Share this article on:

PICK1 Regulates the Expression and Trafficking of AMPA Receptors in Remifentanil-Induced Hyperalgesia

Wang, Zhifen MD; Yuan, Yuan PhD; Xie, Keliang PhD; Tang, Xiaohong MD; Zhang, Linlin MD; Ao, Jiying MD; Li, Nan MD; Zhang, Yu MD; Guo, Suqian MD; Wang, Guolin MD

doi: 10.1213/ANE.0000000000001442
Pain and Analgesic Mechanisms: Original Laboratory Research Report

BACKGROUND: Remifentanil is used widely in clinical anesthesia because it induces more rapid and more common hyperalgesia than other opioid analgesics. Activation of N-methyl-D-aspartate (NMDA) receptors takes a pivotal part in remifentanil-induced hyperalgesia. Like NMDA receptors, the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are excitatory ion glutamate receptors in postsynaptic membrane, which are involved in the transmission of both acute and chronic pain. Protein interacting with C kinase 1 (PICK1) plays an important role in NMDA receptor-mediated internalization of glutamate receptor 2 (GluR2)-containing AMPARs and contributes to the induction and maintenance of inflammation-induced pain. This study aimed to test the hypothesis that PICK1 contributes to remifentanil-induced hyperalgesia by regulating AMPAR expression and trafficking in the spinal cord.

METHODS: Using a rat model of remifentanil-induced hyperalgesia by intravenous infusion of remifentanil, we first measured changes in mechanical and thermal hyperalgesia at 24 hours before remifentanil infusion and 2, 6, 24, and 48 hours after infusion. PICK1 mRNA and protein expression and AMPAR subunit expression and trafficking in the spinal cord were then detected by reverse transcription–qualitative polymerase chain reaction, immunohistochemistry, and Western blot. In addition, we knocked down PICK1 expression by intrathecal administration of PICK1 antisense oligodeoxynucleotide to investigate the effects of PICK1 deficiency on remifentanil-induced hyperalgesia and the expression and trafficking of AMPARs.

RESULTS: A significant time–group interaction was found for nociceptive thresholds (paw withdrawal threshold and paw withdrawal latency; all P < .0001). Remifentanil infusion induced distinct hyperalgesia at different time points (P < .0001), which was partly reversed by PICK1 knockdown (P < .007). Besides, remifentanil infusion increased the expression of PICK1 mRNA and protein (P < .0001) and the membrane GluR1 and GluR2 internalization in spinal dorsal horn neurons (P < .0011). More importantly, PICK1 deficiency could attenuate remifentanil-induced GluR2 internalization in the spinal cord dorsal horn (P < .01) but had no effect on remifentanil-induced membrane GluR1 expression (P ≥ .985).

CONCLUSIONS: These results indicate that PICK1 deficiency might reverse remifentanil-induced hyperalgesia through regulating GluR2-containing AMPAR expression and trafficking in the spinal cord dorsal horn.

From the Department of Anesthesiology, Tianjin Medical University General Hospital, Tianjin Research Institute of Anesthesiology, Tianjin, China.

Accepted for publication May 6, 2016.

Funding: This work was supported by grants from the National Natural Science Foundation of China (grant numbers 81300960, 81371245, 81571077), Science and Technology Foundation of Tianjin Health Bureau (2013KZ124), and Tianjin applied basic and frontier technology research program (14JCQNJC12800).

The authors declare no conflicts of interest.

Zhifen Wang and Yuan Yuan contributed equally to this work, and are cofirst authors.

Reprints will not be available from the authors.

Address correspondence to Guolin Wang, MD, Department of Anesthesiology, Tianjin Medical University General Hospital, Tianjin Research Institute of Anesthesiology, Tianjin 300052, China. Address e-mail to 15602176894@163.com.

Remifentanil is an ultrashort-acting μ-opioid receptor agonist. Because of its reliability, rapid onset, and predictable rapid recovery profile, it has been used widely in clinical practice with little risk of delayed postoperative recovery or respiratory depression.1 However, patients receiving remifentanil to control their pain somewhat paradoxically may become more sensitive to pain as a direct result of remifentanil-induced hyperalgesia, which inevitably aggravates patients’ suffering.2–5

Recently, it is accepted widely that the activation and trafficking of N-methyl-D-aspartate (NMDA) receptors play an important role in remifentanil-induced hyperalgesia. It is suggested that activated NMDA receptors cause cellular calcium influx via promoting the interaction between superoxide and nitric oxide, leading to peroxynitrite (PN) formation. PN activates divalent metal-ion transporter-1 without iron-responsive element (DMT1(−)IRE) and manganese superoxide dismutase (MnSOD), leading to the development of hyperalgesia.6,7 It is indicated that the activation of glycogen synthase kinase (GSK)-3β contributes to remifentanil-induced postoperative hyperalgesia via regulating the function and trafficking of NMDA receptors in the spinal cord8,9; however, the mechanisms of remifentanil-induced hyperalgesia still need to be clarified.

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are homo- or heterotetramers assembled from receptor subunits GluR1–4, whose expression and trafficking are thought to determine its functional properties.10,11 Because GluR1-containing AMPARs have Ca2+ permeability, whereas glutamate receptor 2 (GluR2)-containing AMPARs are impermeable to Ca2+, the increased expression and trafficking of GluR1 to postsynaptic membrane and internalization of GluR2 from the membrane all contribute to the increase of intracellular Ca2+, which may alter excitatory synaptic strength and involve a variety of forms of physiological processes in the central nervous system. Some literature suggested that the AMPARs modulate spinal synaptic plasticity and participate in the induction and development of pain.12–15 Acute morphine application could modify subcellular distribution of AMPAR subunits GluR1 and GluR2 in dendrites of neurons in the mouse.16 However, whether their expression and trafficking play a role in remifentanil-induced hyperalgesia still need to be clarified.

