstrated, via the application of the specific inhibitors, α-cyano-4-hydroxycinnamate (CIN) and 4-acetamido-4'-isothiocyanos-tilbene-2,2'-disulphonate (SITS) in conjunction with L- and D-Iactate kinetics, that the carrier-mediated lactate transporters (Lacˉ/H⁺ symport and Lacˉ/HCO₃-Cl antiport), and free diffusion of HLac were involved in the transmembrane lactate exchange in both the resting and exercised fish white muscle (in vitro). The "lactate retention" theory was supported by this study.
The decrease of ATP in the post-exercise muscle was mirrored by the increase in total ammonia (Tᴀᴍᴍ = NH₃ + NH₄⁺) and IMP. This suggested that adenylate deamination was the souce of ammonia production, and that the retention of ammonia in muscle was used for adenylate resynthesis. Muscle membranes were permeable not only to NH₃, but also to NH₄⁺, and the Tᴀᴍᴍ distribution was affected by both pH and electrical gradients. Membrane potential may be more important in determining ammonia distribution (NH₄⁺ permeable) during the post-exercise condition whereas the pH gradient may be more important (NH₃ permeable) at rest. The lack of change in ammonia flux in response to amiloride treatment revealed that the NH₄⁺/Na⁺ exchanger was not involved in ammonia efflux.
Wang, Yu-Xiang, "The Dynamics of Lactate and Ammonia In Rainbow Trout (Oncorhynchus mykiss) White Muscle" (1996). Open Access Dissertations and Theses. Paper 2380.