NO problem

Two years ago, whenever members of Jon Lundberg's team at Karolinska University wanted to get near their lab mice, they donned sterile gloves and reached into a steel isolator box. Not typical research rodents, these creatures had been bred to be completely germ-free. The technicians in the animal lab delivered the baby mice by cesarean section and kept them in complete isolation to eliminate the chance that bacteria would enter the rodents' bodies.

By comparing the germ-free mice's nitrate metabolism with that of regular bacteria-ridden mice, the researchers were hoping to get a better glimpse of how strongly the mammals depended on the bacteria to get their systemic supply of nitric oxide (NO) - an essential molecule for mammals, which can function as a neurotransmitter, vasodilator, and cytotoxin. But mammals, unable to produce NO's precursor nitrite themselves, depend on two well-established pathways for generating NO. In the first pathway, bacteria reduce nitrate (found in many leafy vegetables) to nitrite in the saliva. In the second, non-reduction pathway, NO synthases catalyze the formation of NO from oxygen and arginine.

As part of the experiment, the researchers put sodium nitrate in the mice's drinking water. They knew that the saliva of the germ-free mice contained none of the bacteria that could pull off one oxygen atom from the nitrate molecule (NO₃-) to produce nitrite (NO₂-).

But when the researchers examined the germ-free mouse blood two hours later, not only did they find nitrite, they found, in a few of the mice, 8- to 10-fold more nitrite than before they drank the water. "We immediately thought there was contamination," says Lundberg. They called up the department that had cared for the animals to ask if the cohort had been contaminated. The department assured them the mice were germ free- the technicians had been checking the animals' feces every three days for any sign of bacteria. Lundberg immediately set to recreating the experiment.

With the next batch of germ-free mice, they injected nitrate intravenously. Sure enough, two hours later, nitrite levels were sky high. The only way this would be possible without bacteria, the researchers thought, was if some mammalian process was reducing the nitrate.

There was evidence this might be possible: Two previous in vitro studies had shown that mammalian cells reduce nitrate in times of extreme hypoxia. But Lundberg's mice were not hypoxic.

Taking the next step, Lundberg and his team tested rodent and human liver samples for their ability to reduce nitrate. His team found that mammalian tissue did indeed convert nitrate to nitrite under normal, non-hypoxic conditions, but even more so under anaerobic conditions and at abnormal pHs. When the researchers replicated conditions of ischemia in the mice, they found that an enzyme called xanthine oxidoreductase was responsible for catalyzing the reduction. This was not all that surprising, since this enzyme happens to share homology with the bacterial enzyme responsible for the reaction. The researchers followed up by showing that mammalian tissue could further reduce nitrite to NO in both aerobic and hypoxic conditions (Nat Chem Biol,4:411-7, 2008).

In addition to the two well-established pathways that produce nitrite and NO, "Now there's a third, probably lesser pathway," says Mark Gladwin, at the National Institutes of Health's National Heart Lung and Blood Institute, referring to Lundberg's results.

Although it remains unclear what proportion of systematic NO is formed by this new pathway, Gladwin adds, the fact that it is most strongly triggered by low oxygen and pH conditions suggests that the pathway is linked to oxygen signaling in the mammalian body, quite a change in thinking: "Up until the last 4 years, nitrite was really not considered to be important at all except in the stomach," where it killed bacteria. Scientists certainly didn't believe nitrate could play a role in other physiological processes like signaling low oxygen conditions in the body, Gladwin says. "Suddenly this opens up an entirely new pathway for NO."