DURHAM, N.C. -- Duke University Medical Center researchers
report that the membranes of red blood cells are actively
involved in storing and releasing nitric oxide, a molecule that
regulates blood flow and oxygen delivery in humans.

The findings, published in the Feb. 1 issue of the journal
Nature, could help improve understanding and treatment of
cardiovascular side effects associated with a number of
diseases, including diabetes and sickle cell anemia, and with
medical treatments such as blood transfusion or the use of
blood substitutes, said Dr. Jonathan Stamler, the study's
principal investigator and a professor of medicine.

"It's clear from our work that there exists a crucial
relationship between nitric oxide (NO), oxygen, hemoglobin and
red blood cells for appropriate dilation of blood vessels and
delivery of oxygen to tissues," said Stamler, also a Howard
Hughes Medical Institute investigator at Duke University
Medical Center. "It's time to look at the role of red blood
cells in these diseases. Faulty interaction of nitric oxide,
hemoglobin and red blood cells may help explain cardiovascular
morbidity."

In particular, Stamler noted that recent studies elsewhere
have shown that blood transfusion and administering a drug
called erythropoeitin, which increases red blood cell
production, are associated with increases in unwanted
cardiovascular side effects. There also are documented changes
in red blood cell structure and function in sickle cell crisis,
high blood pressure and pulmonary vascular disease, he
said.

"In all these instances, the red blood cells are probably
deficient in NO, and we've shown in this study that we can put
NO back," he said.

While the scientists reported in 1996 that hemoglobin binds
NO inside red blood cells, the importance of the red blood cell
membrane in releasing NO from the cell wasn't recognized until
now. The research was funded by the Howard Hughes Medical
Institute.

Due in large part to Stamler's work over the past five
years, the image of NO has changed from merely being a noxious
atmospheric gas to also being one of the most important
molecules in the human body, responsible for such vital
functions as controlling blood pressure and coordinating the
expansion and contraction of blood vessels.

A remaining question had been how NO can move from inside
red blood cells, where it is bound to the hemoglobin molecule,
to outside the blood cells where it can interact with the
smooth muscle cells surrounding the blood vessels to cause
dilation of the vessels. While both oxygen and NO can diffuse
into red blood cells, only oxygen can diffuse back out.

"We've shown that red blood cell membranes are little pumps
for nitric oxide. We've also demonstrated a different view of
the inside of red blood cells," Stamler said. "The findings
point out that red blood cells are unique and complex, and
their normal operation is of vital importance."

To solve the puzzle of NO's mobility in blood, lead author
Dr. John Pawloski and co-author and assistant professor of
medicine Doug Hess examined whole red blood cells. The
laboratory's previous work had been carried out with free
hemoglobin -- hemoglobin molecules without the red

blood cells that normally would contain them.

The researchers first proved that hemoglobin interacts with
NO when inside red blood cells the same way it does when
"free." Most of the hemoglobin binds NO to one of its four iron
atoms -- the same places it binds oxygen -- which renders the
NO non-functional. However, some of the hemoglobin binds the NO
to a sulfur atom at another specific site, creating what they
called S-nitrosothiol (SNO) when Stamler's group first
described it in 1996. SNO is an activated version of NO that
maintains its function.

To localize the two hemoglobin-NO complexes in the cell, the
scientists dismantled the cells, separating the membrane
portion from almost everything else. They reported that the
oxygen-hemoglobin-SNO complex was associated predominantly with
the membrane of the red blood cells, while most of the NO-iron
hemoglobin complex was found in the non-membrane portion.

Additional experiments showed that hemoglobin and the cell
membrane interact via the bound SNO. Upon release of the
hemoglobin's oxygen, the SNO is transferred to the cell
membrane, specifically to a protein called AE1, or anion
exchanger 1, which is known to swap negatively charged species
(anions).

"Red blood cells were thought to be 'sacks' of hemoglobin,"
Stamler said. "Instead, we've shown that there are two
compartments of hemoglobin and that the cell membrane actively
regulates release of NO from the cell. One compartment is in
the middle, the other near the membrane."

Furthermore, the scientists found that the cell membrane
acts as a reservoir of NO. In experiments in which red blood
cells were exposed to nitric oxide and then added to rings of
blood vessel muscle in the laboratory, the researchers found
that the red blood cells could store functional NO and release
it to relax the muscle.

"The idea had been that NO was consumed immediately by
hemoglobin when it went into the red blood cell," Stamler
explained. "The finding that NO has a lifespan inside the red
blood cell shows that things are not happening at all the way
it was thought. Not only are the reactions taking place
differently than expected, but the whole process is
different."