As a post-synaptic density 95/Drosophila discs large/zona-occludens 1 (PDZ) and Bin/amphiphysin/Rvs (BAR) domain-containing scaffolding protein, protein interacting with C kinase 1 (PICK1) expresses in the perinuclear region. In nerve cells, PICK1 locates in synapses,17 and it was reported to bind to numerous proteins, including protein kinase Cα (PKCα)18 and synaptic AMPAR subunit GluR2 in neurons.19,20 Via the PDZ domain, PICK1 is able to recruit intracellular PKCα to synaptic GluR2 and then lead to GluR2 phosphorylation at Ser880 and promote GluR2 internalization in brain neurons.21,22 Many reports showed that PICK1 contributes to the maintenance of inflammation-induced pain by binding to GluR2 and PKCα in the spinal cord.23,24 However, whether PICK1 contributes to remifentanil-induced hyperalgesia is unknown.

Therefore, we hypothesized whether PICK1 takes a part in remifentanil-induced hyperalgesia by regulating the expression and trafficking of AMPARs in the spinal cord. We used PICK1 antisense (AS) oligodeoxynucleotide (ODN) to knockdown the expression of PICK1 to verify the effect.

Back to Top | Article Outline

METHODS

Animals

Adult male Sprague-Dawley rats (240–260 g) were bought from the Laboratory Animal Center of the Military Medical Science Academy of the People’s Liberation Army and maintained in an animal experiment facility with controlled temperature and humidity in a 12-hour light/12-hour dark cycle. Water and food were available ad libitum. The experiments were approved by the Institutional Animal Care and Use Committee of Tianjin Medical University and adhered to the guidelines of the National Institutes of Health. Experimenters were blinded to treatment conditions in the experiments explained subsequently.

Back to Top | Article Outline

Knockdown of PICK1

Figure 1

Figure 1

To knock down the expression of PICK1, the PICK1 AS ODN was injected intrathecally into the spinal cord of rats. The PICK1 AS ODN (5′-C*T*G*GGCCCCTCCTCC*G*A*T-3′) and control missense (MS) ODN (5′-G*C*C*CGCTTCTCCCAG*C*T*G-3′) were designed and synthesized by SBS Genetech Co, Ltd, Beijing, China, where * represented phosphorothioate linkages. The administration of PICK1 AS ODN and MS ODN was conducted according to the previous study.24 In brief, to intrathecally inject the PICK1 AS ODN and MS ODN, an intrathecal polyethylene (PE-10) catheter was inserted into the subarachnoid space at the rostral level of the spinal cord lumbar enlargement segments through an incision at the atlanto-occipital membrane based on the method described previously.23,25 After 1-week recovery, rats that showed any neurologic impairment according to locomotor function testing were discarded. Then, rats in different groups were separately intrathecally injected with saline (10 μL; control), AS ODN (10 μg/10 μL), or MS ODN (10 μg/10 μL) once daily for 4 days. On the fifth until the eighth day, to verify the effect of PICK1 AS ODN, 4 rats were randomly euthanized from each group everyday and the spinal cord segment L4–L6 for Western blot was collected. The remaining rats were entered into the following experiments. An illustration of the experimental design is shown in Figure 1.

Back to Top | Article Outline

Locomotor Function Testing

The testing was conducted in rats as described previously.25 (1) Placing reflex: the rats’ hind limbs were held slightly lower than the forelimbs and the dorsal surfaces of the hind paws were brought into contact with the edge of a table, and whether the hind paws were placed on the table surface reflexively were recorded. (2) Grasping reflex: the rats were placed on a wire grid and recorded whether the hind paws grasped the wire on contact. (3) Righting reflex: the rats’ backs were placed on a flat surface, and whether it immediately reverts to the normal upright position was recorded. Every test was repeated for 6 trials, and the counts of each normal reflex contribute to scores for the testing. Besides, the rats’ general behaviors were also observed, like spontaneous activity.

Back to Top | Article Outline

Behavior Testing

The mechanical and thermal hyperalgesia were determined by paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) as described previously.9

To measure the PWT, each rat was placed in elevated wire chambers (20 × 20 × 20 cm) with a grid bottom and acclimated for 30 minutes before tests. The electronic von Frey filament (BSEVF3; Harvard Apparatus, Holliston, MA) was applied vertically to the sole of right hind paw, and the PWT was defined as the pressure (g) at which the rat flinched, shook, or licked its paw. The test was repeated 3 times at 5-minute intervals. The mean of the 3 trials was regarded as the PWT. To avert tissue damage, we used a cutoff threshold at 60 g.

To measure the PWL, each rat was placed on the hotplate (50°C) (YLS-6B; Zhenghua Biological Instrument Equipment Co, Ltd, Huaibei, Anhui, China) and acclimated for 30 minutes before tests. The PWL was defined as the time (s) when the rat appeared positive response (a clear paw withdrawal). Also, the test was repeated 3 times at 5-minute intervals. The mean of the 3 trials was regarded as the PWL. To avert tissue damage, a cutoff time at 30 seconds was used. PWL was measured 10 minutes after the determination of PWT in the same animal.

Back to Top | Article Outline

Reverse Transcription–Qualitative Polymerase Chain Reaction

To detect the expression of PICK1 mRNA, rats were euthanized and the L4–L6 spinal cord segments were removed. Total mRNA was extracted with TRIzol (Invitrogen, Carlsbad, CA). A cDNA Reverse Transcription Kit and SYBR Green PCR Master Mix (Roche, Mannheim, Germany) were used to perform reverse transcription–qualitative polymerase chain reaction (RT-qPCR). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. PICK1 gene expression was calculated with the delta-delta-Ct method.26 The primers of PICK1 and GAPDH were designed and synthesized by SBS Genetech Co, Ltd (PICK1 forward 5-CATTTGTGAAGTTTGCTGACG-3, reverse 5-TCTTGATGGTGAGGCGTGTA-3, GAPDH forward 5-AACAGCAACTCCCACTCTTC-3, reverse 5-CCTCTCTTGCTCAGTGTCCT-3).

