The results of a number of studies demonstrate that the gas nitric oxide (NO) plays a functional role in the central nervous system. This all originated with the discovery that the so-called endothelium-derived relaxing factor (EDRF), found in blood vessels, and thought to be a peptide, was in fact NO. The potential roles of this freely diffusible gas have subsequently been extended to many other tissues and organs but we will concentrate on the possible neuronal roles of what is obviously a novel mediator. There are also suggestions that the closely related carbon monoxide may also have a function in the central nervous system.
Many brain and spinal cord neurons have the capacity to produce NO and experimental evidence indicates a role for this gas in neuronal transmission in animals. A major issue is that the effects of a gas are not limited to the release site and interpretation of the apparent neuronal actions of NO is complicated by the fact that some of the observed effects may be via changes in local blood flow.
Being a gas, NO can diffuse freely once produced, and so is not constrained by the usual mechanisms of release and uptake that confine most transmitters to the synapse. Likewise, the fact that it is not stored means that the criteria of presence and storage are not met by this highly labile and freely diffusible molecule. Finally, its ability to cross lipid barriers means that it is a transcellular mediator rather than a molecule that acts on a surface receptor close to its release site. Thus while it cannot be considered as a neurotransmitter, NO can still have important actions in the central nervous system.
NO is the product of the oxidation of one of the guanidino nitrogens of the amino acid, L-arginine by the enzyme nitric oxide synthase (NOS). L-arginine is then hydroxylated and a second oxygen atom is incorporated to produce NO and citrulline (see Fig. 13.9). The production of NO occurs in many tissues. There are three isoforms of the enzyme, endothelial (eNOS), inducible (iNOS) and neuronal (nNOS). Whereas nNOS and eNOS are regulated by calcium, iNOS is not. This control stems from the production of a calcium-calmodulin complex that then binds NOS and switches on the production of NO from arginine. Then, as internal calcium levels drop, the production of NO also ceases. In many parts of the central nervous system, NMDA receptors are expressed on neurons with the capacity to produce nitric oxide. Thus, calcium influx through the NMDA receptor channel appears to trigger production of nitric oxide by activation of nNOS. To complicate matters, nNOS is also found outside the CNS in epithelial cells and skeletal muscle. Nitric oxide seems to play a much greater role in neuronal
transmission following excessive stimulation/pathology in some regions of the brain and so although the enzyme is constitutive it can clearly be unregulated.
The main action of NO is on the enzyme-soluble guanylate cyclase. NO activates this enzyme by binding to the heme moiety and so there is an increased conversion of GTP to cGMP. This reduces intracellular calcium and this action, and also partly through activation of cGMP-dependent protein kinases, relaxes smooth muscle. The same mechanism of action occurs in neurons but NO can also inhibit other enzymes with a heme group such as cyclooxygenase and lipoxygenase. How these effects translate into what appear to be mostly excitatory effects in the CNS is unclear. Thus agents that increase NO production cause increases in neuronal excitability and vice versa. The results of a number of studies manipulating the levels of the gas show that NO plays a role as a neuronal communicator. There is, however, the problem of a lack of selective agents that modulate the production and actions of NO.
Application of L-arginine and nitrates and nitrites (that donate NO) has been used to drive the system but, as always, blocking the effects of a potential mediator provides the best approach. There have been reports of a large number of putative inhibitors of NOS but there are two agents that have been widely used, L-NG nitroarginine (L-NAME) together with the closely related L-NG monomethylarginine (L-NMAA). These agents block NOS at the arginine site, acting as false substrates, and have no selectivity for any of the three forms of the enzyme. Thus, any study of the physiological role of NO in neurons based on the use of these compounds will be carried out in animals where the vascular effects of NO are also blocked leading to severe hypertension. This may well lead to problems of interpretation and even local application of these compounds directly within the CNS will change local blood flow.
However, more recently, a functionally selective inhibitor of nNOS has been described—7-nitroindazole (7-NI). It is puzzling that in vitro this compound has no selectivity for nNOS over eNOS but after systemic administration, fails to change blood pressure yet alters neuronal responses that are thought to result from production of NO. A suggested resolution of this action is that 7-NI is metabolised in the periphery but not the CNS, so that once it has crossed the blood-brain barrier, it can only act on nNOS.
