23 November, 1998

November 23, 1998

Not much new to report today. Another busy day and I am totally exhausted. We all have been working extremely hard, pretty-much non-stop, for over a week. The only things I took time out for were food, sleep, and church. It sure will be nice to get back out in the field again sometime. Again the spectacular view from the lab lifts my spirits.


Today, instead of photographing cells in gill filaments I switched the focus of my work to photographing microscopic views of suspensions of gill cells that were scraped off of the gills. These cells were still alive and were also stained with DASPEI, so the chloride cells were easily identified by their flourescence. My job will again be to count and measure the cells on the photographs. This is much easier to do than in the living gill filaments because, in a suspension they are all in focus in a thin film of water on a microscope slide. Though the filament looks fine and thin to the naked eye, it is very thick when it is magnified 200-400 times in the micoscope. This depth makes it impossible to focus on all parts of the filament at once. In addition, the out-of-focus tissue tends to distort the image in the photograph, making it hard to differentiate one glowing chloride cell from an overlapping one slightly below or above it. This also causes fuzzy cell boundaries making it very hard to precisely measure the cells in the photographs.

Question to think about: If this is true, why did I spend so much time photographing the cells in the gill filaments instead of doing it this way? Any ideas? (It's not because we didn't think of it.)


As you recall, the main theme of the research we are doing is how the fish regulate their salt concentrations in their blood and tissue fluids. We know that about half of the ability of these fish to resist freezing in the -1.86 degree Celsius water is due to their high salt concentration, twice as salty as the marine fish near where you live. (The other half is due to the antifreezes in their blood.) This salt concentration is regulated by an enzyme in the membranes of the choride cells called Na-K/ATPase (sodium potassium ATPase) whose job is to parcticipate in the removal of salt from the blood. This enzyme is less active in the cold water, thus keeping a higher amount of salt in the blood, and preventing it from freezing. When this enzyme is active it uses energy from a molecule called ATP, the energy "money" of a cell. Since it is less active in the freezing cold water, it ends up using less ATP energy. That's thrifty, that's nifty, that's neat!

One thing that Dr. Petzel's group has found in the past is that when you warm these fish up to about 4 degrees celsius, the Na-K/ATPase increases its activity and the amount of salt in the fish decreases. What we are trying to find out is if this increase in activity is due to more chloride cells in the gills, bigger chloride cells, more enzyme molecules being manufactured, or an increased activity/efficiency of the individual enzymes. This sounds kind of simple but it really is very complicated and time-consuming work that involves the sacrifice of many fish and many, many, many hours by all of us in the lab. There are several components being done by the people here now.

1. You already know about my work: counting and measuring chloride cells.

2. Ed is trying to find out what hormones regulate the salt concentrations by injecting prolactin, thyroxin, and cortisol into their blood and finding out how that changes the salt concentration. To do this he has to extract a small sample of blood from the fish and use a machine to measure how much salt is in it. He also sacrifices the fish to make a gill suspension so he can count the number of cells. Another thing he does is to add small amounts of a hormone to a known number of cells in another nifty gadget which measures how much oxygen is used up by the cells. Most of you Biostudents know that cells need oxygen to make ATP in their mitochondria. Since these cells use lots of ATP for their Na-K/ATPase to work we are assuming that the more oxygen that is used, the more Na-K/ATPase activity there is. So if he injects a hormone into the suspension of gill cells and it causes a rapid increase in the use of oxygen, he knows that this hormone has some role in helping the enzyme work.

3. Dr Smith (David) is using this same oxygen consumption technique to try to find other unknown hormones, probably found only in fish, that affect the Na-K/ATPase. He knows that when he injects the juice of of an organ found only in fish (called the head kidney)

it causes the cells to use more oxygen. Now the job is to find exactly what chemical in this juice has this effect. The head-kidney juice has hundreds of chemicals in it. He has to use a process call HPLC, high-pressure liquid chromatography, to separate out the complex mixture into smaller fractions containing fewer chemicals, and then test these fractions to find out their effect on oxygen consumption. Then when he finds a fraction that works, he needs to do even more HPLC to separate it into even more finely-separated fractions, until finally he narrows it down to one parcticular chemical. Even then he isn't done, because he still needs to try to figue out what kind of molecule this chemical is. Who knows, he might find a new hormone, unknown to science.

4. Sierra is studying how much Na-K/ATPase is present in the cells and how active it is. One of the tests she is doing uses the radioactive form of a chemical called ouabain, a plant extract, that sticks to the Na-K/ATPase. The amount of radioactivity she finds gives an indication of how much of this enzyme is present and how active it is. She is also doing a test that measures how much protein is present, since enzymes such as Na-K/ATPase are proteins. Then she puts it all together to try to find out if there is more of the enzyme present per cell and if those pumps are more active in the warm fish than in the cold fish.

Why are we doing all these studies? First of all, it's amazing to find out new things about how organisms are adapted to live in their environment, especially extreme ones like this. Second, every kind of animal seems to have this enzyme. In fact in us, its activity uses up a very high percentage of the energy our body consumes. If we can better understand how this enzyme works, this knowledge might help humans, other animals, or these fish in the future.

Question to think about: Is the knowledge we are learning from this research worth the death of a couple hundred experimental fish each year? E-mail your answer to me and explain what you think. It has been a real moral dilemma for me. (Don't worry if your opinion is different from with what you think I think.)



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