Back to Top | Article Outline

Immunohistochemistry

To detect the expression of AMPAR subunits GluR1 and GluR2 in rats’ spinal cord dorsal horn, the L4–L6 spinal segments were removed and fixed in 4% paraformaldehyde at pH 7.4 for 24 hours and subsequently embedded in paraffin. Then, the samples were sliced into 7-μm-thick sections. After deparaffinization and rehydration separate in xylene and graded alcohol, 0.3% hydrogen peroxide was used for 10 minutes to block endogenic peroxidase activity. For epitope retrieval, the tissue slides were immersed in EDTA buffer at pH 8.0 and heated for 2.5 minutes on high power in a microwave oven. After blocking nonspecific reactions by 5% normal goat serum for 30 minutes, the slides were incubated with rabbit antibodies against rat GluR1 and GluR2 (1:200; Abcam, Cambridge, UK) overnight at 4°C. Before detecting with diaminobenzidine (DAB substrate kit; Boster Biological Technology, Ltd, Wuhan, China) and counterstaining with hematoxylin, the slides were incubated with polymerized horseradish peroxidase-labeled antirabbit IgG (1:300; Boster Biological Technology) for 45 minutes at 37°C. After being dehydrated in graded alcohols, cleared in xylene, the slides were coverslipped with gum. Negative control slides in the absence of primary antibody were included for each batch of staining. An Olympus DP73 microscope (Olympus, Tokyo, Japan) was used to acquire images. An observer who was blinded to the experimental design conducted the mean density quantification of GluR1- and GluR2-positive cells with the Image-Pro Plus (version 5.0, Media Cybernetics, Rockville, MD). We analyzed 3 consecutive sections per animal and 4 animals per group to calculate the mean and standard deviation (SD).

Back to Top | Article Outline

Western Blot

According to the experimental design, the rats were euthanized and their L4–L6 spinal cord segments collected. To extract the total protein, samples were homogenized in the buffer containing radio immunoprecipitation assay (RIPA) Lysis Buffer and protease inhibitor phenylmethylsulfonyl fluoride (PMSF) (Solarbio, Inc, Beijing, China) on the ice and centrifuged at 4°C (12,000 rpm, 10 minutes). The supernatant liquor was pipette as the total protein. To extract the membrane and cytoplasmic proteins, the Membrane Protein Extraction Kit (BioChain Institute, Inc, Hayward, CA) was used. According to the instructions, tissues were homogenized in the cytoplasm protein to remove buffer and spun at 18 000 g at 4°C for 20 minutes. Later, the cytoplasm protein (the supernatant) was removed and membrane protein extraction buffer was added to resuspended pellet. After spinning at 9000 g at 4°C for 20 minutes, the membrane proteins were in the supernatant. Before the proteins were transferred to nitrocellulose membrane, they were separated by 10% sodium dodecyl sulfonate–polyacrylamide gel electrophoresis (SDS–PAGE). Then, the membrane was blocked with nonfat milk (5%) for 1.5 hours on the shaker and subsequently incubated with rabbit antibodies against rat GluR1, GluR2, PICK1 (all 1:1000; Abcam) at 4°C overnight. The proteins were detected with horseradish peroxidase-conjugated goat antirabbit IgG antibodies (1:1000; Jackson ImmunoResearch, West Grove, PA) for 1 hour on the shaker at room temperature and visualized by enhanced chemiluminescence detection (Millipore, Billerica, MA) using a Bio-Rad GS-700 imaging system with software (Bio-Rad, Hercules, CA). β-actin was used as a loading control. Gene Tools Match software (Syngene, Cambridge, UK) was used to measure the protein band.

Back to Top | Article Outline

Statistical Analysis

SPSS 18.0 software (SPSS, Inc, Chicago, IL) was used to perform the statistical analysis. All data were expressed as mean ± SD. To determine the sample size, a power analysis was performed based on a pilot experiment. Under test conditions at 48 hours, the mean PWL was 16.0 seconds for control group and 8.1 seconds and 11.9 seconds for treatment groups (group NS + R, group AS + R). Thus, to find a difference of at least 30% (SD = 1.3 seconds) between the control and the treatment group with an alpha risk of 5% and a power (1-beta) of 90%, 4 animals are needed in each group. On the basis of this calculation, and to ensure accurate data, we have chosen to increase the sample size to 8 in behavior testing.

Using the Shapiro-Wilk test of normality, all residuals of each analysis of variance (ANOVA) model were normal distribution (All P ≥ .114). Homogeneity of variance was proved by the Levene test (All P ≥ .145). Behavior testing results were analyzed by 2-way repeated-measures ANOVA. Multiple comparisons in PWT and PWL were adjusted using the Tukey honestly significant difference test. The sphericity for the repeated-measures ANOVA had been tested before using the repeated-measures ANOVA (All P ≥ .201). To analyze the results of Western blot, RT-qPCR, and immunohistochemistry in different groups, the Student t tests and a 1-way ANOVA followed by the Tukey honestly significant difference test were used separately in experiment 1 and experiment 2. A value of P < .01 was considered as statistically significant.

Back to Top | Article Outline

RESULTS

Remifentanil-Induced Mechanical and Thermal Hyperalgesia

Figure 2

Figure 2

A significant time–group interaction was present for nociceptive thresholds (PWT and PWL) (all time × group P < .0001; Figure 2). Before the infusion (−24 hours), baseline numbers of nociceptive thresholds (PWT and PWL) were similar in group C and group R (all post hoc P ≥ .727; Figure 2). Compared with baseline, from 2 to 48 hours, PWT and PWL in group R were decreased significantly (all post hoc P < .0001; Figure 2). Compared with group C, remifentanil infusion did decrease the nociceptive thresholds (PWL and PWT) from 2 to 48 hours (all post hoc P < .0001; Figure 2).

