Better known as an environmental hazard, nitric oxide (NO) is produced in combustion engines and contributes to smog and acid rain and has been implicated in the catalytic destruction of the ozone layer (Lancaster;1992). Though NO is the bad-boy of the environment, it’s roles in the body are extremely diverse and in some instances can be deleterious or beneficial depending on the circumstances. NO has been connected with immune function, control of blood pressure and hypertension, impotence and penile erection, septic shock, insulin-dependent diabetes mellitus, and macrophage mediated destruction of oncogenic cells (Young;1993, Stroh;1992). However, its activities in the nervous system may be the most exciting discovery yet for this eclectic molecule (Koshland;1992).
Due to NO’s radical structure, it is highly reactive and very short-lived within the body (6-15 sec.) making its detection difficult (Lancaster;1992); as such, it can therefore be synthesized only on demand since radicals are known to disrupt cellular homeostasis. NO is small and uncharged and it rapidly diffuses through cellular membranes from its site of synthesis making it in ideal intercellular paracrine-like messenger or poison (Lancaster;1992). In the body, NO reacts with redox metals such as copper, manganese, or the iron heme-like protein centers, and molecular oxygen forming nitrites and nitrates, the latter constituting the principal manner in which NO is inactivated in vivo (Snyder;1992).
The enzyme which synthesizes NO, nitric oxide synthase (NOS), has been cloned and structurally resembles cytochrome P-450 reductase, possessing many sequence homologies and the same physical sites for binding of identical cofactors (FMN, FAD, and NADPH); in addition, NOS also possesses binding sites for calmodulin and a phosphorylation site which may modulate its activity (Bredt;1991) (Snyder;1992). NO is synthesized by NOS from L-arginine and molecular oxygen with the concomitant release of citrulline; NADPH and tetrahydrobiopterin are required as cofactors (Lancaster;1992).
In order to understand NO’s neurobiological roles, some of NO’s biochemistry and physiology must be discussed. NO’s radical structure, fleeting half-life, and rapid diffusibility make it an ideal neurotransmitter, albeit an unconventional one. In the peripheral vascular blood supply, acetylcholine or bradykinin act on endothelial cells in the vessels which causes a release of endothelium-derived relaxation factor (EDRF) which has been verified to be NO (Snyder;1992). Acetylcholine or bradykinin causes an increase in the intracellular Ca2+ concentration through the action of inositol triphosphate; the increased intracellular Ca+ binds the calmodulin site in NOS and stimulates the synthesis of NO which rapidly diffuses away from its site of synthesis to the tunica muscularis of blood vessels causing vasodilation (Murray;1993). In this manner, NO is a sort of second neurotransmitter potentiating the effects of acetylcholine or bradykinin on the muscular layer of blood vessels. In this system, NOS is located in the endothelial cells; however, in the cerebral arteries, NOS is located in the nerve plexus themselves in the outer layer of cerebral arteries, and the cell bodies of these neurons lie in the sphenopalatine ganglion in the neck (Snyder;1992). Relaxation of the cerebral arteries can be blocked by a synthetic arginine derivative N-nitroarginine (Snyder 1992).
In these systems, NO satisfies several common criteria for a neurotransmitter: NOS is located in the neurons themselves, inhibition of synthesis of NO resembles the blockade of the corresponding physiologic nerve, and mimicking NO causes an effect similar to the corresponding physiologic nerve stimulation (Snyder;1992). As is well-known, most neurotransmitters are packaged in vesicles and released at synaptic clefts or upon termination of the action potential at the pre-synapse. NO, however, is not packaged into vesicles but rather synthesized upon demand and diffuses to adjacent areas in the brain to exert its neurophysiological effect (Snyder;1992). NO’s property to diffuse in all directions from its point of synthesis foreshadows many of its unique properties in the brain.
