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*Department of Pharmacology, Faculty of Medicine, Gulhane Military Academy of Medicine, Ankara, Turkey; Department of Pharmacology, University of New England College of Osteopathic Medicine, Biddeford, Maine; and Department of Pharmacology, University of Arizona Health Sciences Center, Tucson, Arizona
Address correspondence and reprint requests to Frank Porreca, PhD, Department of Pharmacology, College of Medicine, University of Arizona, AHSC, 1501 North Campbell Ave., Tucson, AZ 85724. Address e-mail to frankp{at}u.arizona.edu.
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Abstract |
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Introduction |
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Although some investigators suggest that antinociceptive tolerance may be caused by intracellular adaptive mechanisms (8,9), many unrelated drugs have been reported to abolish antinociceptive tolerance to opioids. These substances include N-methyl-d-aspartate (NMDA) antagonists, nitric oxide synthase inhibitors, calcium channel blockers, kinase inhibitors, and cyclooxygenase inhibitors (3,7). These drugs have at least one mechanism in common, and that is to reduce synaptic transmission in the spinal dorsal horn, either by attenuating neurotransmitter release from primary afferent terminals or inhibiting excitability of second-order neurons. Neurotransmitter release is dependent, in part, on the entry of extracellular calcium into the primary afferent nerve terminals through voltage-dependent calcium channels (VDCCs). Three types of VDCCs have been characterized (L-, N-, and T-type), with each channel having distinct electrophysiological characteristics and pharmacological sensitivities (10). Both mixed L- and T-type VDCC blockers and selective L-type VDCC blockers have been shown to enhance morphine antinociception without eliciting antinociception when given alone (11–13). Moreover, the inhibition of the VDCCs have been shown to prevent the development of opioid antinociceptive tolerance (12). In the present investigation, we extend these observations to include the ability of amlodipine, generally considered to be a selective L-type calcium channel blocker, to prevent the development of morphine-induced abnormal hyperesthetic state coincident with antinociceptive tolerance.
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Methods |
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Intrathecal (IT) injections (10 µL) were made into the subarachnoid space in unanesthetized mice, as previously described (14). The mice were held immobile and wrapped in a towel, and the injections were made by inserting a short (half-inch) 30-gauge needle at a shallow angle between the L4 and L5 vertebral processes into the subarachnoid space. Proper placement of the needle was indicated by a reflexive stiffening of the tail. Amlodipine (10 µg) or saline was given 5 min before morphine administration. Injections were completed twice daily (12-h intervals) for 8 consecutive days. Behavioral testing for responses to tactile and thermal stimuli were performed before the first injection of morphine or morphine and amlodipine on the day of testing. Tail-flick determinations were made at the time of peak effect, 30 min after spinal morphine injection in different groups of mice. Separate groups of mice that received repeated spinal injections of morphine were challenged with systemic morphine to explore the effect of amlodipine on systemic cross-tolerance to spinal morphine.
The hotplate test was used to assess paw-withdrawal latency to a thermal nociceptive stimulus. Mice were placed individually on the uniformly heated surface (maintained at 48°C), and the reaction time was measured starting from the time the mouse was placed on the plate until the mouse either demonstrated hindpaw licking or jumping. A cut-off latency of 90 s was used to prevent tissue damage. Paw-withdrawal thresholds averaged 48.2 ± 4.6 s before drug administration (baseline) and were maintained in the control group at roughly this level across the 8-day period of injections and through the postinjection phase of the experiments.
Paw-withdrawal thresholds were determined by probing the hindpaw with a series of finely calibrated von Frey filaments. The strength of the von Frey stimuli ranged from 0.02 g to 6 g on a logarithmic scale. Mice were allowed to acclimate within plexiglas enclosures for approximately 20 min, and then withdrawal thresholds were determined by increasing and decreasing stimulus strength until the minimal stimulus required to elicit a response was determined ("up-down" method). The paw-withdrawal threshold was estimated by the Dixon nonparametric test (15). These data were represented as mean withdrawal threshold ± sem. Paw-withdrawal thresholds averaged 4.5 ± 0.5 g before drug administration (base-line) and were maintained in the control group at approximately this level across the 8-day period of injections and through the postinjection phase of the experiments.
Morphine antinociception was assessed in separate groups of mice with the radiant heat tail-flick analgesia meter (Columbus Instruments Inc., Columbus, OH). Baseline tail-flick latencies (TL) for each mouse were determined 3 times at 5-min intervals, and the mean of these scores was designated as the baseline latency (BL). The mean pretreatment pooled BL was 3.08 ± 0.99 s. Morphine was administered either by IT or subcutaneous (s.c.) injection, and the TL were measured 30 min later. A cut-off time of 8 s was used to prevent tissue damage.
