Nitric Oxide and Bone Cell Formation

Nitric Oxide (NO) is a simple molecule consisting of one atom of nitrogen and one of oxygen, making it even simpler than water. Research into the effects of this small but important molecule began in the 1970’s when scientists started to examine why blood vessels relaxed when certain compounds were added. In 1998, 20-some years after this research first began, a Nobel Prize was awarded to these researchers for their breakthrough discoveries regarding NO. Over the last two decades NO research has continued to grow, and there has been an exponential increase in the number of publications on this fascinating molecule. Some health promoting effects of nitric oxide include increased circulation, lowered blood pressure, enhanced digestion, enhanced libido, improved immune system and stronger bones.

How NO is Produced in the Body

The conventional method of NO synthesis is from the amino acid L-Arginine (see Figure 1). L-Arginine is oxidized via a series of steps involving a family of enzymes called nitric oxide synthases (NOS). There are essentially three common NOS termed isozymes; these are iNOS, eNOS and nNOS. Each of these enzymes plays a different role in the generation of NO in different tissues like the nerves, endothelium or blood vessels or on demand. In each case however, normal oxygen conditions (also called “normoxia”) as well as a neutral to high (alkaline) pH level are required. When these conditions are met the NOS-dependent conversion of L-Arginine occurs efficiently. Production of NO is the primary reason for the dietary intake of L-Arginine. However, under low oxygen conditions (also called “hypoxia”) the conversion of L-Arginine to NO is severely limited. Low oxygen conditions can occur for a variety of reasons. For example, partial or complete blockage of a blood vessel (ischemia), conditions of extreme physical exercise or high altitudes can all result in reduced blood flow and thus reduced oxygen delivery to the body’s tissues and cells. Moreover, such low oxygen conditions are also accompanied by an increased production of lactic acid which reduces pH making the tissue condition both hypoxic and acidic.

Another Way to Produce Nitric Oxide

A novel pathway to NO generation from nitrates has been discovered by researchers at the Karolinska Institute in Stockholm, Sweden and by researchers from the University of London, England. The Swedish and English researchers were trying to discover why certain diets, like the Mediterranean diet, vegetarian diets, Japanese diets and the famed DASH diet (Dietary Approaches to Stop Hypertension) were particularly protective of the heart. Both groups independently reported that the key to the success of these diets was the consumption of leafy green vegetables, and that a key component of all these diets was the high nitrate content. The researchers proposed that the nitrate was converted into NO via a reductive process as follows: essentially, the nitrate is reduced in the mouth by bacteria that are normally present on the back of the tongue. These specialized bacteria use the nitrate to help them make energy in the form of ATP. In return, the bacteria utilize their own nitrate reducing enzyme called Nitrate Reductase to generate nitrite. This special relationship is an interesting example of human-bacteria symbiosis: a mutually beneficial relationship. The nitrite is a much more active molecule than nitrate and is present in high concentrations in the saliva which is swallowed the stomach where conditions of low oxygen (relative to the mouth) and high acid are present. These conditions are ideal for further reduction of nitrite into NO. The entire reduction process of nitrate into nitrite and then into NO occurs without the intervention of NOS enzymes. It should be noted that low oxygen and low pH conditions don’t just occur in the stomach, they can also occur throughout the body in certain situations including extreme physical exercise, heart disease and psychological and physical stress. Both pathways of NO generation are depicted in Figure 2. How Bone Remodeling Occurs Bone is a complex tissue composed of several cell types which is continuously undergoing a process of renewal and repair termed ‘bone remodeling’ (see Figure 3). The two major cell types responsible for bone remodeling are osteoclasts (“bone eaters”), which breakdown bone, and osteoblasts (“bone builders”), which form new bone. During the bone remodeling cycle, old or damaged bone is removed by osteoclasts, which secrete acid and enzymes that digest the bone onto the bone surface. Subsequently the osteoclasts migrate away from the area of bone undergoing resorption and die. They are replaced by osteoblasts, which lay down new bone matrix in the form of osteoid. Later, the osteoid becomes calcified to form mature bone. During bone formation, some osteoblasts become embedded within the bone matrix, and become osteocytes, a third cell type unique to bone. Osteocytes interconnect with one another and with cells on the bone surface via channels in the bone matrix. It is thought that osteocytes act as sensors of mechanical stress in the skeleton, by detecting and responding to changes in fluid flow which run through cannaliculi in the bone.3-5 Bone remodeling is regulated by several systemic hormones, such as parathyroid hormone (PTH), vitamin D, sex hormones (e.g. estrogen) and calcitonin, as well as by local factors including NO, prostaglandins, growth factors and cytokines.6

How is Nitric Oxide (NO) Involved in Bone Health?

Nitric oxide appears to have a two-fold effect on osteoblast activity. Studies in vitro have indicated that the small amounts of NO which are produced by osteoblasts may stimulate their own growth as well as the production of immune modulating proteins.7 Whilst some investigators have shown that slow release NO donors stimulate osteoblast growth and differentiation in vitro,(8-10) other workers reported that NO donors and NOS (Nitric Oxide Synthase) inhibitors had little effect on osteoblast growth or differentiation, except at high concentrations where osteoblast growth actually seemed inhibited.(11,12) The most compelling evidence supporting a role for NO in osteoblast function comes from studies of eNOS (endothelial NOS) in animals. Two groups of investigators have reported major defects in bone formation and osteoblast activity and a reduced growth response to administered estrogen both in vivo and in vitro in eNOS deficient animals. The molecular mechanisms responsible for this remain to be defined, but indicate the existence of an important interaction between eNOS and the molecular pathways involved in osteoblast differentiation and function.(13,14) In addition, a possible mechanism for the inhibition of osteoclast activity by NO is the modification of cathepsin K (a bone regulating enzyme). Cathepsin K is highly expressed in osteoclasts and plays a key role in the bone resorption mechanism, since it degrades bone collagen. NO and several NO donors have been shown to inhibit the activity of this enzyme.15

What You Need to Know

Bone health is a key component to healthy aging. It is imperative that we look at our options, both novel and conventional, when it comes to keeping our bones strong. This article has focused on the novel yet effective nitric oxide molecule and its role in keeping the bone remodeling process robust over time. Our bones are very much alive and it is vital that our system keeps removing the old bone and laying down new bone in its place. Consuming foods rich in nitric oxide producing nitrate in combination with other key bone building nutrients (such as calcium, magnesium, vitamins D, C and K) is a potent recipe for maintaining bone integrity and worth considering for preventing or managing concerns related to poor bone health.

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