The expression of a high level of morphine antinociceptive tolerance in mice involves both PKC and PKA

doi:10.1016/S0006-8993(03)03170-6

Brain Research

Volume 985, Issue 1, 19 September 2003, Pages 78-88

https://doi.org/10.1016/S0006-8993(03)03170-6 Get rights and content

Abstract

We have previously reported that intracerebroventricular (i.c.v.) injection of either a PKC or PKA inhibitor completely reversed the expression of 5- to 8-fold morphine antinociceptive tolerance. We developed a model of 45-fold morphine tolerance that included a 75-mg morphine pellet and twice daily morphine injections. PKC inhibitor doses of bisindolylmaleimide I and Gö-7874 that completely reversed 8-fold tolerance only partly reversed the 45-fold level of antinociceptive tolerance. A component of tolerance was resistant to PKC inhibition, since even higher inhibitor doses failed to further reverse the high level of morphine tolerance. In addition, the 45-fold tolerance was only partly reversed by the PKA inhibitor KT-5720 at a dose previously cited by others to reverse 5-fold tolerance. Another PKA inhibitor 4-cyano-3-methylisoquinoline only partly reversed the morphine tolerance as well. In other experiments PKC and PKA inhibitors were co-administered together to determine their effectiveness for completely reversing the 45-fold level of morphine tolerance. Co-administering either bisindolylmaleimide I with KT-5720, or Gö-7874 with KT-5720, completely reversed the high level of tolerance. The high level of morphine tolerance was also completely reversed by co-administering Gö-7874 with 4-cyano-3-methylisoquinoline. Thus, high levels of morphine tolerance may reflect increases in protein phosphorylation by the terminal kinases of both the adenylyl cyclase and phosphatidylinositol cascades in brain and spinal cord areas critical to the expression of antinociception.

Introduction

The cellular mechanisms responsible for antinociceptive tolerance to opioids have been the subject of numerous research studies for several decades. Low intrinsic efficacy agonists such as morphine appear to produce tolerance in the absence of affecting mu-opioid receptor density and mRNA levels [5], [7], [48]. Instead, morphine may mediate tolerance through downstream Ca²⁺-dependent/independent-PKC and cAMP-PKA pathways, as opposed to high intrinsic efficacy agonists like etorphine that induce mu-opioid receptor desensitization and downregulation through G-protein-coupled receptor kinases and β-arrestin [22], [52]. In support of this, Bernstein and Welch [2] reported the PKA inhibitor KT-5720, but not the PKG inhibitor KT-5823, reversed antinociceptive tolerance in morphine pellet-implanted mice. Recently, it was reported that i.c.v. injections of the anti-sense oligodeoxynucleuotide to PKA mRNA blocked the tolerance induced to morphine [33]. 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 [36]. 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 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 [3], [13], [29], [30]. 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 [15], [28]. These PKC inhibitors were also effective in reversing opioid antinociceptive tolerance [15], [35], [36]. Recent studies demonstrated that spinal infusion of anti-sense oligodeoxynucleuotide to PKCalpha mRNA blocked the tolerance to spinally infused morphine [19].

All of these studies utilized models that induced a moderate 5- to 8-fold level of opioid tolerance. For this study, we developed a model of 45-fold tolerance to determine the effectiveness of PKC and PKA inhibitors to reverse a high level of morphine tolerance. We hypothesized that it would be necessary to co-administer both PKC and PKA inhibitors to completely reverse high levels of tolerance, since both PKC and PKA contribute to tolerance expression. Our results demonstrate that the PKC or PKA inhibitors administered alone only partly reversed the high level of morphine tolerance, but that co-administration of both classes of inhibitors completely reversed the tolerance.

Section snippets

Methods of handling mice

Male Swiss Webster mice (Harlan Laboratories, Indianapolis, IN) weighing 25–30 g were housed six to a cage in animal care quarters 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), marked for identification and allowed 24-h to recover from transport and handling. The Institutional Animal Care and Use Committee (IACUC) at the Virginia Commonwealth University School of Medicine approved

Influence of PKC and PKA inhibitors alone on high morphine tolerance

The antinociceptive tolerance observed following the implantation of a 75-mg morphine pellet and daily supplemental morphine administration resulted in a 45-fold reduction in the potency of morphine. In previous studies, implantation of the 75-mg morphine pellet alone yielded approximately a 5–8-fold level of tolerance [2], [35], [36]. Seventy-two hours after pellet implantation, the mice were tested with vehicle injections i.c.v. or the PKC inhibitor bisindolylmaleimide I. The 11.1 nmol dose

Reversal of moderate versus high morphine tolerance

The efficacy of PKC and PKA inhibitors to reverse morphine tolerance was determined utilizing animal models that express different degrees of tolerance. Each of these inhibitors were completely effective in reversing the 5–8-fold rightward shifts in the morphine dose–response curves [2], [35], [36]. Yet, when the degree of tolerance was increased to 45-fold, a threshold was crossed whereby tolerance reversal by either the PKC or PKA inhibitors alone was impossible. During high level opioid

Acknowledgements

This research was funded by the National Institute on Drug Abuse grants DA-01647-26 and K05-DA-00480; R.J. was supported by T32-DA-07027.

