 . . . Summer 1997
Blame it on Archimedes. He was the guy, remember, who leapt from his bath and ran dripping through the streets shouting, "Eureka!" after discovering the law of hydrostatics. Ever since that story got around, people have thought all great scientific advances happen that way. Well, maybe not always with public nudity. But surely with some startling discovery and flash of insight.
Not so, scientists will tell you. For every Eureka moment, there are countless hours of tedious research that seems to lead nowhere. Scientists navigate a trail that meanders into unfamiliar territory, circles back on itself, and may not end up where it seemed to lead. Their fellow travelers are not always who they'd expect to encounter on the road. Biochemists may cross paths with automotive engineers. A botanist's discovery may hold the long-sought key to some secret of human health. Sometimes, scientists must even travel back in time to move forward. Research results that lay forgotten for decades may make new findings snap sharply into focus.
'Several Huge Surprises' With so many on-ramps, intersections and back roads, it's hard to pinpoint exactly where the route to Marletta's discovery began. So let's just start with the hot dogs. In the 1970s, people started to worry about cancer-causing chemicals called nitrosamines. There was concern about exposure to nitrosamines in the environment, but also about the possibility that nitrite preservatives in hot dogs and other cured meats might be converted into nitrosamines in the body. One scientist investigating this notion was Steven Tannenbaum, a toxicologist at the Massachusetts Institute of Technology. Through his own research and a review of nearly forgotten studies dating back to the early 1900s, Tannenbaum concluded that nitrites weren't the only culprits. Their benign chemical sisters, nitrates--found in drinking water, vegetables and many other foods--were being converted into nitrites by bacteria in saliva. The nitrites, in turn, were converted into nitrosamines in the stomach. Mystery in Urinalysis Where was the excess nitrate coming from? Could the subjects' bodies be producing it? It seemed "inconceivable" to Tannenbaum, given what scientists knew--or thought they knew--about the formation and breakdown of nitrogen compounds in the human body. More likely, the nitrates were being made by bacteria in the subjects' intestines. But that possibility was ruled out by experiments on rats, done by one of Tannenbaum's graduate students. Still, some were skeptical of the results from Tannenbaum's lab. "We were catching a lot of flack," he recalls. Something must be wrong with the experimental methods, critics charged. There was no way people-or rats-could excrete more nitrate than they took in. To gather more evidence, Tannenbaum's team put student volunteers on nitrate-free diets and checked for nitrate in their urine. A Lucky Intestinal Bug Was the intestinal bug making the excess nitrate? Or was the student's immune system producing nitrate in response to the infection? Further experiments ruled out the bug and pointed to the immune system. But just how and where was the nitrate being made? Enter Michael Marletta, who was then beginning his career as a biochemist at MIT and had become a sort of science buddy of Tannenbaum's. The two "just loved to talk science," and did so every morning, Tannenbaum recalls. Science may have been the glue that cemented their friendship, but coffee was the catalyst. Tannenbaum always kept a pot brewing in a little room off his lab. Marletta, a serious coffee drinker whose quarters were one floor below, often ran upstairs to grab a cup and chat awhile. In their coffee room conversations, Tannenbaum kept trying to get Marletta involved in the nitrate research. Marletta, who specializes in studying enzymes, was intrigued but wasn't sure how to tackle the problem. It was just too vast and unwieldy. "If you told me, here's an enzyme that nobody has ever characterized before, and it makes nitrate, I'd probably say, 'Wow, that's pretty cool, I'll work on it.' " says Marletta. "But if you tell me here's a human . . . and in this human of several billion cells of all different kinds, one or some of them are making nitrates, I'm not going to work on that problem, because I don't know where to start." But once Tannenbaum's experiments pointed to the immune system, Marletta "had a handle to investigate the problem." Borrowing techniques from the field of immunology, he plunged into experiments on mice that showed exactly which cells in the immune system were making nitrate. It was the macrophages-immune cells that cruise the body, gobbling up bacteria and other foreign invaders. What Was the Missing Step? Around this time, scientists in unrelated fields were traveling down paths that would soon merge with Marletta's. But Marletta was as unaware of them as they were of him. In their quest to develop new blood pressure drugs, cardiovascular