Jason E. Wright and Dr. Steven W. Graves — Chemistry and Biochemistry

Proposal and hypothesis

Preliminary data suggested that bovine lens epithelial cells (BLEC) are able to increase the amount of sodium-potassium ATPase (sodium pump or SP) in the plasma membrane. I set out to accomplish three objectives: (1) to continue ongoing ouabain inhibition experiments in order to confirm the preliminary data; (2) to examine BLEC’s response to peptide hormones (in other tissues the peptide hormone induced phosphorylation of SP is followed by an observable removal of SP from the membrane); and (3) to confirm our hypothesis that observations of SP redistribution are a manifestation of BLEC’s ability to compensate for the inhibitory effect of ouabain on membrane-bound SP. I expected to report that although the distribution and location of SP fluctuates in response to differing inhibitory treatments, the net SP activity remains unchanged after the cell is allowed sufficient time to shuffle the distribution of its SPs, thereby compensating for the loss of ion exchange activity. My overarching intent was to determine whether two methods of reducing SP activity, ie. via direct inhibition or phosphorylation, went uncompensated by BLEC (further confirming their candidacy for cataract-causing agents) or whether other mechanisms, e.g., genetic regulation, most likely explain the reduced SP activity characteristic of cataracts.

Materials, methods

Bovine eyes are obtained at the Deseret meat packing plant in Spanish Fork. All other materials are available either through biochemical catalogues or the central stockroom (BNSN). After dissection of the bovine eyes, the lens capsule is removed and the epithelial cells transferred to tissue culture plates for incubation in a simulated physiological environment. Once cells have grown to confluence, and a sufficient number are available, they are incubated in the presence or absence of ouabain (in graded doses). After 2, 8, or 24 hours, the cells are harvested from the plates, lysed, and centrifugally separated into cytosolic and membranous samples. Then, after determining the concentration of total protein in the samples, they are prepared for and undergo electrophorectic separation and Western blot analysis using SP a-isoform specific antibodies. This protocol allows one to observe the effect of various types of SP inhibitors on the distribution of SP within cultured BLEC. In comparable experiments, BLEC is incubated in the presence or absence of endothelin (10-8M), a peptide hormone, or the presence or absence of other hormones with cell surface receptors (leading to phosphorylation events) such as epinephrine, norepinephrine, or insulin-like growth factor, all of which occur in the aqueous humor. These experiments can determine if phosphorylation with SP a-isoform removal occurs, as in other tissues, and whether with time it is compensated by SP replacement.

Challenges

Shortly after receiving funds from ORCA in February our population of BLEC was lost to an unidentified contamination. Through August we struggled with eliminating the unknown source of contamination. Two or three times we had enough cells to run inhibition experiments. The contamination is finally under control after a thorough revamping of our cell culturing protocol (filtering media, different antibiotic, etc.).

Results

Of the three objectives stated in the proposal, I was able to accomplish the first completely, make significant progress on the second, and begin preliminary work on the 3rd. (1) With one more experiment we adequately confirmed our preliminary findings. There is more membrane-bound SP in BLEC after incubation in a solution of direct SP inhibitor than before incubation. (2) Two experiments were performed to test the effects of peptide-hormone SP inhibition of BLEC. We used the same protocol as with direct inhibition (ouabain) but were unable to find conclusive results concerning the cell’s reaction to the treatment. The only sure conclusion that came of the two experiments was that our observations were markedly different than the results of the ouabain treatments, to which we were accustomed. (3) The last objective, to test for compensation, held my interest the most out of the three. It is unfortunate that due to the contamination of our lens cells, we did not recuperate in time to run the Rb-exchange experiments in the graphite furnace atomic absorption (GFAA) instrument before I left. During my last week in the lab we had grown enough cells to make decisions as to how many cells would be necessary per experiment using the GFAA. As soon as the kinks have been worked out of the protocol for that part of our research, the lab will be on its way to contributing significant data to biochemical literature.

Conclusion

Unfortunately, due to challenges, I was not able to find all the answers to the questions stated in the proposal. However, others have taken up the project and are on their way to finding those answers. Thanks to BYU’s ORCA I was able to keep working in Dr. Graves’ lab prolonging my research experience and improving my qualification for acceptance into medical school. Thanks to those who fund these research grants. My experiences in the lab have put me ahead of the game here in medical school.