Role Of Intracellular Protein Kinases

Activation of NMDA receptors initiates intracellular processes that may lead to neuroplastic changes. A common feature of NMDA receptor activation is an increase in intracellular Ca2+ concentration, which, in turn, initiates a number of second/third-messenger-mediated intracellular processes. One such process is the redistribution (translocation from cytosolic to membrane-bound form) and activation of the Ca2+-sensitive protein kinase C (PKC). It is known that Ca2+-regulated PKC translocation and activation are associated with a variety of central nervous system (CNS) functional changes that occur by means of protein phosphorylation. The following sections will briefly discuss the role of NMDA receptor-mediated activation of protein kinases, particularly PKC, in the development of opi-oid tolerance.

2.1. Behavioral Evidence

A growing body of evidence indicates an important role of protein kinases, particularly PKC, in the development of antinociceptive tolerance (42-46). It has been shown that antinociceptive tolerance to morphine, butorphanol, DAMGO, or DELT II can be prevented by coadministration with opioids of the nonselective protein kinase inhibitor H7 (morphine, butorphanol) (46,47), the PKC translocation blocker GM1 ganglioside (morphine) (42,44), or the selective PKC inhibitor calphostin C (DELT II, DAMGO) (45). Inhibition of protein kinase A (PKA) with KT5720 has not been effective in preventing the antinociceptive tolerance to DAMGO or DELT II (45). Additional evidence for the involvement of PKC in DELT II-mediated antinociception is that it administration of phorbol 12,13-dibutyrate (PDBu), a PKC activator, produced calphostin C-reversible attenuation of antinoci-ception induced by it DELT II, whereas PDBu alone had no effect on the nociceptive threshold in the mouse tail-flick test (45).

2.2. Electorphysiological Evidence

Further evidence for the intracellular modulation of |-opioid receptor desensitization through the calcium/calmodulin-dependent kinase and PKC was elegantly demonstrated in a recent study using a human ^-opioid receptor cDNA (48). In that study, both ^-opioid receptor (encoded by a cDNA from human ^-opioid receptors) and a cloned G-protein-activated K+ channel (displaying coupling to the opioid receptor) were coexpressed in Xenopus oocytes. The verification of |-opioid receptors was confirmed by binding to selective | -opioid agonists and antagonists. Under these conditions, functional desensitization of | -opioid receptors, as reflected by reduced K+ currents following repeated exposure to | -opioids, was potentiated by both the calcium/calmodulin-dependent kinase and PKC (48). These results provide convincing evidence for the protein-kinase-mediated modulation of functional ^-opioid receptors. Interestingly, a recent study has shown that activation of PKC also decreases ^-opioid receptor mRNA levels (49), suggesting that PKC may also have a role in regulating the ^-opioid receptor turnover. The functional importance of the PKC-mediated ^-opioid receptor turnover in mechanisms of opioid tolerance is yet to be determined.

2.3. Autoradiographic and Immunocytochemical Evidence

Spinal cord levels of membrane-bound PKC increase reliably as morphine tolerance develops (42). This increase in membrane-bound PKC occurs mainly within laminae I-II of the spinal cord dorsal horn, a region showing increased levels of PKC translocation in nerve-injured animals with demonstrable thermal hyperalgesia (50,51). This tolerance-associated increase in membrane-bound PKC was reduced by it treatment with the PKC translocation blocker GM1 ganglioside (42). In another study, however, only cytosolic PKC, but not membrane-bound PKC, was upregulated in the pons and medulla following chronic morphine administration (52), although the affinity of PDBu binding to membrane-bound PKC was increased in the brain under the same condition (47).

More evidence for PKC changes in morphine-tolerant rats was provided by a recent study utilizing an immunocytochemical method. In that study, the development of morphine tolerance was shown to be associated with increases in immunoreactivity of a PKC isoform (PKCy) in laminae I-II dorsal horn neurons (43). Such increases in PKCy immunoreactivity along with behavioral manifestations of morphine tolerance were prevented by it administration of MK-801 (43). Increases in PKCy immunoreac-tivity in similar spinal cord dorsal horn regions also were seen in rats with nerve-injury-induced thermal hyperalgesia, further suggesting the involvement of common regions of the spinal cord dorsal horn in mechanisms of hyperalgesia and morphine tolerance. In contrast, spinal cord PKC levels did not change in saline control rats receiving a single injection of morphine (43), suggesting that activation of NMDA receptors and subsequent intracellular PKC within the spinal cord reflects neuroplastic changes following repeated exposure to opioids.

It should be noted that a different mechanism of ^-opioid receptor desensitization involving G-pro-tein-coupled receptor kinases, but not PKC, also has been proposed (53-55). It has been shown that P-adrenergic receptor kinase 2 and P-arrestin 2, both of which are G-protein-coupled receptor kinases, synergistically desensitize homologous ^-opioid as well as 8-opioid receptors (54). It will be of interest to investigate how different intracellular processes converge to influence the cellular and molecular mechanismas of opioid tolerance.

