Cell Membrane Plays Crucial Role In Releasing Nitric Oxide From Red Blood Cells

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."