Tramadol is a synthetic centrally acting analgesic drug with opioid agonist activity selectively on μ receptors and weakly on κ and σ receptors. It also inhibits re-uptake of norepinephrine and increases extraneuronal serotonin concentrations. Nitric oxide (NO), produced from L-arginine by the enzyme nitric oxide synthase (NOS) increases the intracellular content of cyclic GMP. The activation of the L-arginine-NO-c-GMP pathway is involved in the modulation of pain perception and in the nociceptive transmission at spinal and supraspinal levels . NG-nitro-L-arginine methyl ester (L-NAME) is a non-selective antagonist that inhibits the activity of both endothelial NOS (eNOS) and neuronal NOS (nNOS), while 7-nitro indazole (7-NI) has been described as a nNOS inhibitor in vivo . The role of NOS inhibitors on the systemic analgesic effect of tramadol has not been studied previously. We aimed to investigate the interaction of NOS inhibitors L-NAME and 7-NI with tramadol by using thermal and chemical pain models.
After local animal Ethics Committee approval, 108 adult female Swiss-Webster mice 25-45 g served as the subjects. Hot-plate and acetic acid induced writhing tests were used in order to assess nociception. All drugs were injected intraperitoneally, in a volume of 10 mL kg−1, by the same person and from the same side. Control animals received saline, 36 mice were randomly assigned into six equal groups for the hot-plate test. The first injection was saline, L-NAME 10 mg kg−1 or 7-NI 3 mg kg−1. Twenty minutes after the first intraperitoneal injection, the mice received either saline or Tramadol 40 mg kg−1. Each mouse was placed on a hot-plate, at 55°C 20 min after the second injection. A cut off latency period of 40 s was used in order to prevent tissue damage. Measurements were repeated at the 50th and 80th minutes of injection.
Seventy-two mice were randomly allocated into three groups to receive intraperitoneal saline, L-NAME 10 mg kg−1, or 7-NI 3 mg kg−1. Each main group was divided into four subgroups to make 12 groups. Twenty minutes after the first injection another injection of saline, tramadol 10 mg kg−1, tramadol 20 mg kg−1, tramadol 40 mg kg−1 were applied to the subgroups of these three main groups. Writhing was induced by an intraperitoneal injection of 3% acetic acid at a dose of 300 mg kg−1 20 min after the second injections. The number of writhes per animal was counted during a 15 min test period, starting at the 5th minute after injection of acetic acid. Constriction of abdomen, turning of trunk to one side, and extension of hind legs were accepted as one writhing. Friedman test was used for analysing time effect in hot-plate latencies. Kruskal-Wallis test was used for comparing groups and U-test was used with correction of significance for post-hoc multiple comparisons. P < 0.05 values were accepted as statistically significant (Fig. 1).
Although 7-NI did not show any increase in hot-plate latencies by itself, it increased the analgesic effect of tramadol significantly in the hot-plate tests performed at the 20th and the 50th minutes after injections. 7-NI pretreatment lead to significantly longer hot-plate latencies compared to tramadol alone, the effect was emphasized in the 50th minute after injections. L-NAME pretreatment followed by tramadol administration did not show a significant change in hot-plate latencies compared to tramadol alone. 7-NI or L-NAME pretreatments did not possess an analgesic effect solely on writhing tests (Table 1). No statistically significant differences were observed when the pretreatments were compared in the three doses of tramadol used in the study.
Systemic pretreatment with 7-NI, augmented the analgesic effect of tramadol in the hot-plate test in mice. The same dose of 7-NI itself administered alone, demonstrated no analgesic effect on its own. In contrast to systemic interaction of tramadol with 7-NI in hot-plate test, no interaction between tramadol and L-NAME in both hot-plate and acetic acid induced writhing was observed. 7-NI increased the analgesic effect of tramadol in thermal pain model, not in acetic acid induced writhing test. L-NAME pretreatment did not increase the analgesic effect of tramadol both in hot-plate and acetic acid induced writhing tests.
Our results emphasize the possible involvement of different mechanisms, other than those that have been identified, taking part in the analgesic effect of tramadol. It has been proposed that some non-steroidal anti-inflammatory drugs stimulate NO production, which results in increased c-GMP levels and antinociception . Inhibition of NOS activity also attenuates ketamine antinociception . NO-c-GMP pathway is also involved in the mechanism of benzodiazepine induced antinociception in the writhing test in mice . On the other hand there are several observations indicating that NO-c-GMP pathway plays a hyperalgesic rather than antinociceptive role .
Neuronal NOS inhibitors, inhibit hyperalgesia in the rat regardless of the type of noxious stimuli . L-NAME has a synergistic antinociceptive interaction with morphine in response to thermal stimulation when given intrathecally, epidurally or intravenously in rats but it inhibits the analgesic effect of ketamine in rats . L-NAME potentiates the antinociception induced by sumatriptan and buspiron but does not alter the pain threshold itself in the acetic acid writhing assay at a dose of 20 mg kg−1 . In mice, a 94% inhibition of cortical NOS activity has been reported following administration of L-NAME . So we assumed that this used dose in our study would be enough to inhibit the brain NOS activity. We could not find an increase in the analgesic effect of tramadol when the mice were pretreated with L-NAME.
The mechanism of antinociception in the writhing test in mice involves NO-c-GMP pathway . NOS inhibitors fail to alter acute thermal and mechanical pain, whereas persistent chemical pain associated with writhing or the second phase of formalin assay are very sensitive to inhibition by these drugs . Writhing reflects nociception that unlike the tail-flick and hot-plate tests, is mediated by NMDA receptor activity known to be associated with NO synthesis. Writhing assay is somewhat unique as it involves a noxious stimulus and behavioural responses that persist for a sufficient time interval to reflect sensitivity to the relatively slow generation of NO.
We found that systemic pretreatment with the NOS inhibitor 7-NI augmented the analgesic effect of tramadol in the hot-plate test even though it did not exhibit an analgesic effect by itself. No effects of pretreatments with 7-NI or L-NAME were observed in acetic acid induced writhing. The potentiation of analgesic effect of tramadol by 7-NI might be an indicator of the involvement of NO in the antinociceptive effect of tramadol. This is a preliminary study which suggests that NOS inhibitors may interfere with the analgesic effect of tramadol for the first time. It is also possible that 7-NI may increase the analgesic effect of tramadol other than the inhibition of neuronal NOS inhibition with some indistinct mechanism. In order to find out the overall aspects of this synergism, further studies are required.
This study has been presented at the Turkish Anaesthesiology and Reanimation Congress on November 28th 2003 in Antalya, Turkey and XXII Annual European Society of Regional Anaesthesia & Pain Therapy Congress on September 12th 2003 in St Julian, Malta.
Alper B. Iskit has been supported by the Turkish Academy of Sciences, in the framework of the Young Scientist Award Program (EA-TUBA-GEBIP/2001-2-11).
M. A. Salman
A. E. Salman
A. B. Iskit
*Department of Anaesthesiology and Reanimation, Hacettepe University Faculty of Medicine Hacettepe, Ankara, Turkey
†Department of Pharmacology Hacettepe University Faculty of Medicine Hacettepe, Ankara, Turkey
¶Department of Anaesthesiology and Reanimation, Hacettepe University Faculty of Medicine Hacettepe, Ankara, Turkey
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