Back to Top | Article Outline

The Expression of PICK1 mRNA and Protein Is Upregulated in the Rats’ Spinal Cord in Remifentanil-Induced Hyperalgesia

Figure 3

Figure 3

To explore whether remifentanil has an effect on the expression of PICK1 mRNA and protein in the spinal cord of rats, we conducted the RT-qPCR and Western blot. Compared with group C, the expression of PICK1 in group R was increased not only at the mRNA level (P < .0001; Figure 3A), but also at the protein level (P < .0001; Figure 3, B and C) in the spinal cord of rats.

Back to Top | Article Outline

Remifentanil-Induced Hyperalgesia Affects the Expression and Trafficking of AMPARs in the Spinal Cord of Rats

To investigate the expression and distribution of AMPAR subunits GluR1 and GluR2 in rats’ spinal cord dorsal horn vividly, we conducted the immunohistochemistry staining. As group C showed, the GluR1 and GluR2 expressed in almost all the dorsal horn, but they were concentrated in the superficial dorsal horn (Figure 4, A and B). Compared with group C, remifentanil infusion induced an increase in GluR1 (P = .0092; Figure 4C) and a decrease in GluR2 in the spinal cord dorsal horn at 48 hours after the remifentanil administration (P = .0010; Figure 4C).

Figure 4

Figure 4

Figure 5

Figure 5

To further confirm whether remifentanil-induced hyperalgesia affects the trafficking of AMPAR subunits GluR1 and GluR2, we performed the Western blot analysis separately on membrane and cytoplasm protein at 2 hours and 48 hours after remifentanil infusion. Compared with group C, the levels of GluR1 protein in membrane were approximately elevated by 0.81-fold at 2 hours and by 1.62-fold at 48 hours after remifentanil infusion, whereas the GluR2 protein in membrane was approximately reduced by 28% at 2 hours and by 43% at 48 hours after remifentanil infusion (all P < .0011; Figure 5, A–D). However, the expression of GluR1 and GluR2 in cytoplasm was not affected by the remifentanil infusion (all P ≥ .516; Figure 5, A, B, E, and F).

Back to Top | Article Outline

PICK1 AS ODN Knocks Down the Expression of PICK1 Protein in the Spinal Cord of Rats

Table

Table

Figure 6

Figure 6

Because the intrathecal catheter placement was an invasive procedure, it may disable the rats to affect rats’ behaviors. Before intrathecal injection, we conducted locomotor function testing. All rats exhibited normal locomotor functions (Table). To confirm the effect of PICK1 AS ODN, PICK1 protein expression in rats’ L4–L6 spinal cords was detected by Western blot. Compared with group NS (rats were intrathecally injected with saline) and group MS (rats were intrathecally injected with PICK1 MS ODN), the expression of PICK1 protein in group AS (rats were intrathecally injected with PICK1 AS ODN) was distinctly downregulated (P < .0001, Figure 6, A and B) and compared with group NS, on days 1, 2, 3 (all P < .0001; Figure 6, C and D), and 4 (P = .0028; Figure 6, C and D) after the final ODN injection, the expression of PICK1 protein in group AS remained depressed, which manifested that the PICK1 AS ODN was working until the rats were euthanized.

Back to Top | Article Outline

PICK1 Knockdown Partly Prevents Remifentanil-Induced Mechanical and Thermal Hyperalgesia

Figure 7

Figure 7

For nociceptive thresholds (PWT and PWL), there was a significant time–group interaction (all time × group P < .0001; Figure 7). Before the infusion (−24 hours), baseline numbers of nociceptive thresholds (PWT and PWL) were similar in all groups (all post hoc P ≥ .15; Figure 7, A and B). Remifentanil infusion decreased PWT and PWL from 2 to 48 hours (group NS + R and group AS + R versus group NS + NS, all post hoc P < .003; Figure 7, A and B), which suggests that group NS + R and group AS + R developed remifentanil-induced hyperalgesia. Interestingly, there were no differences between group NS + NS and group AS + NS on the PWT and PWL (all post hoc P ≥ .414; Figure 7, A and B), which illustrates that PICK1 knockdown does not impact normal rats’ nociceptive thresholds. Compared with group NS + R, group AS + R showed an increase of PWT and PWL from 2 to 48 hours (all post hoc P < .007; Figure 7, A and B), which suggests that PICK1 knockdown partly prevents remifentanil infusion-induced mechanical and thermal hyperalgesia.

Back to Top | Article Outline

PICK1 Knockdown Partly Reverses the Expression and Trafficking of AMPARs in Remifentanil-Induced Hyperalgesia

According to immunohistochemistry staining, compared with group NS + NS, there was a significant increase in the expression of GluR1 in rats’ spinal cord dorsal horn in group NS + R and group AS + R (both post hoc P < .0005; Figure 8, A and B), but no obvious differences between group NS + R and group AS + R (P = .7895). It indicates that remifentanil infusion induced an increase in GluR1 in rats’ spinal cord dorsal horn and that PICK1 knockdown has no influence on the expression of GluR1. However, compared with group NS + NS, the expression of GluR2 in group NS + R and group AS + R was diminished (both post hoc P < .0023; Figure 8, A and B), and compared with group NS + R, the expression of GluR2 in group AS + R was upregulated (P = .0037). It suggests that remifentanil infusion induced a decrease in GluR2 in the spinal cord dorsal horn and that PICK1 knockdown could partly reverse the decreased expression of AMPARs GluR2 subunit in the dorsal horn caused by remifentanil.

