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The Discovery of EDRF |
In the meantime, more clues were accumulating from another direction. One of the problems that Robert Furchgott of the State University of New York (SUNY) in Brooklyn set out to solve in the 1950s was how blood vessel dilation works on the molecular level. Furchgott's starting point was the neurotransmitter acetylcholine, which was known to make blood vessels dilate when injected into animals. Presumably the acetylcholine was instructing the muscle cells that surround blood vessels to relax, thus increasing the diameter of the blood vessels. To get at the steps between acetylcholine and dilation, Furchgott tried to replicate the acetylcholine response in the laboratory, using isolated strips of blood vessels and the muscles surrounding them. A lengthening of the strips would indicate that the muscles were relaxing and, by inference, that the blood vessel was dilating. But with acetylcholine he saw the strips shorten (muscle contraction instead of relaxation) every time. This was a puzzle that Furchgott temporarily set to one side.
Many years later Furchgott planned an experiment to determine the relative potencies of several chemicals as relaxing agents of blood vessels. Carbachol, a chemical relative of acetylcholine (which Furchgott's puzzling earlier experiments had shown was a contracting agent), was to be used to contract the blood vessel preparations so that Furchgott could then observe the relaxing effects of the three agents he was investigating.
Furchgott laid out a detailed protocol for his technician, David Davidson, which began with tests to see that the tissue was reacting correctly. First came a test contraction with the neurotransmitter norepinephrine, then a wash with fresh saline solution to remove the norepinephrine, and then a test contraction with carbachol. After another wash to remove the carbachol, the actual experiment would commence. The experiment was planned for May 5, 1978. As it happened, Davidson forgot the first wash. To the blood vessel preparation, still contracted by the norepinephrine, he added the carbachol. But rather than contracting further, the vessel relaxed.
Furchgott had added acetylcholine or carbachol to vessels treated with various chemicals many times before and seen only contraction. The only difference in experimental procedure was that this time he was using rings of blood vessels instead of strips. In further experiments Furchgott took rings, all of which had relaxed in preliminary tests with acetylcholine, cut them into strips, and retested the strips with acetylcholine. Infuriatingly, some of these strips continued to relax, but some of them now contracted. He noticed that the ones that contracted had curled up when they were cut earlier and had required some manipulation. Perhaps the manipulation had done some damage.
Sure enough, rubbing any of the strips on their inner surface took away their ability to relax when acetylcholine was added. Furchgott recognized that the methodical way that he had prepared the strips--always pulling the cut surface over his finger to keep it out of the way--had wiped off something crucial.
Furchgott demonstrated experimentally in 1980 that the missing something was endothelial cells, which form the lining of blood vessels. When he made a sandwich of two blood vessels--one with endothelial cells and one without--both strips would relax in response to acetylcholine. The acetylcholine seemed to instruct the endothelial cells to make a second messenger--which Furchgott dubbed endothelium-derived relaxing factor, or EDRF. The EDRF then directed the relaxation of the surrounding muscle cells in both strips.
Although Furchgott knew that EDRF existed, he could not isolate and identify it. For the moment it was simply defined as the substance produced by acetylcholine treatment of endothelial cells. Meanwhile, for scientists NO remained a separate chemical and medical oddity. Why would the body have a system for responding to this gas?
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