An Investigation into the Mechanism of Ammonia Excretion in Fish

An Investigation into the Mechanism of Ammonia Excretion in Fish

Elliott Sencan

Vertebrate Physiology

5-3-2012

Introduction

Excess ingested amino acids in the body cannot be stored, and are therefore broken down to form ammonia. To prevent this extremely toxic product from interfering with vital bodily functions, it must be eliminated. Aquatic animals excrete it across their gills. The mechanism for this ammonia excretion was thought to involve simple diffusion across the gill epithelium from the blood to the water, but recent research has cast doubt on whether this model fits realistic physiological conditions. The first suggested mechanism involves active transport of NH4. In this model, NH¬4 that is formed inside the gill cell replaces Sodium ions in the Na+/K+ ATPase, and then transferred out of the cell by replacing H+ ions in the H+/Na+ exchanger. This method posits that there are correlations between the unidirectional Na+ influx and the net ammonia excretion rates. The second proposed mechanism posits that diffusion is still the main reason for excretion. NH3 diffuses from the blood to the gill epithelium, and this change in concentration in NH3 in the blood causes a shift to the right in the equilibrium reaction NH4 = NH3 + H+, producing more NH3 in the blood. This works to maintain the gradient from the blood to the gill epithelium. Once this NH3 has passed into the gill epithelium, it enters an unstirred boundary layer immediately adjacent to the cell layer in which it combines with H+ ions to reform NH4, which maintains the internal concentration gradient out of the cell. This proton trap allows for continued excretion of ammonia via a passive diffusion method, which is efficient in its simplicity and take advantage of the fact that at typical physiological pH values of 7.5-8.0 most NH3 in the body is in the form of NH4; as long as pH levels can be maintained, this mechanism should continue to work if it is in fact being used to excrete ammonia. Evidence has been collected for both possibilities, but we hypothesize that the second mechanism is more plausible as it has lower energy requirements than the first proposed new mechanism. To test which method is being used, we tested groups of Carassius auratus to various treatments designed to either positively or negatively affect each mechanism: high external ammonia concentrations, low external pH, high external pH, and sodium free water. By this pattern of inhibition or stimulation we can gain knowledge of which mechanism is excreting ammonia in aquatic vertebrates.

Materials and Methods

To test our hypothesis, we measured the rates of Na+ uptake and the net ammonia flux in five different sets of Carassius auratus (Goldfish). Our first group was tested under control conditions, or a pH of 7.5 and 400 µM concentration of both Na+2 and Ca+2 , to control for changes during the measurement period. We then ran two groups under increasing ammonia concentrations; the second's external ammonia concentration was at 100 µM and the third at 200 µM. This was to determine if high concentration of extracellular ammonia would interfere with the concentration gradient that our second proposed mechanism relies on. Our fourth group was tested under high pH conditions, at around pH 9.5-10, which will impact the concentration gradient by creating more NH3 due to the scarcity of H+ ions, degrading the concentration gradient out of the cell. Our fifth group was tested under low pH conditions, with a pH of 5, which shifts our equilibrium towards NH4+ by increasing the concentration of H+ ions, which will increase the concentration gradient promoting excretion of ammonia out of the cell via proposed mechanism two. Our last group was tested in Na+ free water, which had the same conditions as our control but with a very small amounts of sodium. This will prevent the excretion of ammonia if our first proposed mechanism is working, as low concentrations of Na+ will interfere with the active transport mechanism of the H+/K+ exchangers being used to excrete ammonia via proposed mechanism one.

Results

The values between our two measurement periods in our control group are extremely similar, which demonstrates us that time does not significantly impact sodium uptake or ammonia excretion in our experiment.

Our 100 µM ammonia concentration group resulted in a 45% drop in ammonia excreted and a 7% increase in sodium uptake when compared with our control. Our higher 200 µM resulted in an even higher drop of 87%, while our sodium uptake dropped 7% in relation to our control.

Our High pH group resulted in a 85% reduction in sodium uptake, and an 88% reduction in 85% reduction in sodium uptake when compared with our control.

Our low pH had no significant effect on our ammonia flux resulting in only an 8% increase in ammonia excretion when compared to our control. This treatment group did produce an 88% reduction in sodium uptake when compared with our control.

Our sodium free water resulted in a 93% reduction in sodium uptake when compared with our control, and resulted in a 23% drop in ammonia excretion..

Discussion

Overall, our data is ambiguous concerning our hypothesis. While there is strong evidence from our results supporting our gradient mechanism, there are some results that suggest that the proton pump mechanism is playing some role in ammonia excretion. While the results from both our high ammonia assays and our high pH assay support the gradient mechanism, our low pH mechanism raise some questions regarding the validity of our second proposed mechanism for ammonia excretion.

If our proposed hypothesis is correct, we should see reductions in ammonia that vary directly with increases in ammonia concentration outside the cell. Since our gradient model relies on the strength of the NH3 gradient moving out of the cell, increasing the external concentration weakens the gradient and will inhibit excretion, while if the proton pump model is truly at work, then the exchangers should not be affected by a weakened diffusion gradient. As our two levels of ammonia concentration outside the cell demonstrate, as we double the concentration of ammonia