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
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Abstract |
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Br J Anaesth 2000; 85: 58791
Keywords: analgesics opioid, morphine; rat
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Introduction |
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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.1013 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.
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Materials and methods |
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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 h1 i.t.) for 5 days. MK-801 (10 µg h1 i.t.) co-administered with morphine (10 µg h1 i.t.) infusion was used to inhibit the development of tolerance.9 Control animals were given equal amounts of saline (1 µl h1 i.t.) or MK-801 (10 µg h1 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 Trisacetate 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 Trisacetate 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 Trisacetate 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 (0.160 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 TrisHCl, 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 510 µg protein, were denatured in sample buffer (50 mM TrisHCl, 10% glycerol, 2% sodium dodecyl sulphate (SDS), 100 mM dithiothreitol, pH 6.8) and heated for 5 min. An equal amount of protein (510 µg) was loaded on 67.5% SDSpolyacrylamide 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 buffer0.1% 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 StudentNewmanKeuls post hoc test for multiple comparisons. P<0.05 was taken to indicate a significant difference.
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Results |
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Discussion |
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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 NMDAnitric 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.1013 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.
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Acknowledgements |
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Footnotes |
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References |
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