Figure 8

Figure 8

Figure 9

Figure 9

To further investigate the effect of PICK1 knockdown on the trafficking of AMPARs in remifentanil-induced hyperalgesia, we performed Western blot in all groups. In contrast with group NS + NS, membrane GluR1 protein was upregulated, whereas membrane GluR2 protein was downregulated both in the group NS + R and in group AS + R at 2 hours and 48 hours after remifentanil infusion (all post hoc P < .0020; Figure 9, A–D). Compared with group NS + R, the expression of membrane GluR2 protein in the group AS + R was approximately increased by 20% at 2 hours (P = .0083; Figure 9, A, B, and D) and by 27% at 48 hours (P < .0001; Figure 9, A, B, and D), but the level of membrane GluR1 protein had no obvious differences (both P ≥ .985; Figure 9, A–C). It manifests that PICK1 knockdown could partly reverse increased GluR2 trafficking from membrane to cytoplasm caused by remifentanil but has no effect on the trafficking of GluR1. However, cytoplasmic GluR1 and GluR2 protein exerted no differences in all 4 groups (all P ≥ .700; Figure 9, A, B, E, and F).

Back to Top | Article Outline

DISCUSSION

Our results showed a significant time–group interaction for nociceptive thresholds (PWT and PWL). Remifentanil infusion downregulated the nociceptive thresholds at different time points, which could be partly prevented by PICK1 knockdown. Furthermore, the increase of the PICK1 protein, the expression and trafficking of GluR1 and GluR2 internalization, and the decrease of GluR2 protein expression were seen in rats’ spinal cord dorsal horn. The expression and trafficking of GluR2 were reversed partly by PICK1 knockdown. It indicates that PICK1 knockdown could regulate the expression and trafficking of AMPAR subunit GluR2 to prevent the remifentanil-induced hyperalgesia.

In clinical practice, remifentanil is used extensively; 0.05 to 0.26 μg•kg−1•min−1 is accepted and is the suggested dose for satisfactory analgesia after surgery,27–29 and 0.05 to 2.0 μg•kg−1•min−1 is for the maintenance of anesthesia.30–33 Some articles documented that hyperalgesia would occur when remifentanil was infused at or more than the rate of 0.1 μg•kg−1•min−1.34,35 According to an equivalent dose conversion table between the species,36 the dose of rats is 6.25 times of humans to achieve the same pharmacodynamic effect. In the present study, we adopted 1.2 μg•kg−1•min−1 as the rate of remifentanil infusion according to our early work,9 which when converted to human dose is 0.192 μg•kg−1•min−1 and is within the clinically accepted doses.

Our team’s previous research has found that RIH in rats presents in 2 hours and lasts at least 7 days after remifentanil infusion and reaches the maximum at 24 to 48 hours.7 So we chose 2 hours and 48 hours as the time points to represent the development and maintenance phases of remifentanil-induced hyperalgesia for biochemical studies.

Cumulative literature demonstrates that the expression and trafficking of AMPARs are required for CFA-induced hyperalgesia.23,37–39 However, whether the expression and trafficking of AMPARs are involved in remifentanil-induced hyperalgesia is much less reported. Here, our immunohistochemistry staining results showed the GluR1 and GluR2 expressed in almost all the dorsal horn, whereas both subunits concentrated in the superficial dorsal horn. We found that remifentanil infusion induced an increase in the expression of GluR1 and a decrease in the expression of GluR2 in rats’ spinal cord dorsal horn. In conclusion, the expression of AMPARs may be involved in remifentanil-induced hyperalgesia.

To further verify the effect of AMPARs on remifentanil-induced hyperalgesia, we detected their trafficking by Western blot. We found that the trafficking to membrane of GluR1 and internalization of GluR2 in the spinal cord are related to remifentanil-induced hyperalgesia. The levels of GluR1 protein and GluR2 protein in membrane changed much more at 48 hours than at 2 hours after remifentanil infusion compared with group C, which is in accordance with the changes of remifentanil-induced mechanical and thermal hyperalgesia. It further demonstrates that the trafficking of AMPAR subunits GluR1 and GluR2 may be involved in remifentanil-induced hyperalgesia.

A large number of evidence demonstrates that spinal central sensitization is a key mechanism of remifentanil-induced hyperalgesia.40–42 Until now, the most mature viewpoint is that the activation of glutamatergic system leads to spinal central sensitization and further involves remifentanil-induced hyperalgesia.9,43 The typical example is as follows: upregulated trafficking and expression of N-methyl-d-aspartate receptors (NMDARs) could play an essential role in the remifentanil-induced central sensitization.6,9,44–46 Like NMDARs, AMPARs are one of the excitatory ion glutamate receptors in postsynaptic membrane.43 Functional AMPARs are tetrameric cation channels composed by GluR subunits. GluR2-containing receptors are Ca2+ impermeable.47 Membrane insertion of GluR1 and internalization of GluR2 strengthen conductance properties of AMPAR channels, and the quantity of membrane insertion of GluR1 and internalization of GluR2 is proportional to calcium permeability. The calcium influx plays a pivotal role in spinal central sensitization. The present study showed that remifentanil infusion induced an increase in membrane insertion of GluR1 and internalization of GluR2 and strengthened conductance properties of AMPAR channels and then led to the calcium influx, which contributed to spinal central sensitization and was further included in remifentanil-induced hyperalgesia.

