The modulatory neurotransmitter circuits offer particularly useful pharmacological targets. The most intensively studied system is the glutamate/NMDA/NO cascade (Figure ). The importance of NMDA receptors in tolerance was first recognized over 15 years ago (
11,
12) and was followed almost immediately by the discovery of a role for NO in morphine tolerance (
13,
14) (see “Selected tolerance-related actions of NO”). Since these initial observations, many laboratories have confirmed the importance of the glutamate system and NO in opioid tolerance using a vast array of NMDA antagonists and neuronal NOS (nNOS) inhibitors (
15,
16). There is even a mouse strain that does not develop tolerance to morphine due to a deficit at the level of the NMDA receptor (
17), illustrating the potential importance of the genetic background of patients in the clinical development of tolerance.
Despite the extensive literature over the past 15 years documenting the importance of NO in morphine tolerance, its molecular actions remain unclear. NO is an uncharged free radical gas, making it unique in the realm of neurotransmitters (
18). It can readily diffuse through membranes and therefore can chemically attack many targets. It is synthesized upon demand since it cannot be stored, and its selectivity among targets is likely due to limiting its site of action by very localized stimulation of its synthetic enzyme NOS. One of the first NO actions identified was its ability to stimulate cGMP formation by binding to the heme moiety in the enzyme guanylyl cyclase, but it also will interact with other heme-containing proteins. NO is chemically reactive and S-nitrosylates a wide range of proteins under physiological conditions, including both subunit 1 and subunit 2 of the NMDA receptor (
19) (see “Selected tolerance-related actions of NO”).
In their study in the current issue of the
JCI (
20), Muscoli and coworkers explore another mechanism of NO action in which exposure of NO to superoxide generates peroxynitrite, which then nitrates tyrosine residues. Using a murine model of chronic morphine administration, these authors confirmed the ability of the NO synthase inhibitor l-NAME to prevent morphine-induced tolerance and then demonstrated a similar effect with the superoxide scavenger MnTBAP
3–, consistent with a role for peroxynitrite in the development of morphine tolerance. Evidence for this proposed role of peroxynitrite was strengthened by their observation of the accumulation of nitrotyrosine immunoreactivity in the dorsal horn of the spinal cord, a region important in opioid action. Immunoprecipitation followed by western blots revealed the presence of nitrotyrosine in mitochondrial superoxide dismutase, the glutamate transporter GLT-1, and the enzyme glutamine synthase following chronic morphine administration — proteins important in glutamate actions and in the regulation of superoxide formation. The same paradigms that blocked tolerance in vivo diminished the nitration of these proteins, as did inducing the decomposition of peroxynitrite.
These are interesting studies in that they provide a strong association between morphine tolerance and the nitration of tyrosine residues in a number of proteins associated with the glutamate/NMDA receptor/NO signaling cascade. However, establishing a causal relationship is not simple. Although blocking NO production prevents tolerance, not all NO is involved in tolerance. In fact, NO also enhances opioid analgesia (
21). Neuronal NOS undergoes splicing. Although the predominant splice variant is involved in the development of morphine tolerance, a second splice variant, lacking exons 9 and 10, generates NO that has an opposite effect in vivo, facilitating analgesia, presumably through different targets (
21). This may make it difficult to separate NO targets important in tolerance as opposed to those important to analgesia.
It also is important to integrate these findings with the prior observation that blockade of guanylyl cyclase also prevents morphine tolerance (
22) and the demonstration that two different subunits of the NMDA receptor are endogenously S-nitrosylated in the brain (
19). Any or all of these mechanisms of NO action may be important in opioid tolerance. The challenge in the future will be to establish causal relationships for these mechanisms. Muscoli and colleagues have provided strong evidence correlating the generation of nitrotyrosine proteins in morphine tolerance, and this needs to be examined further (
18). The potential involvement of proinflammatory cytokines and the nuclear enzyme poly(ADP-ribose) polymerase, as suggested by these authors, seems more tenuous, but certainly deserving of additional investigation. The involvement of so many diverse components in the expression of morphine tolerance makes development of a comprehensive, integrated model difficult. However, this diversity also expands the number of potential pharmacological targets and will hopefully lead to new therapies to facilitate the use of opioids in the management of chronic pain.
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