Response thresholds and latencies over time were compared with the preexposure baseline values by analysis of variance followed by the post hoc Fisher’s least significant difference test. A significant reduction in paw-withdrawal threshold to tactile stimuli indicated tactile hyperesthesia, and significantly reduced latencies to respond to thermal stimuli indicated thermal hypersensitivity. Significance level was set at P = 0.05. Data were converted to % MPE (maximal possible effect) by the formula % MPE = 100 x (TL – BL)/(8 – BL) to generate dose-response curves. Linear regression analysis of the log dose-response curves was used to determine the 50% antinociceptive value (A50) and the 95% confidence intervals. The relative potency of the dose-response curve before and after chronic morphine exposure was determined with FlashCalc software. A potency ratio value more or less than one, with a 95% confidence limit that does not include unity, indicated a significant shift in the dose-response curve.
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Results |
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BL were 3.1 ± 0.14 s and 2.9 ± 0.18 s in saline-treated and morphine-treated mice (tested on Day 8). Thermal hyperalgesia is usually not detected with the tail-flick reflex because it is a fast spinal reflex (about 3 s), and significantly faster TL are not usually observed. The antinociceptive A50 value for IT morphine for the saline-treated control group was 0.33 µg (0.29–0.52 µg; Fig. 2A). Repeated IT injections of morphine produced a significant (P < 0.05) 17.4-fold shift to the right of the antinociceptive dose-response curve for IT morphine. The A50 value was 5.77 µg (3.45–9.66 µg; Fig. 2A). The administration of amlodipine blocked the development of antinociceptive tolerance to morphine (Fig. 2A). The calculated A50 values for morphine was 0.31 µg (0.15–0.61 µg), which was not significantly different from the A50 value for morphine in the group that received repeated saline injections. Acute IT injection of amlodipine (10 µg) marginally increased the antinociceptive potency of IT morphine. The IT morphine A50 value was 0.16 µg (0.08–0.22 µg). Tolerance to the repeated administration of IT morphine was reversed within 12 days of cessation of morphine administration. On Day 20 of the experiment, the A50 value for IT morphine in the mice previously pretreated with IT morphine was 0.31 µg (0.20–0.45 µg; Fig. 2B). In mice that received both IT amlodipine and morphine, the IT morphine A50 value was 0.21 µg (0.13–0.31 µg; Fig. 2B).
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Separate groups of mice that received spinal injections of morphine were challenged with systemic morphine to generate dose-effect curves. The antinociceptive dose-response for s.c. morphine was significantly (P < 0.05) shifted to the right by 6.6-fold in mice that received repeated spinal injections of morphine (Fig. 3). The calculated A50 value was increased from 2.26 mg/kg (1.41–3.63 mg/kg) to 15.0 mg/kg (9.61–23.3 mg/kg; Fig. 3). Mice that received both spinal amlodipine and morphine did not demonstrate antinociceptive tolerance to systemic morphine (Fig. 3). The A50 values for systemic morphine was 2.84 mg/kg (2.57–3.19 mg/kg), which was not significantly (P > 0.05) different from that of the saline-pretreated group (Fig. 3).
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Discussion |
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Early studies have shown that a single spinal injection of morphine may elicit an allodynic syndrome that includes spontaneous biting and scratching of the back at the dermatomal level of the injection and distress vocalization to light brush (19). However, these behavioral observations were manifest only after extremely large doses (150 µg) of spinal morphine and did not occur after injection of equimolar concentrations of methadone or sufentanil (19). Moreover, this hyperesthetic response was not reversed by opioid antagonists and was partially stereospecific (19). It was suggested that this hyperesthetic response is a non-opioid excitatory effect mediated by certain phenanthrene alkaloids that contain a free 3-OH position, an ether bridge, and without an extension of the N-methyl group and are thus subject to conjugation (19). Importantly, this property is not shared by many other opioid agonists, including meperidine, alfentanil, sufentanil, oxycodone, levorphanol, and oxymorphone (19). There are several indications that the tactile and thermal hyperesthesias observed in the present investigation are not caused by this non-opioid excitatory effect of spinal morphine. This syndrome itself is qualitatively different from, and more intense than, the tactile and thermal hyperesthesias observed after prolonged opioid administration. The doses of morphine used in the present study are only a fraction of the extremely large doses used in the studies reported above. Opioid agonists that do not produce the behavioral excitation, including DAMGO and fentanyl, still produce tactile and thermal hyperesthesias upon prolonged administration (7,10,16,20). Finally, we have recently found that prolonged administration of the active (-) stereo-isomer of oxymorphone produces tactile and thermal hyperesthesia, whereas sustained administration of the non-opioid isomer (+) is without effect (unpublished observations). This observation suggests that thermal and tactile hyperesthesias are mediated through long-term opioid receptor activation.
