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British Journal of Anaesthesia, 2000, Vol. 85, No. 4 587-591
© 2000 The Board of Management and Trustees of the British Journal of Anaesthesia

Morphine tolerance increases [3H]MK-801 binding affinity and constitutive neuronal nitric oxide synthase expression in rat spinal cord

Chih-Shung Wong1,*, Ming-Man Hsu1, Yen-Yen Chou1, Pao-Luh Tao2 and Che-Se Tung3

1Department of Anesthesiology, National Defense Medical Center and Tri-Service General Hospital, 2Departments of Pharmacology and 3Physiology and Biophysics, National Defense Medical Center, Taipei, Taiwan

Accepted for publication: April 4, 2000


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
N-Methyl-D-aspartate (NMDA) receptor antagonists and nitric oxide synthase (NOS) inhibitors inhibit morphine tolerance. In the present study, a lumbar subarachnoid polyethylene (PE10) catheter was implanted for drug administration to study alterations in NMDA receptor activity and NOS protein expression in a morphine-tolerant rat spinal model. Antinociceptive tolerance was induced by intrathecal (i.t.) morphine infusion (10 µg h–1) for 5 days. Co-administered (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) (10 µg h–1 i.t.) with morphine was used to inhibit the development of morphine tolerance. Lumbar spinal cord segments were removed and prepared for [3H]MK-801 binding assays and NOS western blotting. The binding affinity of [3H]MK-801 was higher in spinal cords of morphine-tolerant rats (mean (SEM) KD=0.41 (0.09) nM) than in control rats (1.50 (0.13) nM). There was no difference in Bmax. Western blot analysis showed that constitutive expression of neuronal NOS (nNOS) protein in the morphine-tolerant group was twice that in the control group. This up-regulation was partially prevented by MK-801. The results suggest that morphine tolerance affects NMDA receptor binding activity and increases nNOS expression in the rat spinal cord.

Br J Anaesth 2000; 85: 587–91

Keywords: analgesics opioid, morphine; rat


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Two possible mechanisms of opioid tolerance, receptor uncoupling and receptor down-regulation, have been demonstrated.1 2 Larcher and colleagues3 proposed that activation of the NMDA-dependent pain-facilitatory systems may also play another possible mechanism of morphine tolerance. There is a growing body of evidence that opioid tolerance can be inhibited or attenuated by both competitive and non-competitive NMDA receptor antagonists in several animal models.4–7 It has been proposed, therefore, that NMDA receptor activation plays a role in the development of morphine tolerance.5 8 Recently, we have shown that NMDA receptor antagonists inhibit the development of morphine tolerance and affect µ-opioid receptor binding.9 However, the effect of development of morphine tolerance on NMDA receptor-binding activity has not been studied.

Nitric oxide, an intracellular messenger linked to NMDA receptor activation, also plays a role in morphine tolerance.8 NOS inhibitors attenuate or prevent morphine tolerance.10–13 NOS activity is increased in chronic morphine-treated mouse brains14 15 and the NOS mRNA level is greater in morphine-tolerant rat spinal cords than in controls.16 However, it is not clear which NOS protein isoform is involved in the morphine tolerance.

In the present study, we examined the involvement of NMDA receptors and NOS in the development of morphine tolerance in the rat spinal cord.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Animal preparation and intrathecal catheter implantation
With institutional approval, male Sprague–Dawley rats weighing 300–350 g were anaesthetized with chloral hydrate 400 mg kg–1 i.p., and a PE10 intrathecal (i.t.) catheter implantation was performed as previously described.9 Rats were housed in the Animal Facility of the National Defense Medical Center for recovery after surgery. Three days after catheterization, a mini-osmotic pump (model 2001; Alzet, Palo Alto, CA, USA) with a pump rate of 1 µl h–1 for 7 days was first filled with morphine or other test drugs and implanted subcutaneously under chloral hydrate anaesthesia (400 mg kg–1 i.p.) between the scapulae. Rats with gross neurological injury or those in which fresh blood was found in the cerebrospinal fluid were excluded from the study.