The ubiquitous protein PICK1 is mainly expressed in the central nervous system and testes.17 Previous studies suggested that PICK1 plays a role in CFA-induced inflammatory pain.23,24 However, whether PICK1 has a relationship with remifentanil-induced hyperalgesia (RIH) is unexplored. Here, our research suggested that remifentanil infusion upregulated the expression of PICK1 in the spinal cord. PICK1 knockdown partly prevented thermal and mechanical hyperalgesia caused by remifentanil infusion. It indicates that PICK1 is involved in RIH. Via its PDZ domain, PICK1 has an interaction with the C-termini of AMPAR GluR2, GluR3, and GluR4 in the mammal nervous system.48 Our present study indicated that PICK1 deficiency caused by knockdown of PICK1 could partly reverse the expression and internalization of GluR2, which prevented thermal and mechanical hyperalgesia caused by remifentanil infusion. It has been shown previously that the activation of NMDARs may trigger PKCα activation in RIH.44,45 Then, GluR2 was phosphorylated at the site of Ser880 by PKCα, removed from AMPA Receptor Binding protein/Glutamate Receptor Interacting Protein (ABP/GRIP), and reconnected to PICK1. With the help of PICK1, GluR2 was internalized in neurons.23,37,38 As a result of GluR2 internalization, elevation of calcium in dorsal horn neurons resulted in central sensitization and further induced RIH.49 The current research suggested that PICK1 is required for regulating the expression and trafficking of GluR2 in the spinal cord in RIH.

However, PICK1 has no effect on the expression and trafficking of GluR1, because the GluR1 plays an important role in RIH. It is reported that, in inflammatory pain, brain-derived neurotrophic factor-p-tropomyosin-related kinase B (BDNF-TrKB) signaling is upregulated to initiate the trafficking of GluR1 to membrane via PLC-PKC signaling and accompanied with the phosphorylation of GluR1 Ser-831.50 However, how the GluR1 participates in RIH needs further research in the future.

Besides GluR2, PICK1 also could interact with several proteins, including mGluR7a, GluR5, GluR6, EphB2, DAT, NET, and so on.51–54 These proteins might be related to RIH. What is more, PICK1 has been found in the dorsal root ganglion (DRG).55 Thus, whether PICK1 is involved in remifentanil-induced peripheral sensitization remains to be explored.

In conclusion, we demonstrated that remifentanil-induced hyperalgesia is related to the expression of PICK1 protein and the expression and trafficking of GluR1 and GluR2 in spinal dorsal horn neurons. PICK1 knockdown could partly prevent hyperalgesia caused by remifentanil. In addition, we found that PICK1 knockdown could partly reverse remifentanil-induced GluR2 expression and internalization in dorsal horn but has no effect on remifentanil-induced membrane GluR1 expression. So it suggests that spinal cord PICK1 might participate in remifentanil-induced hyperalgesia through promoting GluR2 internalization in dorsal horn neurons. Although remifentanil provides perfect analgesia during clinical practice, hyperalgesia after remifentanil administration inevitably aggravates patient suffering and affects postoperative recovery, which might be a challenge to anesthesiologists. The PICK1 might be a potential therapeutic target to treat remifentanil-induced hyperalgesia. It should be noted that knockdown approaches might be impractical in the clinical setting. Thorsen et al56 identified a small-molecule inhibitor (FSC231) that specifically bound to the PICK1 PDZ domain, inhibited PICK1 interaction with GluR2, and accelerated recycling of GluR2 after internalization. Thus, FSC231 may represent a promising novel strategy for treating remifentanil-induced hyperalgesia.

Back to Top | Article Outline

DISCLOSURES

Name: Zhifen Wang, MD.

Contribution: This author helped design the study and write the manuscript.

Name: Yuan Yuan, PhD.

Contribution: This author helped design the study, analyze the data, and write the manuscript.

Name: Keliang Xie, PhD.

Contribution: This author helped design the study, analyze the data, and write the manuscript.

Name: Xiaohong Tang, MD.

Contribution: This author helped conduct the study.

Attestation: Xiaohong Tang has seen the original study data and approved the final manuscript.

Name: Linlin Zhang, MD.

Contribution: This author helped analyze the data and write the manuscript.

Name: Jiying Ao, MD.

Contribution: This author helped conduct the study.

Attestation: Jiying Ao has seen the original study data and approved the final manuscript.

Name: Nan Li, MD.

Contribution: This author helped analyze the data.

Name: Yu Zhang, MD.

Contribution: This author helped conduct the study.

Attestation: Yu Zhang has seen the original study data and approved the final manuscript.

Name: Suqian Guo, MD.

Contribution: This author helped conduct the study.

Name: Guolin Wang, MD.

Contribution: This author helped design the study and write the manuscript.

This manuscript was handled by: Jianren Mao, MD.