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Orexin neuropeptides are implicated in the expression of morphine dependence. Locus coeruleus (LC) nucleus is an important brain area involving in the development of withdrawal signs of morphine and contains high expression of orexin type 1 receptors (OX1Rs). Despite extensive considerations, effects of immediate inhibition of OX1Rs by a single dose administration of SB-334867 prior to the naloxone-induced activation of LC neurons remains unknown. Therefore, we examined the direct effects of OX1Rs acute blockade on the neuronal activity of the morphine-dependent rats which underwent naloxone administration. Adult male rats underwent subcutaneous administration of 10 mg/kg morphine (two times/day) for a ten-day period. On the last day of experiment, intra-cerebroventricular administration of 10 μg/μl antagonist of OX1Rs, SB-334867, was performed just before intra-peritoneal injection of 2 mg/kg naloxone. Thereafter, in vivo extracellular single unit recording was employed to evaluate the electrical activity of LC neuronal cells. The outcomes demonstrated that morphine tolerance developed following ten-day of injection. Then, naloxone administration causes hyperactivity of LC neuronal cells, whereas a single dose administration of SB-334867 prior to naloxone prevented the enhanced activity of neurons upon morphine withdrawal. Our findings indicate that increased response of LC neuronal cells to applied naloxone could be prevented by the acute inhibition of the OX1Rs just before the naloxone treatment.
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Continuous use of crack induces hyperalgesia which is related to drug tolerance. Despite cumulative evidence based on the growth rate of crack abuse, no serious study has been focused on the mechanisms of crack-induced hyperalgesia. This study aimed to elucidate whether extracellular signal-regulated kinases (Erk1/2)/β-arrestin pathways are involved in the crack-induced hyperalgesia. Fifty adult male Wistar rats were randomly divided into five groups: normal saline (NS), crack (0.9 mg/kg/day), heroin (1 mg/kg/day), crack + barbadin (100 μM), and heroin + barbadin groups, which received their intraperitoneal (i.p) treatments for four weeks. The thermal sensitivity was assessed using the hot-plate test. Moreover, phosphorylation of the Erk1/2 and JNK, as well as expression of protein kinase C-alpha (PKC-α), Mu-receptor (MOR), and β-arrestin 2 were determined in the whole lysate and membrane fraction using immunoblotting assay in the periaqueductal gray (PAG) area. The results demonstrated that chronic administration of crack and heroin significantly decreased hind-paw withdrawal latency compared to the NS group. Furthermore, crack as well as heroin administration increased phosphorylated Erk1/2 and JNK in the PAG. In addition, membrane β-arrestin 2 and PKC-α were significantly increased in the crack and heroin-received groups, while membrane MOR expression was decreased in the PAG. Nevertheless, co-administration of barbadin, an inhibitor of β-arrestin, and crack or heroin reversed all these changes. Our findings may partially confirm the role of β-arrestin 2 and PKC rearrangements, Erk1/2 and JNK phosphorylation in crack-induced hyperalgesia and provide potential therapeutic targets to attenuate crack-induced hyperalgesia.
The role of orexin type-1 receptors in the development of morphine tolerance in locus coeruleus neurons: An electrophysiological perspective
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Long-term exposure to opioid agonists results in tolerance to their analgesic effects, so the effectiveness of opioid agonists in the management of pain becomes limited. The locus coeruleus (LC) nucleus has been involved in the development of tolerance to opiates. Orexin type-1 receptors (OX1Rs) are highly expressed in LC nucleus. Orexin plays a noteworthy role in the occurrence of morphine tolerance. The purpose of the present study is to investigate the role of orexin type-1 receptors in the development of morphine tolerance in LC neurons.
In this study, adult male Wistar rats weighing 250–300 g were utilized. Induction of morphine tolerance was obtained by single injection of morphine per day for 6 successive days. An orexin type-1 receptor antagonist (SB-334867) was injected into the lateral ventricle instantly prior to morphine injection. On day 7, the effect of morphine on the electrical activity of LC neurons was studied using in vivo extracellular single unit recording.
The results demonstrate that morphine injection for 6 consecutive days led to the development of morphine-induced tolerance in LC neurons. In other words, there was a significant decrease in LC neuronal responsiveness to morphine injection. Inhibitory responses of LC neurons to intraperitoneally applied morphine can be observed with the treatment of the SB-334867 prior to morphine injection.
This study showed that OX1R blockade by SB-334867 prevents the development of morphine tolerance in LC neurons. We hope that further studies will lead to considerable progress in understanding the molecular adaptations that contribute to morphine tolerance.
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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.