3. MECHANISMS OF NMDA RECEPTOR-MEDIATED OPIOID TOLERANCE 3.1. Critical Issues Regarding NMDA Receptors and Opioid Tolerance

A critical issue concerning NMDA receptors and the development of opioid tolerance is how exogenous opioid administration leads to the activation of NMDA receptors. This remains the core issue for understanding the involvement of NMDA receptors in the cellular and molecular mechanisms of opioid tolerance and dependence. Several points should be considered in view of the data discussed above. First, in a well-controlled experimental model of opioid tolerance, the primary variable is the prolonged exposure of opioid receptors to an opioid agonist. Yet, NMDA receptors are critically recruited during the process, leading to the development of opioid tolerance, and there is no reliable evidence suggesting that opioids may directly activate the NMDA receptor. Second, NMDA receptor activation is not required for the expression of tolerance, nor does the acute blockade of NMDA receptors reverse an established tolerance status. Third, the NMDA receptor is a unique volt-age/ligand-gated receptor and its activation requires the removal of the Mg2+ blockade following a partial membrane depolarization. It appears difficult to envision that the NMDA receptor would be activated following exposure to opioids given the overwhelming hyperpolarization induced by opioids under most circumstances. Fourth, NMDA receptor-mediated intracellular events such as PKC activation are crucial for the development of opioid tolerance, as inhibition of PKC activation effectively prevents the development of opioid tolerance. Thus, a cellular model of NMDA receptor-mediated opioid tolerance should include these important experimental observations.

Although in an intact system the involvement of a neural circuitry cannot be ruled out in mechanisms of NMDA receptor-mediated opioid tolerance, studies do support interactions between NMDA and opioid receptors at a single-cell level (56,57). In a study utilizing an in vitro trigeminal dorsal horn neuron preparation, NMDA receptor-mediated inward membrane current (depolarization) was initially induced by glutamate (56). The magnitude of this inward membrane current was paradoxically enhanced by the addition of exogenous ¡-opioid agonists. A critical intracellular component that mediates this ¡-opioid effect must be PKC, because activating and inhibiting PKC, respectively, facilitated and blocked the enhancement by ¡-opioid agonists of this NMDA-mediated inward membrane current (56). This PKC effect was subsequently shown to result from a PKC-mediated removal of the Mg2+ blockade of the NMDA receptor (57), making it possible that the NMDA receptor could be activated even under overwhelming inhibitory opioid actions. A necessary condition for this to happen is that NMDA and (¡)-opioid receptors are colocalized in a single neuron. Indeed, such a colocalization has been shown in both supraspinal regions (58-60) and the spinal cord dorsal horn (61). These lines of evidence are the building blocks for a cellular model of | -opioid tolerance shown next.

3.2. A Spinal Cord Model of ¡-Opioid Tolerance

Given the interactions between ¡-opioid and NMDA receptors and the behavioral evidence indicating a role of NMDA receptors, PKC, and nitric oxide (NO) in ¡-opioid tolerance, we previously proposed that the development of tolerance to the analgesic effects of | -opioids is a consequence of a series of cellular and intracellular events initiated by opioid administration, and at least one central locus of such action is in the superficial laminae of the spinal cord dorsal horn (42-44,62,63). The following discussion is based on the frame of a cellular model involving interactions between NMDA and ¡-opioid receptors (42,44,62).

As shown in Fig. 1, ¡-opioid receptor occupation by an exogenous ligand such as morphine may initiate second-messenger (G-protein)-mediated PKC translocation and activation. The involvement of a second messenger is suggested by the observation that PKC-mediated NMDA receptor sensiti-zation takes about 2-4 min to occur after the addition of exogenous ¡-opioid agonists in an in vitro preparation (56). PKC activation removes the Mg2+ blockade from the NMDA receptor-channel complex via PKC-mediated phosphorylation of receptor-channel sites (57). With this blockade removed, even the physiological level of endogenous NMDA receptor ligands may activate the NMDA receptor (64) and allow a localized NMDA receptor/Ca2+ channel opening leading to an increase in intracellular Ca2+ concentration. An elevation of the intracellular Ca2+ level may result in the activation of additional PKC, production of NO via Ca2+ calmodulin-mediated activation of NO synthase, and/or regulation of gene expression via MAP kinases (MAPKs). Although PKC can be activated following repeated ¡-opioid activation, the NMDA receptor activation is required in this process to ensure sufficient and enduring activation of PKC, via Ca2+ actions and/or de novo PKC production and other intracellular moleculars.

