by Tony Fitzpatrick
When Mae West pronounced that too much of a good thing sometimes is a good thing, she probably didn't have the versatile molecule nitric oxide in mind. The molecule is found throughout the biomedical world, playing a vital, though often baffling, role in everything from blood pressure, to bone loss, to rheumatoid arthritis, to male erection.
Now, biologists at Washington University in St. Louis have confirmed the legendary West's observation by showing that a high concentration of nitric oxide in large bone cells called osteoclasts may serve to prevent osteoclasts from eating too much bone away, thus preventing bone loss associated with diseases such as osteoporosis.
Nitric oxide (NO) is not to be confused with nitrous oxide, or laughing gas. Nitric oxide was Science magazine's "Molecule of the Year" in 1992, a biochemical poster boy for ambiguous behavior. Like a character in an old Western, at times it wears the good guy's white hat, only to switch and don the villain's black hat. For instance, human cells produce one form of nitric oxide as a weapon against invading bacteria, but very high concentrations of the molecule can make NO a killer of the very cells that produce it because NO can act as a free radical and destroy cells.
Philip Osdoby, Ph.D., professor of biology in Arts and Sciences at Washington University, and his team, including his wife Patricia Collin-Osdoby, Ph.D., added a monoclonal antibody the team isolated and developed to osteoclast cells believing that the antibody would hone in on a target molecule it recognizes in the osteoclast cells. This target molecule is called an antigen. The antibody (Mab 121F) binds to the antigen, interfering with its function. In testing the antigen/antibody reaction, the biologists found that introducing the antibody into the osteoclasts halted the process of bone resorption, the work of osteoclasts whereby, when the cells are too active or numerous, bone is gnawed away. This leads to pits in the bone matrix and weakening of bone.
After adding antibody, biochemical tests revealed that another free radical, superoxide, was elevated, and, following the burst of superoxide production, there was an increase in NO. This was followed by decreases in bone resorption and production of acid phosphatase, an enzyme produced by osteoclasts that helps degrade bone.
Previous analysis of the novel antigen indicated that it functions on the osteoclast cell surface to protect the cell from high levels of superoxide generated by the cell as part of the resorption process. Osdoby and his colleagues believe if this antigen protection is lost, the osteoclast will stop resorbing by a process that uses NO as a feedback signal to turn the cells off.
"Whatever mechanism is involved, the discovery gives us a whole new number of strategies to pursue," Osdoby says. "Processes that regulate bone loss and osteoclast function are complex and include hormones such as estrogen and local factors such as nitric oxide. One of the earliest events in post-menopausal women is increased osteoclast cell numbers and activity due to diminished circulating estrogen. With a better understanding of the antigen recognized by Mab 121F and how NO is regulated in osteoclasts, we may be able to develop new strategies to prevent bone loss."
The discovery also may have applications for inflammatory conditions such as periodontal disease, and the bone loss problems associated with orthopedic implants by developing strategies to prevent the loosening of artificial bone implants, such as hip replacements.
Nitric oxide is a known scavenger of superoxide, so the investigators are certain it makes a major contribution in halting destruction of the bone matrix.
Osdoby's group has published other studies indicating that if osteoclasts release too much calcium from bone, the nitric oxide system is activated to turn the cell off. More studies are under way to understand these processes. Osdoby, whose work is supported by grants from the National Institutes of Health, published his results in the January 1998 issue of Journal of Bone and Mineral Research.
Dual-natured
The latest publication is part of an array of studies Osdoby and his colleagues have conducted over the past four years on the nitric oxide system of osteoclasts. The researchers have made significant discoveries in the regulation of nitric oxide synthase, the enzyme responsible for making NO; the relationship of osteoclasts to defense mechanism cells called inflammatory cytokines; and the role of transforming growth factor beta (TGF beta) in the bone resorption process.
"What we really want to do is refine our understanding of the nitric oxide mechanism in osteoclasts and pinpoint more precisely how NO synthases are actually regulated," Osdoby says. "Our working hypothesis is that NO acts as a self-regulating mechanism in osteoclasts."
Large, multinucleated and recruited from blood cells, osteoclasts thrive in the bone environment. They work in an odd tandem with bone-forming cells called osteoblasts in the bone remodeling process. This is much like the erosion/deposition process of a river bank where material is removed and then deposited once again. The osteoblasts giveth; the osteoclasts taketh away. Osteoclasts, like NO, are dual-natured. While they quite literally eat holes in bones, in their absence, bone marrow cavities and nerve canals don't form, leading to osteopetrosis. People with this malady often have a compromised immune system and/or neurological problems.
"The osteoclasts and osteoblasts have to work together to remodel bone," Osdoby says. "Bone is turning over all the time, and if you don't have that situation, you can have old bone matrix, where the matrix becomes brittle and susceptible to fracture."
But if the osteoclasts get the upper hand, the skeleton loses too much bone, leading to osteoporosis or inflammatory bone disease.
As for the naughty (and nice) nitric oxide, there are actually three different enzymes that produce the molecule, and these are regulated in different ways. One is called constitutive nitric oxide synthase, which makes NO to regulate blood pressure and vascular processes, and has recently been found to play a role in male erection. Another is neuronal NO synthase, primarily associated with nerve tissues. A recent study on mice with the NO neuronal synthase gene knocked out reported extremely aggressive male mice that were overly sexually aggressive. A third form is inducible NO synthase, the kind involved in bone loss and associated with the activity of defense mechanism cells.
"Nitric oxide, as a free radical, tends to destroy proteins and DNA and RNA, but it also acts as a signal molecule," Osdoby says. "We hope the information we're gathering will help in developing new strategies to buffer the overly ambitious osteoclasts associated with a variety of musculoskeletal disorders."