pharmacologists had been searching for something they called EDRF (short for endothelium derived relaxing factor). They didn't know what EDRF was; they only knew what it did. Produced in the cells that line blood vessels, it acted on the smooth muscle that makes up the outside of blood vessels, making the muscle relax and the blood vessel dilate. When blood vessels dilate, blood pressure goes down. Clearly, EDRF was something drug researchers wanted to know more about. EDRF had been discovered quite by accident in a Brooklyn scientist's lab. The scientist, Robert Furchgott, had developed a way to keep pieces of blood vessels alive in the lab, allowing them to be studied outside the body. He noticed that when the inner lining of the blood vessels was accidentally scraped off, it became impossible to make the vessels relax. Furchgott wrote up his observation, proposing the existence of EDRF and coining the term. His paper, published in 1980, set off the race to identify EDRF. Unexpected Help From England Five years later, when Moncada wrapped up his prostacyclin research and started looking around for a new project, he remembered Furchgott's work and decided to follow up on it. In the process, he came across provocative new research on nitroglycerin, used for more than a century to relieve chest pain by widening blocked arteries supplying the heart. Scientists in Germany had discovered that nitroglycerin was chemically converted to nitric oxide in the body. It was the nitric oxide that dilated the arteries. Nitric oxide made blood vessels dilate. EDRF made blood vessels dilate. Moncada began to wonder whether EDRF and nitric oxide could be one and the same. So did Furchgott and another researcher in the EDRF field, Louis Ignarro. But how could that be, the researchers all asked themselves. Nitric oxide, a toxic gas, was not the sort of chemical you'd expect the human body to make. Still, Moncada's experiments told him he was on the right track. In lab tests, EDRF was destroyed by superoxide, a highly reactive form of oxygen. So was nitric oxide. Applying Some Automobile Technology At this point, the research trail became a sort of cloverleaf, with branches circling around and feeding into each other. Results from Moncada's work held a key Marletta had been searching for. And Marletta's work, in turn, gave Moncada the clue he needed to fill in the details of the EDRF story. During a layover at Denver's Stapleton airport, Marletta read Moncada's paper showing that nitric oxide was the elusive EDRF. Suddenly, it was obvious to Marletta that nitric oxide was the missing piece in the macrophage pathway--the arginine-to-nitrate sequence. He couldn't wait to do the experiments that would confirm his idea, but it looked as if he might have to. Marletta had recently moved from MIT to the University of Michigan and hadn't yet set up his lab. Equipment was still in boxes, and he hadn't built a team of graduate students and post-doctoral fellows to help with experiments. Rushing to an airport pay phone, Marletta called his old science buddy Tannenbaum. "Steve, I figured it out!" he told his friend. "And I don't even have time to set up my lab and do the experiments." By the next morning, Marletta was making plans to go MIT and do the work with members of Tannenbaum's team. There, working night and day for a week, using a monstrous machine like the one Moncada had adapted from the auto industry, Marletta confirmed that macrophages, too, produced nitric oxide. 'The Whole Field Exploded' "We now know that there are two principle functions for nitric oxide in biological systems," explains Marletta. "One is when it acts as a signaling agent--it's the way one cell talks to another cell." That's what goes on in blood vessels, in the brain and in other parts of the nervous system. Nitric oxide's other role is as a poison produced by the immune system to kill invading microorganisms. Scientists now believe that nitric oxide holds promise for treating an assortment of medical conditions, including impotence, stroke, arthritis, Alzheimer's disease and diabetes. Already, knowledge about nitric oxide has led to treatments for high blood pressure in the lungs (pulmonary hypertension) and septic shock--a potentially fatal drop in blood pressure resulting from injury or infection. Thousands of research papers on nitric oxide are published every year, exploring both basic science and potential applications. Brain cells and blood vessels. Hot dogs and flu bugs. Who could have predicted the common threads that would link them? Or that those threads would neatly tie together such diverse medical mysteries. Marletta couldn't have guessed, and yet he's not entirely surprised. He knows how science works. "It's an unpredictable process," he says, "because we're asking questions about the unknown." Nancy Ross-Flanigan is a freelance science writer who lives in Belleville, Michigan.
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