FUNCTION — EXCITOTOXICITY
The proposal that NO or its reactant products mediate toxicity in the brain remains controversial in part because of the use of non-selective agents such as those listed above that block NO formation in neuronal, glial, and vascular compartments. Nevertheless, a major area of research has been into the potential role of NO in neuronal excitotoxicity. Functional deficits following cerebral ischaemia are consistently reduced by blockers of NOS and in mutant mice deficient in NOS activity, infarct volumes were significantly smaller one to three days after cerebral artery occlusion, and the neurological deficits were less than those in normal mice. Changes in blood flow or vascular anatomy did not account for these differences. By contrast, infarct size in the mutant became larger after eNOS inhibition by L-NAME administration. Hence, after middle cerebral artery occlusion neuronal NO production appears to exacerbate acute ischemic injury, whereas vascular NO protects. The data emphasise the importance of developing selective inhibitors of the neuronal isoform.
Behavioural studies are generally unable to find a role for spinal NO in nociceptive reflexes in normal animals, whereas NO inhibitors are highly effective in blocking these same reflexes following the induction of peripheral inflammation or neuropathy. Although a complication is that NO may also play a role in peripheral vascular events during inflammation, these results do suggest that the gas is produced only under certain conditions.
The nNOS inhibitor 7-NI causes a greater inhibition of the wind-up and hyper-excitability of dorsal horn neurons (see Chapter 21) than the immediate response due to direct afferent C-fibre stimulation in normal animals. The preferential inhibition of the NMDA receptor-mediated neuronal hyperexcitability and wind-up of the neurons by 7-NI conforms to the idea that the NO generated in the spinal cord during the transmission of nociceptive information is a consequence of NMDA receptor activation. This also agrees well with a number of other observations, including electrophysiological studies in which block of NO production reduces the excitatory effects of NMDA on neurons and behavioural studies where block of NO production reduced the behavioural effects of NMDA. It seems that following the development of peripheral inflammation and consequent hyperalgesia the NMDA receptor is able to participate in spinal nociceptive reflexes providing a mechanism whereby NO is generated. Thus NOS inhibitors do block nociceptive reflexes in behavioural studies in animals with peripheral inflammation. However, once NO is generated in the spinal cord, the mechanism by which it produces its effects, such as the role played by NO in the wind-up process, has yet to be confirmed. Although NO can act in the neuron in which it is produced to increase levels of cGMP, NO can also diffuse to other neurons to produce its effects. It has been shown that activation of NMDA receptors in the cord can produce an NO-mediated release of glutamate, some of which may represent release from primary afferent terminals following the retrograde diffusion of NO. Nitric oxide can also evoke the release of CGRP and substance P from the dorsal horn of the spinal cord. An NO-evoked release of glutamate, CGRP and substance P may operate as a positive feedback system to further generate wind-up and centrally mediated hyperalgesia. Thus, the development of clinically useful neuronal NOS inhibitors could provide a novel approach to indirectly controlling NMDA receptor-mediated transmission. As with agents directly acting at the NMDA receptor-channel complex, side-effects may preclude their use.
The idea of retrograde messengers such as NO has also been advanced with regard to hippocampal LTP (Chapter 20). There is a marked lack of consensus on whether NO plays a role in LTP and much discussion on why different groups find different results. The importance of the need for a diffusible messenger in the initiation of long-term changes comes from the fact that LTP is induced by activation of postsynaptic NMDA receptors yet maintained by presynaptic changes. Thus, there is a requirement for a mediator to be generated by NMDA receptor activation and then diffuse back to the presynaptic terminals. NO could fulfil this role. Unfortunately, some studies have shown that NOS inhibition blocks LTP whereas others have failed to show this.
SUMMARY AND PERSPECTIVES
NO differs from the more conventional NTs like the amino acids and monoamines in that it is not released from nerve terminals by arriving action potentials. Thus unlike them it is not a primary messenger. It could be regarded as a second messenger except that its effects appear to be mediated by the production of cGMP, itself an established second messenger. In that sense, NO is more like a G-protein. The fact that its synthesis and release from neurons, and so its actions, are dependent on and stimulated by Ca2+ influx, often after NMDA receptor activation, inevitably links NO to more extreme excitatory effects such as LTP, excitotoxicity, pain and possibly also epilepsy. Whether blocking its synthesis will be a more effective therapeutic approach than the use of NMDA receptor antagonists is problematic in that even if really specific NOS inhibitors are developed these effects will potentially be at least as widespread as block of NMDA receptors. Where NO inhibition may have the advantage is that it should only operate under conditions of NMDA action that are above normal and so may only affect adverse but not normal neuronal function. This should only occur in those brain areas and pathways showing that extreme level of activity. Time will tell . . .
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