Neuroscience Online

Section I: Cellular and Molecular Neurobiology 14. Neuropeptides and Nitric Oxide Part 3 of 4 Neal Waxham, Ph.D.

Nitric Oxide (NO)

Characteristics of NO

Summary of NO's Properties

1. Gas that freely diffuses through membranes
2. Short-lived with a half-life measured in seconds
3. Highly reactive free radical
4. Toxic at high concentrations

NO is a short-lived gas not to be confused with the relatively stable anesthetic gas nitrous oxide (laughing gas). NO is actually a free radical and is therefore a highly reactive compound. Some of its toxic effects are likely due to NO reacting with superoxide to produce the destructive radical peroxynitrate. NO is considered an unconventional neurotransmitter because it is not released by exocytosis and its action does not occur through conventional receptor molecules.

As mentioned previously, the typical description of neuronal communication considers transmission to be unidirectional. A presynaptic neurotransmitter is released that produces changes in the postsynaptic neuron. Several compounds (like neuropeptides and NO) produced in postsynaptic neurons diffuse into the local environment and affect the surrounding cells. Since NO is a freely diffusible gas it has the potential to travel quickly in any direction from its point of production. For example, if produced in a postsynaptic cell because of glutamate receptor stimulation, NO could be released into the local environment and send a signal back to the presynaptic neuron (Figure 14.5). This type of activity is referred to as retrograde signaling since the signal travels in a retrograde direction from the postsynaptic to the presynaptic neuron.

Figure 14.5

Figure 14.6

Figures 14.5, 14.6, and 14.7 summarize the main aspects of NO synthesis. In this example, glutamate is released from the presynaptic terminal that binds to NMDA receptors on the postsynaptic membrane, causing them to open and permitting Ca2+-influx. The Ca2+ activates calmodulin which binds to and activates the enzyme nitric oxide synthetase (NOS). Using arginine as a substrate, NOS produces NO and a second reaction product citrulline. The NO is then free to diffuse into the environment and interact with the presynaptic terminal which initially released the glutamate or any other cell in the local environment. Recognize that any process that elevates intracellular Ca2+ will potentially activate NOS. Glutamate activation of NMDA receptors is just one well-documented example.

Figure 14.7

Synthesis by Nitric Oxide Synthase (NOS) and Release

NO is produced by the enzyme nitric oxide synthetase (NOS). This enzyme is found in a subpopulation of neurons (1-2% of neurons in cortex) and is found in most all endothelial cells. At least one form of NOS in these cells is dependent on calcium and calmodulin for activation as indicated in Figures 14.5, 14.6, and 14.7. It also contains cofactors similar to cytochrome P-450. These cofactors are NADPH (nicotinamide adenine dinucleotide phosphate, FAD (flavin adenine mononucleotide) and FMN (flavin mononucleotide). These cofactors are essential for the transfer of electrons that produces the unstable and short-lived product NO. The substrate in this reaction is the common amino acid arginine and the products are citrulline and NO (Figure 14.5). Because of NO's short lifetime it is extraordinarily difficult to measure directly. However, in experimental preparations, there is an excellent correlation between the application of NMDA, which increases intraneuronal Ca²⁺ and activates NOS, with the production of the additional product of NOS's enzymatic activity, citrulline (see Figure 14.8). Citrulline production is a reliable indicator of NO production. It is also possible to block the production of citrulline (and NO) by feeding cells the non-metabolizable substrate for NOS termed methyl arginine (Figure 14.9), and such compounds have been used to reduce the production of NO and terminate its biological effects.

Figure 14.8

Figure 14.9

"Receptors" for NO

Figure 14.10 One of the main targets for NO appears to be the enzyme guanylyl cyclase. This discovery was made indirectly by monitoring the accumulation of the NOS product citrulline while also monitoring for the production of cGMP, the product of the enzyme guanylyl cyclase (Figures 14.8 and 14.9). In this experiment NMDA was added to a neuronal preparation to activate NOS. NOS utilizes arginine as a substrate to produce NO and as mentioned, if a non-hydrolyzable analog like methyl-arginine is added to the system, citrulline and cGMP accumulation were found to terminate in the same dose-dependent fashion. These strong correlations led to the discovery that guanylyl cyclase is a main NO target. Guanylyl cyclase is an unusual enzyme because it has a heme ring with an associated iron molecule as part of its structure. NO activates this enzyme by binding to iron in the heme, initiating production of cyclic GMP from GTP (Figure 14.10) through conformational changes in the enzyme. cGMP then activates a cGMP-dependent protein kinase and other enzymes described below. The spectrum of effects produced by the activation of cGMP-dependent protein kinase are only now becoming understood.