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 Howard Hughes Medical Institute (HHMI) and
Duke University Medical Center 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 Science
(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. 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.