Tail-flick test and induction of morphine tolerance
The tail-flick test was used to measure antinociception. All tests were performed daily for 5 days and 30 min before osmotic pump implantation. The tail-flick test was performed every 90 s, and the latencies were expressed as the average of four measurements over a 10 min test period in each rat. The heat intensity used was the average latency (3.1±0.2 s) of 40 opioid-naive rats.

Tolerance, defined by a loss of the antinociceptive effect of morphine infusion as shown by the tail-flick test, was induced by morphine infusion (10 µg h–1 i.t.) for 5 days. MK-801 (10 µg h–1 i.t.) co-administered with morphine (10 µg h–1 i.t.) infusion was used to inhibit the development of tolerance.9 Control animals were given equal amounts of saline (1 µl h–1 i.t.) or MK-801 (10 µg h–1 i.t.).

Membrane preparation and [3H]MK-801 binding assays
On day 5, rats were killed and the lumbosacral segments of the spinal cords were collected. The segments of spinal cords were homogenized with a PT20 polytron in 50 mM Tris–acetate buffer (pH 7.4 at room temperature), then centrifuged (35 000g for 15 min at 4°C) twice. The pellets were resuspended in 50 mM Tris–acetate buffer, frozen in a methanol bath chilled with dry ice, thawed at room temperature and centrifuged again to remove excess endogenous excitatory amino acids. The pellets were resuspended in 50 mM Tris–acetate buffer, incubated at 37°C for 20 min and centrifuged again (35 000g for 20 min).

The method of [3H]MK-801 saturation binding was modified from a previous study17 and is briefly described below. [3H]MK-801 (0.1–60 nM) was used as the radiolabelled ligand. Equilibrium binding was conducted in the presence of unlabelled NMDA (100 µM), glycine (10 µM) and spermidine (50 µM) at room temperature for 4 h. Nonspecific binding was determined in the presence of unlabeled MK-801 (10 µM). The incubation volume was 1 ml and the protein concentration was adjusted to 150 µg per tube. To stop binding, the rapid filtration method was used with a semi-automatic cell harvester (model 11021; Skatron Instruments, Sterling, VA, USA). All samples were incubated with scintillation cocktail overnight and then counted in a ß-counter.

Western blot analysis of NOS protein expression
The frozen dorsal part of lumboscaral spinal cord segments were also prepared in cold extraction buffer (50 mM Tris–HCl, 1 mM EDTA, 1 mM phenylmethylsulphonyl fluoride, pH 7.4) homogenized and centrifuged (13 500 rpm, 45 min, 4°C) for NOS western blotting. Samples, containing 5–10 µg protein, were denatured in sample buffer (50 mM Tris–HCl, 10% glycerol, 2% sodium dodecyl sulphate (SDS), 100 mM dithiothreitol, pH 6.8) and heated for 5 min. An equal amount of protein (5–10 µg) was loaded on 6–7.5% SDS–polyacrylamide gel to separate NOS by electrophoresis (120 V, 4°C) and then transferred (250 mA, 4°C) to a nitrocellulose membrane (Millipore). Membranes were then blocked with 5% non-fat milk in phosphate buffer–0.1% Tween (PBST) for 1 h at room temperature and incubated with mouse monoclonal antibody to nNOS or of inducible NOS (iNOS) (1:2500 dilution; Transduction Laboratories, Lexington, KY, USA) in PBST (0.1% Tween) overnight at 4°C. The membranes were then washed with PBST (0.2% Tween) and incubated with anti-mouse IgG conjugated to horseradish peroxidase (1:10 000) for 1 h at room temperature. NOS proteins were visualized with a horseradish peroxidase enhanced chemiluminescence (ECL) kit (Amersham, Arlington Heights, IL, USA). Prestained protein standards (Bio-Rad) and positive control for nNOS or iNOS (Transduction Laboratories, Lexington, KY, USA) were used for molecular weight determinations and protein identification. The density of NOS bands was quantified by densitometric scanning (Image-Pro Plus software); the density of the other bands on a gel were expressed relative to that of the nNOS band (150 kDa) in the control group, which was defined as 100%.

