130-Year-Old Mysteries Solved: How Nitroglycerin Works; Why Patients Develop Tolerance

Jonathan Stamler, M.D.

DURHAM, N.C. -- For more than 130 years, doctors have prescribed nitroglycerin for relief of chest pain without a clear knowledge of how it actually worked. Now, not only have researchers from Duke University Medical Center and the Howard Hughes Medical Institute (HHMI) solved this age-old riddle, they have also shed light on the second major mystery surrounding nitroglycerin -- why patients eventually develop a tolerance to the drug's effects. Shortly after heart patients take nitroglycerin, the blood vessels supplying the heart muscle relax, allowing oxygen-rich blood to nourish the heart and relieve the pain. While it is known that nitric oxide -- a breakdown product of nitroglycerin -- plays a critical role regulating blood vessel relaxation, scientists still did not know the mechanism by which nitric oxide is generated from the nitroglycerin molecule, which in fact shows little resemblance to nitric oxide. The research team led by Jonathan Stamler, M.D., HHMI investigator at Duke, found an enzyme that not only breaks down nitroglycerin and releases a nitric oxide-related molecule, but whose action is suppressed in blood vessels made tolerant after repeated doses of nitroglycerin. While researchers in the past have searched for such an enzyme in different tissues, the Duke team found that the biochemical reaction that breaks down nitroglycerin takes place in mitochondria, a compartment within cells commonly known as the cell's "powerhouse." The enzyme is called mitochondrial aldehyde dehydrogenase (mALDH), and only in mitochondria can the nitric-oxide-related product of the enzyme get further processed to blood vessel-relaxing nitric oxide. "For more than 100 years, doctors have been prescribing nitroglycerin without a clue how it works," Stamler said. "And for the past 30 years scientists have been looking unsuccessfully for an enzyme that can release nitric oxide from nitroglycerin. "Additionally, there is no data from clinical trials showing that nitroglycerin actually improves outcomes for heart patients, and there is reason to believe that nitroglycerin may even adversely affect these patients," Stamler said. "The results of this study should make it much easier for researchers to design new studies whose goals would be to maximize the benefits of nitroglycerin and lessen its side effects." The results of Stamler's research were published today (June 4, 2002) in the Proceedings of the National Academy of Sciences (PNAS). The results of this study "teaches us that mitochondrial aldehyde dehydrogenase is at least partially responsible for the bioactivation of nitroglycerin and is likely the target of nitroglycerin tolerance," writes Louis Ignarro, Ph.D., in an accompanying commentary in PNAS. Ignarro, a University of California at Los Angeles School of Medicine researcher, won the 1998 Nobel Prize in medicine for his research into to the role of nitric oxide in the cardiovascular system. "Moreover, by understanding the molecular mechanism of nitroglycerin bioactivation and tolerance, it may now be possible to design and develop novel nitrovasodilator drugs that do not cause tolerance." Nitroglycerin, first manufactured by the Swedish industrialist Alfred Nobel, is a common treatment for angina (chest pain) and heart failure. While the drug can be effective, it tends to lose it effectiveness over time, a situation that has for years frustrated physicians, who often take their patients off the drug for periods of time, leaving them at risk for angina and heart attacks. According to Stamler, the key breakthrough in solving the puzzle came in the development of complex biochemical processes used by the researchers to identify where the mALDH broke down the nitroglycerin. Instead of looking for the reaction in blood vessel tissue as had other researchers, the Duke team screened alternative tissue types and surprisingly found that macrophages generated similar biochemical reactions. Macrophages are large immune system cells that can be grown in the laboratory in vast quantities, while blood vessel cells can be difficult to grow in useful quantities. The team then subjected these macrophages to a long series of complex purifications and found that the key reaction took place in the mitochondria of the macrophages. The experiments were conducted in a number of animal models.

Jonathan Stamler, M.D. and protein biochemist Zhiqiang Chen
According to Stamler, protein biochemist Zhiqiang Chen, HHMI post-doctoral fellow at Duke, designed the complicated processes responsible for this key breakthrough. With the knowledge that mitochondria appeared to be the center of the biochemical reactions, the researchers then looked at mitochondria within blood vessel cells and found that indeed, mALDH caused the nitric oxide to be released from the nitroglycerin. "In general, cells don't work as well after being exposed to nitroglycerin," Stamler explained. "It appears that after several reactions, the enzyme is used up and over time, the mitochondria become totally depleted of active enzyme and are therefore unable to break down nitroglycerin. That is why patients eventually become tolerant to its effects. Additionally, by damaging mitochondria, nitroglycerin can actually damage the precious heart cells it is being given to protect. Interestingly, according to Stamler, these findings shed light on many other disorders and diseases. "Our studies suggest that certain classes of drugs such as sulfonylureas used by diabetics, chloral hydrates used for sleep disorders and acetaminophen (Tylenol) inhibit mALDH activity," he said. "For that reason, heart patients who take nitrate drugs such as nitroglycerin may do better if they did not take those drugs." In addition, alcohol will interfere with nitroglycerin, so these patients probably shouldn't drink, Stamler said. "I'm not sure if that glass a day of red wine is good or bad for patients taking nitroglycerin," he added. Even more intriguing, according to Stamler, are the possibilities of a genetic influence. It is known that a certain variation, or polymorphism, in the mALDH gene has been linked to impairments in the ability of the body to metabolize alcohol, as is seen in some people of Asian descent. "Additionally, the mALDH gene variation has been identified as a possible risk factor for the development of cancer and dementia," Stamler continued. "It also follows that testing a patient's genetic status to see if they have the variation may be helpful in predicting the effectiveness of nitroglycerin therapy." Duke cardiologist Jian Zhang, M.D., was also a member of the research team.

Note to editors: Jonathan Stamler, M.D., can be reached at (919) 684-6933 or staml001@mc.duke.edu. A color photograph of Stamler (shown below) is available at http://www.dukemednews.duke.edu/gallery/detail.php?id=655. A color photograph of Stamler and first author Zhiqiang Chen, Ph.D., (shown below) is available at http://www.dukemednews.duke.edu/gallery/detail.php?id=663.