PKC may then modulate the ¡-opioid-activated G-protein-coupled K+ channel or uncouple G-pro-teins from the ¡-opioid receptor, resulting in decreased responsiveness of ¡-opioid receptors and behavioral manifestations of opioid tolerance. Changes in the responsiveness of opioid receptors and the dissociation between G-proteins and opioid receptors have already been suggested to be a contributory factor to the cellular mechanisms of opioid tolerance (26,65-69). This hypothesis is in agreement with previous autoradiographic data showing that morphine tolerance develops without concurrent downregulation (decreases in receptor numbers and/or binding affinity) of ¡-opioid receptors (70,71) and is supported by observations that protein kinases can indeed modulate the ¡-opioid

Fig. 1. A spinal cord model of |l-opioid tolerance. Postsynaptic opioid (|) receptor occupation by an exogenous ligand such as morphine may initiate G-protein-mediated PKC translocation and activation. PKC translocation/acti-vation facilitates the removal of the Mg2+ blockade from NMDA receptors. With this blockade removed, the NMDA receptor could be activated in the absence of excessive release of glutamate from presynaptic sources. An elevation of the intracellular Ca2+ level leads to the activation of additional PKC and production of NO via Ca2+ calmodulin-mediated activation of NO synthase. PKC may modulate the | -opioid-activated G-protein-coupled K+ channel or attenuate G-protein activation. PKC may also regulate gene expression and contribute to the NMDA receptor-mediated neurotoxic process, either directly or indirectly via MAPKs. In addition, NO may activate various protein kinases by means of increasing cGMP and thus participate in the modulation of the | -opioid-activated G-protein-coupled K+ channel within the same cell. NO could also diffuse out of the neuron that produces it, thereby enhancing presynaptic release of glutamate/aspartate resulting in a positive feedback; that is, opioids may increase the basal level of presy-naptic glutamate release via the NO mechanism initiated by the postsynaptic opioid action. The role of presynaptic |-opioid, NMDA and metabotropic glutamate receptors as well as postsynaptic NK-1, non-NMDA receptors, and metabotropic glutanmate receptors in the development of opioid tolerance remains to be elucidated. Because many of the intracellular steps following activation of the NMDA receptor in this proposed model are similar to those that occur following nerve-injury-induced hyperalgesia, it has been shown that NMDA receptor-mediated intracellular changes initiated by peripheral nerve injury may lead to both the development of hyperalgesia and the reduced effectiveness of opioid antinociception, mimicking the status of pharmacological tolerance. Collectively, this model shows interactions between the cellular and molecular mechanisms of opioid tolerance and hyperalgesia.

receptor responsiveness (45,46,48,72). In particular, PKC has been shown to facilitate the desensitiza-tion of ^-opioid receptor coupling through a G-protein to an inwardly rectifying K+ channel (73). More recently, a PKC isoform, PKCy, has been shown convincingly to attenuate ^-opioid receptor-mediated G-protein activation following chronic administration of DAMGO, further indicating a critical role of the NMDA receptor/PKC pathway in the cellular mechanisms of ^-opioid tolerance (74).

It is conceivable that NO may participate in the modulation of the |-opioid-activated G-protein-coupled K+ channel within the same cell by activating various cGMP-associated protein kinases (75). NO may also diffuse out of the neuron that produces it and influence the presynaptic glutamate/aspar-tate release (76), resulting in a positive feedback. Such NO actions may then counteract presynaptic inhibitory effects of opioids on neurotransmitter release, further diminishing the analgesic effects of opioids. In addition, spinal cord endogenous opioids and adrenergic agonists could be released via the action of extracellular NO (77) and their role in mechanisms of |-opioid tolerance remains to be determined. Further, the downstream activation of intracellular pathways such as MAPKs may result in translational and posttranscriptional modifications leading to prolonged neuroplastic changes. Perhaps more importantly, neurotoxic consequences may occur following prolonged and excessive activation of NMDA receptors and the associated intracellular pathways, causing potentially irreversible changes within the central nervous system (78-80).

3.3. Limitations of the Proposed Cellular Model

Although much effort has been made to formulate a cellular model that may incorporate the experimental data accumulated over the last decade (44,62,81-83), it becomes clear that the cellular and molecular mechanisms of NMDA receptor-mediated opioid tolerance and dependence are much more complex across subtypes of opioid receptors. It should be noted that although interactions between postsynaptic |-opioid and NMDA receptors are emphasized in this model, this consideration does not exclude a possible role of presynaptic glutamate receptors in the spinal cord mechanisms of | -opioid tolerance. Activation of presynaptic glutamate receptors (the metabotropic type) has been shown to enhance glutamate release from cerebrocortical nerve terminals (84). Indeed, metabotropic glutamate receptors (particularly type II/III) and their interactions with 8-opioid receptors have been suggested to be involved in the mechanisms of NMDA receptor-mediated | -opioid tolerance primarily for the initiation of PKC activation following opioid administration (81).

In addition, several studies utilizing a cellular model (in the locus coeruleus) of morphine tolerance have implicated the upregulation of the cAMP-PKA system in mechanisms of morphine tolerance (68,85). It will be of interest to determine whether similar PKA upregulation and their interactions with PKC occur at the spinal cord level of tolerant animals. Similarly, investigations on the role of NMDA receptors in supraspinal mechanisms of | -opioid tolerance would be expected to provide valuable information regarding the generality of the NMDA receptor-mediated cellular and intracellular mechanisms in |-opioid tolerance. Finally, the involvement of NMDA receptors in mechanisms of 8-and K-opioid tolerance remains to be elucidated.

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