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We previously demonstrated that intracerebroventricular (i.c.v.) administration of protein kinase C (PKC) or protein kinase A (PKA) inhibitors reversed morphine antinociceptive tolerance in 3-day morphine-pelleted mice. The present study aimed at evaluating whether pre-treating mice with a PKC or PKA inhibitor prior to pellet implantation would prevent the development of morphine tolerance and physical dependence. Antinociception was assessed using the warm-water tail immersion test and physical dependence was evaluated by quantifying/scoring naloxone-precipitated withdrawal signs. While drug-naïve mice pelleted with a 75 mg morphine pellet for 3 days developed a 5.8-fold tolerance to morphine antinociception, mice pre-treated i.c.v. with the PKC inhibitors bisindolylmaleimide I, Go-7874 or Go-6976, or with the myristoylated PKA inhibitor, PKI-(14-22)-amide failed to develop any tolerance to morphine antinociception. Experiments were also conducted to determine whether morphine-pelleted mice were physically dependent when pre-treated with PKC or PKA inhibitors. The same inhibitor doses that prevented morphine tolerance were evaluated in other mice injected s.c. with naloxone and tested for precipitated withdrawal. The pre-treatment with PKC or PKA inhibitors failed to attenuate or block the signs of morphine withdrawal including jumping, wet-dog shakes, rearing, forepaw tremor, increased locomotion, grooming, diarrhea, tachypnea and ptosis. These data suggest that elevations in the activity of PKC and PKA in the brain are critical to the development of morphine tolerance. However, it appears that tolerance can be dissociated from physical dependence, indicating a role for PKC and PKA to affect antinociception but not those signs mediated through the complex physiological processes of withdrawal.
Evidence for an important role of protein phosphatases in the mechanism of morphine tolerance
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Citation Excerpt :
Shen et al. (2000) demonstrated that i.c.v. administration of the anti-sense oligodeoxynucleuotide to PKA mRNA blocked the antinociceptive tolerance to morphine in mice (Shen et al., 2000). We previously reported that i.c.v. administration of the inhibitors of PKA, PKI-(14-22)-amide and 4-cyano-3-methylisoquinoline and KT-5720 completely reversed 5- to 8-fold and only partly 45-fold morphine antinociceptive tolerance (Smith et al., 2003; Javed et al., 2004; Dalton et al., 2005). We also demonstrated that PKA inhibition reinstated antinociception, hypothermia and Straub tail in mice implanted with morphine pellets 72-h earlier (Smith et al., 2006).
Acute morphine antinociception has been shown to be blocked by very low picogram doses of okadaic acid indicating that inhibition of protein phosphatase PP2A allows for increases in phosphorylation to inhibit antinociception. Comparative studies in morphine tolerant animals have not been reported. In the present study, we showed a significant increase in the total phosphatase activity in the periaqueductal gray matter (PAG) from morphine-pelleted versus placebo-pelleted mice, 72-h after pellet implantation. This supports our hypothesis that phosphatase activity is increased in tolerance as a compensatory mechanism for the increase in kinase activity during the development of tolerance. We also demonstrated that i.c.v. administration of the phosphatase inhibitor okadaic acid (3 pmol/mouse; a dose tested to be inert in placebo-pelleted mice) enhanced the level of morphine antinociceptive tolerance assessed by the tail immersion test, 72-h following pellet implantation. This was supported by the fact that the same treatment with okadaic acid blocked the increase in phosphatase activity in PAG of morphine tolerant mice indicating that selective inhibition of PP2A contributes to enhanced levels of morphine tolerance. We have previously reported that PKC or PKA inhibitors reversed morphine antinociceptive tolerance in mice. The current study shows that i.c.v. administration of the PKC inhibitors bisindolylmaleimide I or Go6976 reversed the enhanced level of morphine tolerance induced by okadaic acid treatment to the same level of tolerance observed in non-okadaic acid-treated tolerant mice. However, the PKA inhibitor PKI-(14-22)-amide only partially reversed the enhancement of morphine tolerance induced by okadaic acid. Our data suggest an important role for the balance between kinases and phosphatases in modulating tolerance levels. Further studies will be directed towards a better understanding of the role of different phosphatase isoforms in morphine tolerance.

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Research reportThe expression of a high level of morphine antinociceptive tolerance in mice involves both PKC and PKA

Abstract

Introduction

Section snippets

Methods of handling mice

Influence of PKC and PKA inhibitors alone on high morphine tolerance

Reversal of moderate versus high morphine tolerance

Acknowledgements

J. Biol. Chem.

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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
The expression of a high level of morphine antinociceptive tolerance in mice involves both PKC and PKA