When glutamate binds to NMDA receptors, the intracellular Ca2+ of the cell rises; intracellular Ca++ binds calmodulin which activates NOS to produce NO from arginine. The NO is then free to diffuse to surrounding areas due to its high lipid solubility and rapid diffusibility (see fig. 1) (Snyder;1992). NO has at least two fates after its synthesis: (1)
it is inactivated via its interaction with superoxide hydroxide, or nitrous oxide free radicals or (2) it binds the heme center of guanylate cyclase and initiates the synthesis of cGMP from GTP, initiating a cellular cascade of events since cGMP is a 2nd messenger (Snyder;1992).
NO was first localized in the brain when researcher’s discovered that nerve activity in the cerebellum increased local levels of cGMP, indicating that perhaps NO itself was responsible for the rise in cGMP. NO was later found to be produced when NMDA receptors were stimulated using glutamate, an excitatory neurotransmitter located in most areas of the brain (Young;1993).
Researchers started to look for rising levels of intracellular cGMP as evidence of NOS activity in the brain and found some exciting happenings. Glutamate is released by the pre-synaptic neuron in response to an action potential and binds to the post-synaptic NMDA receptor; through the Ca++/calmodulin pathway previously discussed, NO is synthesized by NOS in response to the high intracellular Ca++ concentration in the post-synaptic neuron (see fig 2) (Lancaster;1992). Scientists discovered that cGMP levels rose in the pre-synaptic neuron and not the post-synaptic neuron indicating that NO had little or no effect on the neuron
Not only can NO act as a retrograde neurotransmitter in the brain but it can also act as a killer. During a stroke, a specific area of the brain supplied by a specific blood vessel is not getting enough oxygen; in the meantime, a cascade of glutamate is causing the neurons in that area to continue to fire. This added stress of having to fire under conditions of anoxia may kill neurons; however, the NO produced from the action of glutamate on the NMDA receptors creates a situation in which NO cannot be rapidly deactivated (remember, NO is a radical and radicals are known to interrupt cellular homeostasis). The excess NO may bind to several requisite metalloproteins necessary for the function of adjacent cells and inactivate them, whereas under normal oxygen tensions in the brain, NO can react with oxygen in its final deactivation step (Snyder;1992, Lancaster;1992). The excess NO diffuses to neighboring cells and destroys them.
A peculiar staining technique was developed by British histochemist Anthony Pearse which is available to study the distribution of NO synthesizing neurons. When brain slices are stained with nitro-blue tetrazolium (a dye) and then NADPH is added as a co-factor, NO synthesizing neurons become bright blue (Snyder;1992). These diaphorase neurons, as they are called, represent only about 2% of the neurons in the brain. In neurodegenerative diseases such as Alzheimer’s and Huntington’s Chorea and in vascular strokes, a large majority of neurons are destroyed due to the postulated inability of the brain to inactivate excess NO production (Snyder;1992). However, very few of the diaphorase-staining neurons are destroyed but neurons adjacent to the diaphorase neurons are inevitably dead (Lancaster;1992, Snyder;1992). This indicates that excess NO production might be a cause of the immense neuronal damage resulting from CVA’s or neurodegenerative diseases. If neuronal damage is indeed the cause of NO-mediated damage, then perhaps infusion of an NOS inhibitor might minimize the damage associated with strokes (Snyder;1992).
There remains much work to be done in determining the neurophysiological roles of NO. For example, the NO model of LTP is a superior model provided there is indeed a pre-synaptic component to memory formation (Barinaga;l991). If there is no pre-synaptic component, then the NO model of LTP holds little merit. Perhaps the most exciting breakthrough is the possibility of diminishing the damage caused by strokes by using NOS inhibitors. NOS inhibitors have already proven useful in septic shock by suppressing the excess NO production induced by bacterial lipopolysaccharide and raising blood pressure and NOS inhibitors have also been used in promoting bronchial dilation in neonates with severe bronchial constriction (Bai;1993). If therapeutic NOS inhibitors can be exploited in the brain, then conceivably strokes may not be as associated with loss of function and ability as in the past. Such an understanding of NO and its functions can only lead to a better comprehension of ourselves and our maintenance, especially our neurobiology.
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