Back to Top | Article Outline

REFERENCES

1. Derrode N, Lebrun F, Levron JC, Chauvin M, Debaene B. Influence of perioperative opioid on postoperative pain after major abdominal surgery: sufentanil TCI versus remifentanil TCI. A randomized, controlled study. Br J Anaesth. 2003;91:842–849.
2. Angst MS, Koppert W, Pahl I, Clark DJ, Schmelz M. Short-term infusion of the mu-opioid agonist remifentanil in humans causes hyperalgesia during withdrawal. Pain. 2003;106:49–57.
3. Koppert W, Angst M, Alsheimer M, et al. Naloxone provokes similar pain facilitation as observed after short-term infusion of remifentanil in humans. Pain. 2003;106:91–99.
4. Luginbühl M, Gerber A, Schnider TW, Petersen-Felix S, Arendt-Nielsen L, Curatolo M. Modulation of remifentanil-induced analgesia, hyperalgesia, and tolerance by small-dose ketamine in humans. Anesth Analg. 2003;96:726–732.
5. Shin SW, Cho AR, Lee HJ, et al. Maintenance anaesthetics during remifentanil-based anaesthesia might affect postoperative pain control after breast cancer surgery. Br J Anaesth. 2010;105:661–667.
6. Shu RC, Zhang LL, Wang CY, et al. Spinal peroxynitrite contributes to remifentanil-induced postoperative hyperalgesia via enhancement of divalent metal transporter 1 without iron-responsive element-mediated iron accumulation in rats. Anesthesiology. 2015;122:908–920.
7. Zhang L, Shu R, Wang H, et al. Hydrogen-rich saline prevents remifentanil-induced hyperalgesia and inhibits MnSOD nitration via regulation of NR2B-containing NMDA receptor in rats. Neuroscience. 2014;280:171–180.
8. Li Y, Wang H, Xie K, et al. Inhibition of glycogen synthase kinase-3β prevents remifentanil-induced hyperalgesia via regulating the expression and function of spinal N-methyl-D-aspartate receptors in vivo and vitro. PLoS One. 2013;8:e77790.
9. Yuan Y, Wang JY, Yuan F, Xie KL, Yu YH, Wang GL. Glycogen synthase kinase-3β contributes to remifentanil-induced postoperative hyperalgesia via regulating N-methyl-D-aspartate receptor trafficking. Anesth Analg. 2013;116:473–481.
10. Rosenmund C, Stern-Bach Y, Stevens CF. The tetrameric structure of a glutamate receptor channel. Science. 1998;280:1596–1599.
11. Collingridge GL, Olsen RW, Peters J, Spedding M. A nomenclature for ligand-gated ion channels. Neuropharmacology. 2009;56:2–5.
12. Dickenson AH, Chapman V, Green GM. The pharmacology of excitatory and inhibitory amino acid-mediated events in the transmission and modulation of pain in the spinal cord. Gen Pharmacol. 1997;28:633–638.
13. Garry EM, Fleetwood-Walker SM. A new view on how AMPA receptors and their interacting proteins mediate neuropathic pain. Pain. 2004;109:210–213.
14. Wang Y, Wu J, Wu Z, Lin Q, Yue Y, Fang L. Regulation of AMPA receptors in spinal nociception. Mol Pain. 2010;6:5.
15. Liu X, Liu Y, Zhang J, et al. Intrathecal administration of roscovitine prevents remifentanil-induced postoperative hyperalgesia and decreases the phosphorylation of N-methyl-D-aspartate receptor and metabotropic glutamate receptor 5 in spinal cord. Brain Res Bull. 2014;106:9–16.
16. Beckerman MA, Ogorodnik E, Glass MJ. Acute morphine associated alterations in the subcellular location of the AMPA-GluR1 receptor subunit in dendrites of neurons in the mouse central nucleus of the amygdala: comparisons and contrasts with other glutamate receptor subunits. Synapse. 2013;67:692–704.
17. Xia J, Zhang X, Staudinger J, Huganir RL. Clustering of AMPA receptors by the synaptic PDZ domain-containing protein PICK1. Neuron. 1999;22:179–187.
18. Staudinger J, Zhou J, Burgess R, Elledge SJ, Olson EN. PICK1: a perinuclear binding protein and substrate for protein kinase C isolated by the yeast two-hybrid system. J Cell Biol. 1995;128:263–271.
19. Dev KK, Nishimune A, Henley JM, Nakanishi S. The protein kinase C alpha binding protein PICK1 interacts with short but not long form alternative splice variants of AMPA receptor subunits. Neuropharmacology. 1999;38:635–644.
20. Dev KK, Nakajima Y, Kitano J, Braithwaite SP, Henley JM, Nakanishi S. PICK1 interacts with and regulates PKC phosphorylation of mGLUR7. J Neurosci. 2000;20:7252–7257.
21. Perez JL, Khatri L, Chang C, Srivastava S, Osten P, Ziff EB. PICK1 targets activated protein kinase C alpha to AMPA receptor clusters in spines of hippocampal neurons and reduces surface levels of the AMPA-type glutamate receptor subunit 2. J Neurosci. 2001;21:5417–5428.
22. Lu W, Ziff EB. PICK1 interacts with ABP/GRIP to regulate AMPA receptor trafficking. Neuron. 2005;47:407–421.
23. Park JS, Voitenko N, Petralia RS, et al. Persistent inflammation induces GluR2 internalization via NMDA receptor-triggered PKC activation in dorsal horn neurons. J Neurosci. 2009;29:3206–3219.
24. Atianjoh FE, Yaster M, Zhao X, et al. Spinal cord protein interacting with C kinase 1 is required for the maintenance of complete Freund’s adjuvant-induced inflammatory pain but not for incision-induced post-operative pain. Pain. 2010;151:226–234.
25. Tao YX, Huang YZ, Mei L, Johns RA. Expression of PSD-95/SAP90 is critical for N-methyl-D-aspartate receptor-mediated thermal hyperalgesia in the spinal cord. Neuroscience. 2000;98:201–206.
26. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008;3:1101–1108.
27. Bowdle TA, Ready LB, Kharasch ED, Nichols WW, Cox K. Transition to post-operative epidural or patient-controlled intravenous analgesia following total intravenous anaesthesia with remifentanil and propofol for abdominal surgery. Eur J Anaesthesiol. 1997;14:374–379.
28. Schüttler J, Albrecht S, Breivik H, et al. A comparison of remifentanil and alfentanil in patients undergoing major abdominal surgery. Anaesthesia. 1997;52:307–317.
