Prolonged reversal of morphine tolerance with no reversal of dependence by protein kinase C inhibitors

doi:10.1016/S0006-8993(02)03394-2

Brain Research

Volume 958, Issue 1, 20 December 2002, Pages 28-35

https://doi.org/10.1016/S0006-8993(02)03394-2 Get rights and content

Abstract

The phosphatidylinositol (PI) cascade plays a pivotal role in mediating behavioral tolerance to the antinociceptive effects of morphine. Earlier we reported that antinociceptive tolerance was completely reversed 30 min after the administration of inhibitors of each step in the PI cascade. The aim of this study was to determine whether injection of a single dose of protein kinase C (PKC) inhibitor would elicit a prolonged reversal of morphine tolerance for up to 24 h. Three days after implantation of placebo- or 75-mg morphine pellets, mice received intracerebroventricular (i.c.v.) injections of vehicle or PKC inhibitor drug. Morphine challenge doses were then administered 4, 8 and 24 h later to test for tolerance reversal. In non-tolerant mice, Gö-7874 and sangivamycin had no effect on the potency of morphine. However, Gö-7874 and sangivamycin significantly reversed morphine tolerance at 4, 8 and 24 h. In addition, the role of PKC in morphine physical dependence was determined. Gö-7874 and sangivamycin by themselves did not precipitate spontaneous morphine withdrawal. Therefore, experiments were conducted to determine whether the PKC inhibitors would block naloxone-precipitated withdrawal. However, neither a 30-min nor a 24-h pretreatment with Gö-7874 or sangivamycin blocked naloxone withdrawal. Our results along with other publications indicate that PKC is a pivotal kinase essential for maintaining animals in an opioid tolerant state. Finally, the use of persistent PKC inhibitors that lasted for 24 h demonstrated that the neuronal systems in these animals did not adapt by increasing the activity of other protein kinase cascades to re-establish morphine tolerance.

Introduction

The significance of phosphorylation events in acute and chronic opioid exposure has been investigated using primarily in vitro methodological approaches. The conclusion of most studies is that signal transduction events are changed in response to the chronic presence of an opioid. Behavioral measures have been increasingly used to implicate specific signal transduction events in mediating opioid tolerance and dependence. Bernstein and Welch [1] reported that an inhibitor of cyclic-AMP-dependent protein kinase (PKA) (KT-5720), but not the PKG inhibitor KT-5823, reversed antinociceptive tolerance in morphine pellet-implanted mice. Phospholipid signal transduction systems have also been implicated in opioid tolerance. We reported that inhibitors of phosphatidylcholine- and phosphatidylinositol-specific phospholipase C reversed morphine tolerance when injected 30 min before testing [22]. Furthermore, the inositol tris-phosphate (IP₃) receptor antagonist low molecular-weight heparin also reversed tolerance in the same study. Studies have also focused on the role of protein kinase C (PKC) in tolerance. For example, PKC inhibitors such as chelerythrine chloride, H7 and calphostin C were able to prevent or reverse acute antinociceptive tolerance to mu- and delta-opioid agonists [2], [7], [15], [17]. In other studies, tolerance following chronic opioid administration was prevented by concomitantly infusing PKC inhibitor i.c.v. or spinally at the time opioids were administered [8], [13], [18]. Finally, physical dependence could be prevented if the PKC inhibitors were infused when the opioids were administered [7], [23].

Based on these previous studies, experiments were conducted to determine whether a single injection of PKC inhibitor would elicit a prolonged reversal of morphine tolerance. Our results demonstrate that tolerance was still reversed 24 h after the injection of either Gö-7874 or sangivamycin. The hypothesis was also tested that the PKC inhibitors would reverse morphine dependence by blocking naloxone-precipitated withdrawal. However, neither Gö-7874 nor sangivamycin blocked naloxone withdrawal.

Section snippets

Methods of handling mice

Male Swiss Webster mice (Harlan Laboratories, Indianapolis, IN) weighing 25–30 g were housed five to a cage in animal care quarters and maintained at 22±2 °C on a 12-h light–dark cycle. Food and water were available ad libitum. The mice were brought to a test room (22±2 °C, 12-h light–dark cycle), housed six to a cage, marked for identification and allowed 18 h to recover from transport and handling. The Institutional Animal Care and Use Committee (IACUC) at Virginia Commonwealth University

Duration of the reversal of morphine tolerance

In an earlier study, morphine tolerance was reversed when Gö-7874 (1.0 nmol) and sangivamycin (8.1 nmol) were acutely administered 30 min before the tail-flick test [22]. These were the minimally effective doses needed to reverse tolerance, and had no effect on non-tolerant mice. In fact, other non-tolerant mice were injected i.c.v. with 4-fold higher doses of Gö-7874 (4.0 nmol) and sangivamycin (32.4 nmol). The animals exhibited no significant antinociception (<5% MPE) or spontaneous activity,

Role of PKC in non-tolerant mice

In the neurons that mediate opioid antinociception in non-tolerant mice, PKC appears to be quiescent since the PKC inhibitors Gö-7874 and sangivamycin failed to affect the potency of morphine. Furthermore, even 4-fold higher doses had no effect on antinociception, spontaneous activity, and general observations of behavior or toxicity. Others have previously reported a similar lack of effect of other PKC inhibitors to affect antinociception in non-tolerant animals [2], [7], [15], [17], [22].