Morphine induces antinociception by acting synergistically at spinal and supraspinal sites. Systemic morphine antinociceptive potency depends on antinociceptive synergy between spinal and supraspinal actions (21). Animal studies and clinical reports have shown that there is cross-tolerance between systemic and intrathecal morphine (21,22). We also observed that repeated IT morphine induced cross-tolerance to systemic morphine. An important feature of the antinociceptive tolerance development to morphine relates to the loss of synergy in supraspinal and spinal synergy in the central nervous system (22). It seems reasonable to suggest that the observed decreased antinociceptive potency of systemic morphine in spinal morphine tolerant animals may result from a loss of spinal and supraspinal synergy. In our study, spinal L-type VDCC blockade prevented the development of tolerance to the antinociceptive effects of spinal morphine and also blocked the development of antinociceptive tolerance to systemic morphine by restoring systemic morphine potency. Therefore, the blockade of the development of tolerance to the antinociceptive effects of morphine at the spinal level by a L-type VDCC blocker also may have restored the supraspinal-spinal synergy, which dictates the potency of systemic s.c. morphine.
Earlier investigations have shown that persistent opioid exposure may elicit the activation of a descending pain facilitatory system arising from the rostroventromedial medulla (3,7,16,18). This pronociceptive system, along with an increase in spinal dynorphin content, has been shown to enhance the capsaicin-evoked neurotransmitter release from primary afferent terminals (3,6,7,16,18). Pharmacologic and surgical manipulations that abolished descending facilitation from the rostroventromedial medulla also abolished morphine-induced abnormal pain, antinociceptive tolerance, and enhanced evoked neurotransmitter release (3,6,7,16,18). It has also been shown that spinal dynorphin enhances evoked neurotransmitter release from primary afferent terminals and that antiserum to dynorphin abolishes this enhanced release, along with opioid-induced abnormal pain and antinociceptive tolerance (3,6,7,16,18). This pronociceptive enhanced release of neurotransmitters from primary afferent terminals might be mitigated by the presence of the L-type VDCC blocker amlodipine, thus abolishing a mechanism through which enhanced pain, and thus tolerance, might be mediated.
The L-type VDCC, like the N-type and the T-type VDCC, is present in the dorsal horn of the spinal cord and participates in the spinal processing of nociceptive impulses (10,23). Moreover, because the L-type VDCC is found on cell bodies and dendrites rather than axon terminals, it is more likely to participate in postsynaptic events (23). Electrophysiologic and behavioral studies with blockers of the L-type VDCC demonstrated that, although this VDCC is not critical to processing of acute nociceptive signals, it is an important component of spinal sensitization (23). For example, behavioral signs of sensitization induced by s.c. formalin or capsaicin, and the enhanced behavioral responses to noxious and innocuous sensory stimuli elicited by inflammation or nerve injury, were attenuated by blockers acting at the L-type VDCC, whereas normal acute nociceptive responses were not affected (23). Moreover, the enhanced responses of spinal dorsal horn neurons to noxious and innocuous stimuli, indicative of central sensitization, were reduced by the spinal administration of diltiazem or nifedipine (23). Prolonged opioid exposure seems to produce signs of central sensitization, including enhanced behavioral responses to sensory stimuli, enhanced capsaicin-evoked release of calcitonin gene-related peptide and increased touch-evoked FOS expression in the spinal cord (6,24). Manipulations that have been shown to abolish central sensitization, including treatment with NMDA antagonists and disruption of descending pain facilitatory systems, also abolish opioid-induced hyperesthesias and antinociceptive tolerance (3,7,8,19,20). Accordingly, it is possible that amlodipine may abolish opioid-induced tactile and thermal hyperesthesias and antinociceptive tolerance by attenuating the component of spinal sensitization mediated through activation of the L-type VDCC.
An alternate interpretation might be that persistent exposure to morphine results in an increase in extracellular calcium, which would lead to enhanced neuronal excitability and transmitter release (25). Increased levels of basal-free intracellular calcium have been observed in synaptosomes taken from brain and spinal cord tissues of mice chronically exposed to morphine and may indicate a neuronal adaptation to the exposure to the opioid (25). In this situation, it may be possible that amlodipine, by virtue of its VDCC blocking ability, normalizes the excitatory state of the primary afferent terminals and returns it to a basal level of activity, thus abolishing enhanced pain and antinociceptive tolerance.
In conclusion, repeated IT injections of morphine resulted in the development of antinociceptive tolerance to morphine that coincided with the development of tactile hypersensitivity and thermal hyperalgesia. Pretreatment with a L-type calcium channel blocker, amlodipine, prevented the development of the abnormal pain states and reversed antinociceptive tolerance to spinal morphine. Thus, blockade of these channels may ultimately reduce stimulus-induced excitatory neurotransmitter release, blocking the opioid-induced hyperalgesia that contributes to spinal, and ultimately systemic, morphine antinociceptive tolerance.
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Footnotes |
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Accepted for publication July 11, 2005.
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