Digitized by Kansas Correctional Industries

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The distribution and transfer of ammonia between intracellular and extracellular compartments of fish and the external water environment was investigated. In vivo and in vitro experiments were performed on the freshwater rainbow trout (Salmo gairdneri) and the intact, seawater lemon sole (Parophrys vetulus). The distribution of ammonia and H⁺ ions were compared between red cells and plasma (in vivo and in vitro) taken from rainbow trout at rest and during hypercapnia. At rest (in vivo and in vitro) measured intracellular ammonia levels were equal to those predicted by the plasma to red cell pH gradient. The same was not true during hypercapnia, where measured red cell ammonia levels were greater than predicted levels. The addition of the Na⁺/K⁺ ATPase inhibitor, ouabain, had no effect on ammonia accumulation during hypercapnia. It was concluded that ammonia is passively distributed according to plasma-to-red cell H⁺ ion distribution in blood at resting pH values, but under hypercapnic conditions, ammonia accumulation must be due to some other active uptake mechanism. The distribution of ammonia and ¹⁴C-DMO were compared in white muscle, heart, brain, red cells, and plasma of lemon sole (in vivo) at rest, during hypercapnia, and following exercise. The red cell ammonia distribution at rest and during an extracellular acidosis (hypercapnia and exercise) was similar to that found in rainbow trout. Red cells are unusual in that H⁺ ions are passively distributed according to membrane potential (Em), whereas in other tissues, this is not the case. In white muscle, heart, and brain under all experimental conditions, intracellular ammonia levels far exceeded those predicted by transmembrane pH gradients. Calculated ENH₄₊ values in these tissues were very close to published resting values of Em. It was concluded that NH₄₊ is permeable across cell membranes and that intracellular ammonia stores are not determined by transmembrane pH gradients in lemon sole. The pH of interlamellar water was investigated in rainbow trout by following changes in the downstream pH of expired water using a stopped-flow method. As water flowed over the gills of control fish, there was a significant decrease in water pH. Acetazolamide (carbonic anhydrase (CA) inhibitor) added to the water increased the CO₂ disequilibrium, while CA eliminated the CO₂ disequilibrium relative to control water. Mucus excreted by the fish was found to contain CA activity by the pH-stat technique. It was concluded that water acidification is due to the conversion of excreted CO₂ to HCO₃₋ and H+ at the gill surface. A possible function of CA at the external gill surface is to facilitate carbon dioxide and ammonia excretion. Acetazolamide or CA added to the water did not alter carbon dioxide (MCO₂) or ammonia (MAmm) excretion in intact rainbow trout. Methazolamide (CA inhibitor) or methazolamide + amiloride (Na⁺ uptake inhibitor) added to the water had no effect on plasma NH₃ tensions (PNH₃), but increased MAmm slightly compared to control fish. In general, methazolamide resulted in an increase in the diffusing capacity of ammonia. The interpretation of these results was complicated by the fact that rapid serial blood sampling resulted in a universal blood alkalosis. The intact resting fish is unsuitable for studying the interaction between water boundary layer chemistry and excretion across the gill. With the blood-perfused trout head preparation it was demonstrated that MC0₂ and MAmm are linked through chemical reactions in the external water boundary layer adjacent to the gill. Pre-incubation of blood with acetazolamide reduced MC0₂ and MAMM in the blood-perfused head. Increasing the buffering capacity of inspired water, significantly reduced MAMM, but MC0₂ was unaffected. Each of these experimental treatments significantly reduced the acidification of ventilatory water flowing over the gills. It is proposed that the catalysed conversion of excreted C0₂ to form HCO₃₋ and H⁺ ions in the gill boundary layer provides a continual supply of H⁺ ions needed for the removal of NH₃ to NH₄₊, which reduces water NH₃ levels and facilitates ammonia excretion. Gas transfer variables in the blood-perfused head preparation were compared to intact cannulated fish with and without oral masks. Oxygen uptake (MO₂) and MCO₂ were lower, and MAMM, higher in the blood-perfused head compared to in, vivo values. these discrepancies were due to differences in venous O₂, CO₂, and ammonia levels, which determine mean gradients across the gills. It was concluded that the blood-perfused head is a suitable preparation for studying the interaction between MCO₂ and MAMM because the overall efficiency of transfer of NH₃ CO₂ was very similar between in. vitro and in vivo preparations, / Science, Faculty of / Zoology, Department of / Graduate

Graduation date: 1997