Acknowledgements

Thus research was funded by the National Institute on Drug Abuse grant DA-01647-25; R.J. was supported by T32-DA-07027.

References (25)

M.A. Bernstein et al.
Effects of spinal versus supraspinal administration of cyclic nucleotide-dependent protein kinase inhibitors on morphine tolerance in mice
Drug Alcohol Depend.
(1997)
M.E. Fundytus et al.
Chronic inhibition of intracellular calcium release of protein kinase C activation significantly reduces the development of morphine dependence
Eur. J. Pharmacol.
(1996)
V. Granados-Soto et al.
Spinal PKC activity and expression: role in tolerance produced by continuous spinal morphine infusion
Pain
(2000)
C.R. Loomis et al.
Sangivamycin, a nucleoside analogue, is a potent inhibitor of protein kinase C
J. Biol. Chem.
(1988)
J. Mao et al.
Increases in protein kinase C gamma immunoreactivity in the spinal cord of rats associated with tolerance to the analgesic effects of morphine
Brain Res.
(1995)
G. Martiny-Baron et al.
Selective inhibition of protein kinase C isozymes by the indolocarbazole Go 6976
J. Biol. Chem.
(1993)
M. Narita et al.
Inhibition of protein kinase C, but not protein kinase A, blocks the development of acute antinociceptive tolerance to an intrathecally administered mu-opioid receptor agonist in the mouse
Eur. J. Pharmacol.
(1995)
M. Narita et al.
A protein kinase inhibitor, H-7, inhibits the development of tolerance to opioid antinociception
Eur. J. Pharmacol.
(1994)
M. Narita et al.
Influence of chronic morphine treatment on protein kinase C activity: comparison with butorphanol and implication for opioid tolerance
Brain Res.
(1994)
M. Ohsawa et al.
Possible involvement of protein kinase C in the attenuation of [d-Ala², NMePhe⁴, Gly-ol⁵]enkephalin-induced antinociception in diabetic mice
Eur. J. Pharmacol.
(1997)

S. Tokuyama et al.

Possible involvement of protein kinases in physical dependence on opioids: studies using protein kinase inhibitors, H-7 and H-8

Eur. J. Pharmacol.

(1995)

E.J. Bilsky et al.

Effects of naloxone and d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen Thr-NH2 and the protein kinase inhibitors H7 and H8 on acute morphine dependence and antinociceptive tolerance

J. Pharmacol. Exp. Ther.

(1996)

Cited by (44)