Data analysis
All data are presented as mean (SEM) for the given number of rats or binding assays performed. All the receptor-binding assay data were analysed as described previously;9 the program used involves a nonlinear, least-squares curve-fitting algorithm and assumes the simultaneous contribution of one or more independent binding sites. For statistical analysis, all data were initially analysed by analysis of variance, followed by the Student–Newman–Keuls post hoc test for multiple comparisons. P<0.05 was taken to indicate a significant difference.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
As in our previous study,9 continuous morphine infusion (10 µg h–1 i.t.) induced antinociceptive tolerance on day 2. Continuous MK-801 infusion (10 µg h–1 i.t. for 5 days) alone did not produce any antinociception, but co-administration of MK-801 (10 µg h–1 i.t.) with morphine attenuated morphine tolerance (Fig. 1). In [3H]MK-801 saturation binding assays, which measure NMDA receptor-gated ion channel activity, [3H]MK-801 binding affinity in tolerant rat spinal cords (KD=0.41 (0.09) nM) was four times that in control rats (KD=1.50 (0.13) nM) (Fig. 2). A tendency to reduction in the receptor density, Bmax, of [3H] MK-801 binding was observed in tolerant rats, but the difference was not significant (Table 1). In the nNOS western blot analysis, a significant increase in constitutive expression of nNOS protein was observed in morphine-tolerant rats; this was partially attenuated by MK-801 co-administration (10 µl h–1 i.t.). Normal saline (1 µl h–1 i.t.) and MK-801 (10 µl h–1 i.t.) infusion alone did significantly affect nNOS expression (Fig. 3). In the present study, expression of iNOS protein was not detectable.



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  		Fig 1 The effects of MK-801 on the development of morphine tolerance. Morphine tolerance (MO) was induced by continuous infusion (10 µg h–1 i.t.) for 5 days. The effect of MK-801 on morphine tolerance was examined by co-administering MK-801 10 µg h–1 i.t. (MO+MK) with morphine for 5 days. The antinociceptive effect of continuous MK-801 10 µg h–1 i.t. infusion (MK) was also examined. The control group was infused with the same amount of normal saline. Tail-flick latencies were measured daily for 5 days. All data points are averages of results from >=10 rats, and the results are expressed as means±SEM. *P<0.05, **P<0.01, ***P<0.001 compared with the morphine-infused group.

 


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  		Fig 2 Scatchard analysis of [3H]MK-801 binding in rat spinal cord membranes with various i.t. drug infusions. Rats were continuously infused with saline (control), morphine 10 µg h–1 (MO), MK-801 10 µg h–1 (MK) or a combination of morphine and MK-801 10 µg h–1 (MO+MK) for 5 days. Rats were killed and their spinal cords removed on the sixth day. Membranes were prepared for [3H]MK-801 binding assays. [3H]MK-801 (0.1–60 nM) was used as the radiolabelled ligand in the presence of glycine (10 µM) and NMDA (100 µM). Unlabelled MK-801 (10 µM) was used to determine nonspecific binding. Data points are from one of the six experiments, each with three-pooled lumbosacral segments of spinal cords and performed in triplicate.