29. Yarmush J, D’Angelo R, Kirkhart B, et al. A comparison of remifentanil and morphine sulfate for acute postoperative analgesia after total intravenous anesthesia with remifentanil and propofol. Anesthesiology. 1997;87:235–243.
30. Bowdle TA, Camporesi EM, Maysick L, et al. A multicenter evaluation of remifentanil for early postoperative analgesia. Anesth Analg. 1996;83:1292–1297.
31. Bürkle H, Dunbar S, Van Aken H. Remifentanil: a novel, short-acting, mu-opioid. Anesth Analg. 1996;83:646–651.
32. Hall AP, Thompson JP, Leslie NA, Fox AJ, Kumar N, Rowbotham DJ. Comparison of different doses of remifentanil on the cardiovascular response to laryngoscopy and tracheal intubation. Br J Anaesth. 2000;84:100–102.
33. Sneyd JR, Camu F, Doenicke A, et al. Remifentanil and fentanyl during anaesthesia for major abdominal and gynaecological surgery. An open, comparative study of safety and efficacy. Eur J Anaesthesiol. 2001;18:605–614.
34. Angst MS, Chu LF, Tingle MS, Shafer SL, Clark JD, Drover DR. No evidence for the development of acute tolerance to analgesic, respiratory depressant and sedative opioid effects in humans. Pain. 2009;142:17–26.
35. Cabañero D, Campillo A, Célérier E, Romero A, Puig MM. Pronociceptive effects of remifentanil in a mouse model of postsurgical pain: effect of a second surgery. Anesthesiology. 2009;111:1334–1345.
36. Lu JT. Wei W, Wu XM, Li yJ. Appendix. Experimental Methodology of Pharmacology. 2010:4th ed. China: People’s Medical Publishing House; 1698.
37. Katano T, Furue H, Okuda-Ashitaka E, et al. N-ethylmaleimide-sensitive fusion protein (NSF) is involved in central sensitization in the spinal cord through GluR2 subunit composition switch after inflammation. Eur J Neurosci. 2008;27:3161–3170.
38. Park JS, Yaster M, Guan X, et al. Role of spinal cord alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in complete Freund’s adjuvant-induced inflammatory pain. Mol Pain. 2008;4:67.
39. Su C, D’amour J, Lee M, et al. Persistent pain alters AMPA receptor subunit levels in the nucleus accumbens. Mol Brain. 2015;8:46.
40. Wanigasekera V, Lee MC, Rogers R, Hu P, Tracey I. Neural correlates of an injury-free model of central sensitization induced by opioid withdrawal in humans. J Neurosci. 2011;31:2835–2842.
41. Sahbaie P, Shi X, Li X, et al. Preprotachykinin-A gene disruption attenuates nociceptive sensitivity after opioid administration and incision by peripheral and spinal mechanisms in mice. J Pain. 2012;13:997–1007.
42. Xia WS, Peng YN, Tang LH, et al. Spinal ephrinB/EphB signalling contributed to remifentanil-induced hyperalgesia via NMDA receptor. Eur J Pain. 2014;18:1231–1239.
43. Lee M, Silverman SM, Hansen H, Patel VB, Manchikanti L. A comprehensive review of opioid-induced hyperalgesia. Pain Physician. 2011;14:145–161.
44. Zhao M, Joo DT. Enhancement of spinal N-methyl-D-aspartate receptor function by remifentanil action at delta-opioid receptors as a mechanism for acute opioid-induced hyperalgesia or tolerance. Anesthesiology. 2008;109:308–317.
45. Gu X, Wu X, Liu Y, Cui S, Ma Z. Tyrosine phosphorylation of the N-Methyl-D-Aspartate receptor 2B subunit in spinal cord contributes to remifentanil-induced postoperative hyperalgesia: the preventive effect of ketamine. Mol Pain. 2009;5:76.
46. Zhang L, Shu R, Wang C, Wang H, Li N, Wang G. Hydrogen-rich saline controls remifentanil-induced hypernociception and NMDA receptor NR1 subunit membrane trafficking through GSK-3β in the DRG in rats. Brain Res Bull. 2014;106:47–55.
47. Hollmann M, Heinemann S. Cloned glutamate receptors. Annu Rev Neurosci. 1994;17:31–108.
48. Bassani S, Folci A, Zapata J, Passafaro M. AMPAR trafficking in synapse maturation and plasticity. Cell Mol Life Sci. 2013;70:4411–4430.
49. Cui W, Wang S, Han R, Wang Q, Li J. CaMKII phosphorylation in primary somatosensory cortical neurons is involved in the inhibition of remifentanil-induced hyperalgesia by lidocaine in male Sprague-Dawley rats. J Neurosurg Anesthesiol. 2016;28:44–50.
50. Tao W, Chen Q, Zhou W, Wang Y, Wang L, Zhang Z. Persistent inflammation-induced up-regulation of brain-derived neurotrophic factor (BDNF) promotes synaptic delivery of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor GluA1 subunits in descending pain modulatory circuits. J Biol Chem. 2014;289:22196–22204.
51. Torres GE, Yao WD, Mohn AR, et al. Functional interaction between monoamine plasma membrane transporters and the synaptic PDZ domain-containing protein PICK1. Neuron. 2001;30:121–134.
52. Torres R, Firestein BL, Dong H, et al. PDZ proteins bind, cluster, and synaptically colocalize with Eph receptors and their ephrin ligands. Neuron. 1998;21:1453–1463.
53. Duggan A, Garcia-Anoveros J, Corey DP. The PDZ domain protein PICK1 and the sodium channel BNaC1 interact and localize at mechanosensory terminals of dorsal root ganglion neurons and dendrites of central neurons. J Biol Chem. 2002;277:5203–5208.
54. Hruska-Hageman AM, Wemmie JA, Price MP, Welsh MJ. Interaction of the synaptic protein PICK1 (protein interacting with C kinase 1) with the non-voltage gated sodium channels BNC1 (brain Na+ channel 1) and ASIC (acid-sensing ion channel). Biochem J. 2002;361:443–450.
55. Wang W, Petralia RS, Takamiya K, et al. Preserved acute pain and impaired neuropathic pain in mice lacking protein interacting with C Kinase 1. Mol Pain. 2011;7:11.
56. Thorsen TS, Madsen KL, Rebola N, et al. Identification of a small-molecule inhibitor of the PICK1 PDZ domain that inhibits hippocampal LTP and LTD. Proc Natl Acad Sci U S A. 2010;107:413–418.
© 2016 International Anesthesia Research Society