Comparison of morphine, oxycodone and the biased MOR agonist SR-17018 for tolerance and efficacy in mouse models of pain
2021, Neuropharmacology
The mu opioid receptor-selective agonist, SR-17018, preferentially activates GTPγS binding over βarrestin2 recruitment in cellular assays, thereby demonstrating signaling bias. In mice, SR-17018 stimulates GTPγS binding in brainstem and produces antinociception with potencies similar to morphine. However, it produces much less respiratory suppression and mice do not develop antinociceptive tolerance in the hot plate assay upon repeated dosing. Herein we evaluate the effects of acute and repeated dosing of SR-17018, oxycodone and morphine in additional models of pain-related behaviors. In the mouse warm water tail immersion assay, an assessment of spinal reflex to thermal nociception, repeated administration of SR-17018 produces tolerance as does morphine and oxycodone. SR-17018 retains efficacy in a formalin-induced inflammatory pain model upon repeated dosing, while oxycodone does not. In a chemotherapeutic-induced neuropathy pain model SR-17018 is more potent and efficacious than morphine or oxycodone, moreover, this efficacy is retained upon repeated dosing of SR-17018. These findings demonstrate that, with the exception of the tail flick test, SR-17018 retains efficacy upon chronic treatment across several pain models.
Inflammatory mediators of opioid tolerance: Implications for dependency and addiction
2019, Peptides
Each year, over 50 million Americans suffer from persistent pain, including debilitating headaches, joint pain, and severe back pain. Although morphine is amongst the most effective analgesics available for the management of severe pain, prolonged morphine treatment results in decreased analgesic efficacy (i.e., tolerance). Despite significant headway in the field, the mechanisms underlying the development of morphine tolerance are not well understood. The midbrain ventrolateral periaqueductal gray (vlPAG) is a primary neural substrate for the analgesic effects of morphine, as well as for the development of morphine tolerance. A growing body of literature indicates that activated glia (i.e., microglia and astrocytes) facilitate pain transmission and oppose morphine analgesia, making these cells important potential targets in the treatment of chronic pain. Morphine affects glia by binding to the innate immune receptor toll-like receptor 4 (TLR4), leading to the release of proinflammatory cytokines and opposition of morphine analgesia. Despite the established role of the vlPAG as an integral locus for the development of morphine tolerance, most studies have examined the role of glia activation within the spinal cord. Additionally, the role of TLR4 in the development of tolerance has not been elucidated. This review attempts to summarize what is known regarding the role of vlPAG glia and TLR4 in the development of morphine tolerance. These data, together, provide information about the mechanism by which central nervous system glia regulate morphine tolerance, and identify a potential therapeutic target for the enhancement of analgesic efficacy in the clinical treatment of chronic pain.
Activation of P2X<inf>7</inf> receptors in the midbrain periaqueductal gray of rats facilitates morphine tolerance
2015, Pharmacology Biochemistry and Behavior
Opiates such as morphine exhibit analgesic effect in various pain models, but repeated and chronic morphine administration may develop resistance to antinociception. The purinergic signaling system is involved in the mechanisms of pain modulation and morphine tolerance. This study aimed to determine whether the P2X₇ receptor in the ventrolateral midbrain periaqueductal gray (vlPAG) is involved in morphine tolerance. Development of tolerance to the antinociceptive effect of morphine was induced in normal adult male Sprague–Dawley (SD) rats through subcutaneous injection of morphine (10 mg/kg). The analgesic effect of morphine (5 mg/kg, i.p.) was assessed by measuring mechanical withdrawal thresholds (MWTs) in rats with an electronic von Frey anesthesiometer. The expression levels and distribution of the P2X₇ receptor in the vlPAG was evaluated through Western blot analysis and immunohistochemistry. The acute effects of intra-vlPAG injection of the selective P2X₇ receptor agonist Bz-ATP, the selective P2X₇ receptor antagonist A-740003, or antisense oligodeoxynucleotide (AS ODN) targeting the P2X₇ receptor on morphine-treated rats were also observed. Results demonstrated that repeated morphine administration decreased the mechanical pain thresholds. By contrast, the expression of the P2X₇ receptor protein was up-regulated in the vlPAG in morphine tolerant rats. The percent changes in MWT were markedly but only transiently attenuated by intra-vlPAG injection of Bz-ATP (9 nmol/0.3 μL) but elevated by A-740003 at doses of 10 and 100 nmol/0.3 μL. AS ODN (15 nmol/0.3 μL) against the P2X₇ receptor reduced the development of chronic morphine tolerance in rats. These results suggest that the development of antinociceptive tolerance to morphine is partially mediated by activating the vlPAG P2X₇ receptors. The present data also suggest that the P2X₇ receptors may be a therapeutic target for improving the analgesic effect of morphine in treatments of pain when morphine tolerance occurs.
Role of FK506 binding protein 12 in morphine-induced μ-opioid receptor internalization and desensitization
2014, Neuroscience Letters
Citation Excerpt :
Morphine, which is widely used in the treatment of pain, has high potential to develop antinociceptive tolerance and physical dependence. Unlike other opioids such as [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) and etorphine which mainly induce receptor phosphorylation, morphine induces less receptor phosphorylation and internalization, but mainly activates protein kinase C (PKC) and subsequent signaling molecules, which contributes to its analgesic tolerance [3–6]. On the other hand, it has been widely demonstrated that phosphorylation-induced receptor internalization facilitates receptor recycling and subsequent reactivation at cell surface, and thus reduces the development of opioid tolerance [7–9].