 

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  		Table 1 Scatchard analysis of [3H]MK-801 saturation binding in the rat spinal cord lumbosacral segment of rats treated with various drugs. The rat spinal cord lumbosacral segments were removed on the sixth day after continuous intrathecal drug infusions and prepared for receptor binding assays. [3H]MK-801 (0.1–60 nM) was used as the radiolabelled ligand for saturation binding experiments in the presence of glycine (10 µM) and NMDA (100 µM). Unlabelled MK-801 (10 µM) was used to determine nonspecific binding. The results are expressed as mean (SEM) of at least six separate assays. Each assay was performed with three pooled spinal cords and in triplicate. *P<0.01 (compared with control group)
 


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  		Fig 3 nNOS protein expression in the dorsal lumbosacral segments of rat spinal cords after various drug treatments, measured by western blot analysis. Rat spinal cords were obtained and treated as in Fig. 1. The dorsal lumbosacral segments of rat spinal cords were used for western blot analysis as described in Materials and methods. The 155 kDa nNOS protein band revealed by monoclonal nNOS antibody on a typical western blot are shown at the top. The positive control of nNOS (+nNOS; 1 µg) is shown in the right-most lane. The optical density of each protein band was quantified by densitometry and the relative optical density was calculated by taking the density of the control band as 100%. The mean intensity of the nNOS protein bands in the four groups is shown below. Data are expressed as mean±SEM of results from 10 rats in each group. **P<0.01 (control group compared with MO group), *P<0.05 (MO group compared with MO+MK group). C, Control (normal saline); MO, morphine; MK, MK-801; MO+MK, morphine co-administered with MK-801.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The present study shows that long-term intrathecal infusion of morphine produced antinociceptive tolerance accompanied by an increase in the KD of [3H]MK-801. Up-regulation of nNOS expression was also observed in the dorsal horn of morphine-tolerant rats. Co-administration of MK-801 attenuates morphine tolerance assessed using the tail flick test in the rat spinal model as reported in previous studies.4 5 7 9 Changes in [3H]MK-801 binding and nNOS expression can be partially inhibited by MK-801 co-administration. These results suggest that activation of the NMDA receptors and their downstream signal transduction messenger, nitric oxide, are probably involved in another mechanism of morphine tolerance besides the µ-opioid receptor uncoupling from the G-protein and receptor down-regulation.1 2

From the results of the NMDA receptor-binding assays, morphine tolerance was associated with a significant increase in the probability of [3H]MK-801 binding to the ligand-gated NMDA receptor channel. It may be reflected by an increase in the [3H]MK-801 binding affinity in morphine tolerant rats, and this was prevented by MK-801 co-administration during the induction of morphine tolerance. The results suggest that continuous i.t. morphine infusion may make NMDA receptor channels open for longer or more often, thus enhancing the downstream production of nitric oxide. Collectively, these findings suggest that the activated NMDA–nitric oxide system may be involved in pain facilitation or may indirectly affect the µ-opioid receptor conformation, thus reducing the antinociceptive effect of morphine. However, Gudehithlu, Reddy and Bhargava18 did not find any changes in the [3H]MK-801 binding of tolerant rat spinal cords in the absence of glutamate and glycine in the binding assay buffer. The difference might result from receptor binding assay conditions; in our study, NMDA and glycine were added for [3H]MK-801 saturation binding. It has been shown that NMDA and glycine are necessary for [3H]MK-801 saturation binding but do not alter receptor density.19 Therefore, it is reasonable to suggest that the NMDA receptor channel blocker, MK-801, attenuates the development of morphine tolerance by inhibiting the activity of NMDA receptor channels, which is seen as a prevention of an increase in [3H]MK-801 binding affinity.

Western blot analysis showed that nNOS protein expression was greater in the spinal cords of morphine-tolerant rats than in control rats, and that MK-801 prevented this up-regulation. This finding is in agreement with a previous report, and supports the involvement of nNOS, via NMDA receptor activation, in the development of morphine tolerance.20 Many previous studies using the antisense approach, in situ hybridization and immunohistochemiical techniques have shown increases in mRNA level and in the activity of nNOS,14 16 but not in iNOS in morphine-tolerant animals.21 In our western blot study, iNOS was undetectable in the spinal cords of both control and morphine-tolerant rats. Even when we loaded 100 µg protein (20 times more than the amount of nNOS) and prolonged the time of transfer; we still could not detect iNOS. In an immunocytochemical study, Wu and colleagues22 showed that iNOS was only distributed in the ependymal cell layer around the central canal in naive animals and was more densely stained, in the same area, after induction of arthritis. Our samples were collected from the dorsal horn of the spinal cord and did not include the central canal, which may explain why we could not detect iNOS expression. NOS inhibitors also reduce morphine tolerance.10–13 It has been suggested that morphine tolerance leads to activation of NMDA receptors followed by the subsequent release of nitric oxide. Mayer and colleagues23 reported that opioid tolerance was associated with NMDA receptor activation. Blockade of nitric oxide production, the consequence of NMDA receptor activation, resulted in the prevention or retardation of development of opioid tolerance.12 24

