Duke Researchers Discover Central Role Of Nitric Oxide In Hemoglobin Action

DURHAM, N.C. -- Duke University Medical Center researchers have found that nitric oxide, combined with hemoglobin, is a major regulator of gas exchange, as well as blood pressure, in the circulatory system. The finding appears to have solved the long-standing mystery of how blood carries oxygen to body tissues and extracts waste carbon dioxide while keeping vessels open and blood pressure steady.

Scientists say the discovery, detailed in the March 21 issue of the British journal Nature, could quickly pay off in developing the first effective blood substitute, and may ultimately change the way many diseases are treated.

"We now know that nitric oxide is involved in the blood's major functions," said cardiologist and pulmonologist Dr. Jonathan Stamler in an interview. "Oxygen delivery is essential to life and a deficiency in oxygen is associated with diseases of every organ. The same picture is gradually emerging for nitric oxide (NO). Understanding delivery of both in concert could have profound therapeutic implications.

"The duet of hemoglobin and NO is fantastically symbiotic in carrying out the machinery of life," he said. "Hemoglobin uses a spritz of the NO it carries to help get oxygen into tissues. And NO helps hemoglobin carry away the trash of carbon dioxide. It's fantastic."

The work was funded by the National Institutes of Health and the Pew Charitable Trusts. Working with Stamler was first author, Duke research associate Li Jia, and Joseph Bonaventura and Celia Bonaventura, from Duke's Nicholas School of the Environment and the Marine Biomedical Center.

Nitric oxide, long known as a noxious gas in the atmosphere, has been found over the past several years to play a major role in numerous biological systems. For example, scientists discovered that NO worked in the circulatory system to dilate blood vessels. "Free" NO is released by endothelial cells on the inside of vessel walls where it migrates to nearby muscle cells and relaxes them, opening the vessel and lowering blood pressure.

At the same time, researchers observed that this free nitric oxide was inactivated by hemoglobin, as the iron molecule in hemoglobin essentially consumes NO.

Adding these bits of knowledge together -- that NO keeps vessels open, but that hemoglobin destroys NO -- produced a major paradox that no one could solve, Stamler said. How can blood vessels maintain a constant pressure when the hemoglobin that flows through them destroys NO on contact?

Stamler suspected that NO had to exist in some other form in the blood, apart from the "free" NO that is made in the vessel and destroyed by hemoglobin. So he and Jia worked with the Bonaventuras, who are experts on hemoglobin, designing investigations using blood from humans and rats.

After a series of experiments, the team discovered that a NO-containing hemoglobin molecule is synthesized in the lungs and that the NO attached to it differs from that produced in vessel walls. Hemoglobin is a large protein complex containing "heme" groups, which include a central iron molecule that serves as a site on which to bind and carry oxygen. The same site also destroys "free" NO. Stamler and his team found the "new" NO attaches itself to the hemoglobin-oxygen complex on a cysteine residue that keeps the NO away from the hemes.

Specifically, in its new form, this NO is attached to a thiol and is called SNO (for S-nitrosothiols). SNO retains its NO-like properties, but is "a souped-up cousin," Stamler said. SNO is protected from inactivation by hemes, unlike NO produced in vessel walls, and it has a wider range of functions than NO. Not many SNOs have been found in the body to date, but the ones that have been discovered are powerful forms of NO, he said. For example, SNO can kill invading bacteria or microbes. Free NO cannot, said Stamler.

"We always knew that the hemoglobin complex had two reactive arms, a heme and a cysteine, to which other molecules could attach, but no one knew what the cysteine's function was," he said. "We now know that it serves to bind NO."

Further experimentation by the group uncovered the intricate interplay between hemoglobin, oxygen, NO and carbon dioxide:

• After hemoglobin loads up oxygen and SNO in the lung, the hemoglobin-trio travels down arteries through the heart and into the rest of the body to deliver its load of oxygen. That process has to happen with open vessels and in a constant pressure. So as the hemoglobin complex inevitably devours the free NO gas that endothelial cells produced to dilate the vessel, hemoglobin simultaneously releases the SNO molecule it had been carrying. One "ineffective" NO is exchanged for another "protected" NO. Blood pressure remains constant and blood flow maintained in order to promote oxygen transport. Hemoglobin detects how much NO has been removed from the bloodstream and compensates with SNO, the researchers believe. Still, the hemoglobin-SNO molecules are very abundant, so there is still plenty of SNO still available as the hemoglobin complex enters tissues.
  
  
• In tissue, the hemoglobin undergoes a major structural change to release its load of oxygen. This same "conformational" change also releases SNO, presumably to increase the efficiency of oxygen utilization, Stamler said. "Mitochondria in tissue make energy from oxygen, and the SNO may help regulate the rate at which the mitochondria respire, or use the oxygen." It is also possible that SNO regulates capillary blood flow, he said.
  
  
• When the hemoglobin has released both its oxygen and SNO, it can attract molecules of carbon dioxide. Carbon dioxide (CO2) is the waste gas produced from oxygen respiration. Hemoglobin binds CO2 and carries it to the lungs, where it is exhaled. At that point, the "free" NO consumed by hemoglobin is also released and exhaled. Then, the oxygenation process is repeated.