Agonist-activated μ-opioid receptor (OPRM1) undergoes robust receptor phosphorylation by G protein-coupled receptor kinases and subsequent β-arrestin recruitment, triggering receptor internalization and desensitization. Morphine, a widely prescribed opioid, induces receptor phosphorylation inefficiently. Previously we reported that FK506 binding protein 12 (FKBP12) specifically interacts with OPRM1 and such interaction attenuates receptor phosphorylation and facilitates morphine-induced recruitment and activation of protein kinase C. In the current study, we demonstrated that the association of FKBP12 with OPRM1 also affects morphine-induced receptor internalization and G protein-dependent adenylyl cyclase desensitization. Morphine induced faster receptor internalization and adenylyl cyclase desensitization in cells expressing OPRM1 with Pro³⁵³ mutated to Ala (OPRM1P353A), which does not interact with FKBP12, or in the presence of FK506 which dissociates the receptor–FKBP12 interaction. Furthermore, knockdown of cellular FKBP12 level by siRNA accelerated morphine-induced receptor internalization and adenylyl cyclase desensitization. Our study further demonstrated that peptidyl prolyl cis–trans isomerase activity of FKBP12 probably plays a role in inhibition of receptor phosphorylation. In the view that internalized receptor recycles and thus counteracts the development of analgesic tolerance, receptor's association with FKBP12 could also contribute to the development of morphine tolerance through modulation of receptor trafficking.
Persistent peripheral inflammation attenuates morphine-induced periaqueductal gray glial cell activation and analgesic tolerance in the male rat
2013, Journal of Pain
Morphine is among the most prevalent analgesics prescribed for chronic pain. However, prolonged morphine treatment results in the development of analgesic tolerance. An abundance of evidence has accumulated indicating that central nervous system glial cell activity facilitates pain transmission and opposes morphine analgesia. While the midbrain ventrolateral periaqueductal gray (vlPAG) is an important neural substrate mediating pain modulation and the development of morphine tolerance, no studies have directly assessed the role of PAG glia. Here we test the hypothesis that morphine-induced increases in vlPAG glial cell activity contribute to the development of morphine tolerance. As morphine is primarily consumed for the alleviation of severe pain, the influence of persistent inflammatory pain was also assessed. Administration of morphine, in the absence of persistent inflammatory pain, resulted in the rapid development of morphine tolerance and was accompanied by a significant increase in vlPAG glial activation. In contrast, persistent inflammatory hyperalgesia, induced by intraplantar administration of complete Freund’s adjuvant (CFA), significantly attenuated the development of morphine tolerance. No significant differences were noted in vlPAG glial cell activation for CFA-treated animals versus controls. These results indicate that vlPAG glia are modulated by a persistent pain state, and implicate vlPAG glial cells as possible regulators of morphine tolerance.
The development of morphine tolerance represents a significant impediment to its use in the management of chronic pain. We report that morphine tolerance is accompanied by increased glial cell activation within the vlPAG, and that the presence of a persistent pain state prevented vlPAG glial activation and attenuated morphine tolerance.
Agonist-dependent μ-opioid receptor signaling can lead to heterologous desensitization
2010, Cellular Signalling
Desensitization of the µ-opioid receptor (MOR) has been implicated as an important regulatory process in the development of tolerance to opiates. Monitoring the release of intracellular Ca²⁺ ([Ca²⁺]_i), we reported that [D-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin (DAMGO)-induced receptor desensitization requires receptor phosphorylation and recruitment of β-arrestins (βArrs), while morphine-induced receptor desensitization does not. In current studies, we established that morphine-induced MOR desensitization is protein kinase C (PKC)-dependent. By using RNA interference techniques and subtype specific inhibitors, PKCε was shown to be the PKC subtype activated by morphine and the subtype responsible for morphine-induced desensitization. In contrast, DAMGO did not increase PKCε activity and DAMGO-induced MOR desensitization was not affected by modulating PKCε activity. Among the various proteins within the receptor signaling complex, Gαi2 was phosphorylated by morphine-activated PKCε. Moreover, mutating three putative PKC phosphorylation sites, Ser⁴⁴, Ser¹⁴⁴ and Ser³⁰² on Gαi2 to Ala attenuated morphine-induced, but not DAMGO-induced desensitization. In addition, pretreatment with morphine desensitized cannabinoid receptor CB1 agonist WIN 55212-2-induced [Ca²⁺]_i release, and this desensitization could be reversed by pretreating the cells with PKCε inhibitor or overexpressing Gαi2 with the putative PKC phosphorylation sites mutated. Thus, depending on the agonist, activation of MOR could lead to heterologous desensitization and probable crosstalk between MOR and other Gαi-coupled receptors, such as the CB1.

View all citing articles on Scopus

View full text

Research reportProlonged reversal of morphine tolerance with no reversal of dependence by protein kinase C inhibitors

Abstract

Introduction

Section snippets

Methods of handling mice

Duration of the reversal of morphine tolerance

Role of PKC in non-tolerant mice

Acknowledgements

Drug Alcohol Depend.

Eur. J. Pharmacol.

Pain

J. Biol. Chem.

Brain Res.

J. Biol. Chem.

Eur. J. Pharmacol.

Eur. J. Pharmacol.

Brain Res.

Eur. J. Pharmacol.

Eur. J. Pharmacol.

Effects of naloxone and d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen Thr-NH2 and the protein kinase inhibitors H7 and H8 on acute morphine dependence and antinociceptive tolerance

J. Pharmacol. Exp. Ther.

Research report
Prolonged reversal of morphine tolerance with no reversal of dependence by protein kinase C inhibitors