In conclusion, our results suggest that morphine tolerance may be associated with NMDA receptor activation and subsequent nNOS up-regulation. Inhibition of NMDA receptor activity may inhibit morphine tolerance and thus preserve the antinociceptive effect of morphine. The underlying mechanism may involve inhibition of NMDA receptor channel activity, subsequent attenuation of nNOS activity and inhibition of pain facilitation.


   Acknowledgements
 
This work was supported by grants from the National Science Council (NSC 88-2314-B-016-090) and the National Health Research Institute (NHRI-GT-EX 89B909P) of the Republic of China.


   Footnotes
 
* Corresponding author Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 Wong CS, Su YF, Chang KJ, Watkins WD. Intrathecal pertussis toxin treatment attenuates opioid antinociception and reduces high-affinity state of opioid receptors. Anesthesiology 1992; 77: 691–9[Web of Science][Medline]

2 Wong CS, Su YF, Watkins WD, Chang KJ. Continuous intrathecal opioid treatment abolishes the regulatory effects of magnesium and guanine nucleotides on mu opioid receptor binding in rat spinal membranes. J Pharmacol Exp Ther 1992; 262: 317–26[Abstract/Free Full Text]

3 Larcher A, Laulin JP, Celerier E, Le Moal M, Simonnet G. Acute tolerance associated with a single opiate administration: involve ment of N-methyl-D-aspartate-dependent pain facilitatory systems. Neuroscience 1998; 84: 583–9[Web of Science][Medline]

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5 Gutstein HB, Trujillo KA. MK-801 inhibits the development of morphine tolerance at spinal sites. Brain Res 1993; 626: 332–4

6 Marek P, Ben-Eliyahu S, Gold M, Liebeskind JC. Excitatory amino acid antagonists (kynurenic acid and MK-801) attenuate the development of morphine tolerance in the rat. Brain Res 1991; 547: 77–81[Web of Science][Medline]

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12 Elliott K, Minami N, Kolesnikov YA, Pasternak GW, Inturrisi CE. The NMDA receptor antagonists, LY274614 and MK-801, and the nitric oxide synthase inhibitor, NG-nitro-L-arginine, attenuate analgesic tolerance to the mu-opioid morphine but not to kappa opioids. Pain 1994; 56: 69–75[Web of Science][Medline]

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17 Sharif NA, Nunes JL, Whiting RL. Pharmacological characterization of the N-methyl-D-aspartate (NMDA) receptor-channel in rodent and dog brain and rat spinal cord using [3H]MK-801 binding. Neurochem Res 1991; 16: 563–9[Web of Science][Medline]

18 Gudehithlu KP, Reddy PL, Bhargava HN. Effect of morphine tolerance and abstinence on the binding of [3H]MK-801 to brain regions and spinal cord of the rat. Brain Res 1994; 639: 269–74[Web of Science][Medline]

19 Wong EH, Knight AR, Woodruff GN. [3H]MK-801 labels a site on the N-methyl-D-aspartate receptor channel complex in rat brain membranes. J Neurochem 1988; 50: 274–81[Web of Science][Medline]

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22 Wu J, Lin Q, Lu Y, Willis WD, Westlund KN. Changes in nitric oxide synthase isoforms in the spinal cord of rat following induction of chronic arthritis. Exp Brain Res 1998; 118: 457–65[Web of Science][Medline]

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