Involvement Of P2X3 Receptor In The Development Of Morphine-Induced Anti-Nociception Tolerance

PART I Blockade and reversal of morphine tolerance by P2X3 receptor antagonist in mice Objective1. To investigate the effect of the selective P2X3 receptor antagonist A-317391 on the development of anti-nociceptive tolerance to chronic morphine administration in mice.2. To investigate the effect of selective P2X3 receptor antagonist A-317491 on the naloxone withdrawal symptoms in mice.3. To observe the reversal effect of P2X3 receptor antagonist A-317491 on spinal morphine tolerance in mice. MethodsThe animals were randomly divided into four groups:1. Group M(n=14) was treated intraperitoneally(i.p.) with 10 mg/kg morphine twice daily at 12 h intervals for 7 days;2. Group AM(n=35; withdrawal test n=7; dose-effect test n=7 for each dose; bolus test n=7) received 10mg/kg A-317491, the selective P2X3 receptor antagonist(Sigma; 1% solution in saline, i.p.), 30 min before each morphine injection for 7 days;3. Group A(n=14; withdrawal test n=7; bolus test n=7) received 10 mg/kg A-317491(1% solution in saline, i.p.) alone for consecutive 7 days;4. Group S(n=35; withdrawal test n=7; dose-effect test n=7 for each dose; bolus test n=7) was given with saline alone i.p. for 7 days;Mechanical thresholds and morphine analgesia was evaluated by von Frey filaments and tail-flick test 30 min after the last injection. Results1. A-317491 reduced the degree of morphine-induced anti-nociceptive tolerance;2. Bolus A-317491 on day 8 could restore the morphine anti-nociceptive efficacy in tolerant mice;3. Daily injection of the P2X3-selective antagonist A-317491 reduced symptoms on the naloxone withdrawal tests in mice; ConclusionsRats treated with intraperitoneal morphine(10mg /kg) twice for 7 consecutive days developed morphine tolerance. A-317491 can partially prevent the development of anti-nociceptive tolerance and morphine-induced hyperalgesia.PART II Selective antagonist of P2X3 receptor, A-317491,inhibits tolerance to analgesic effects of morphine in rats Objective1. To investigate the expressions of P2X3 receptors and PKCε in DRGs after intrathecal morphine treatment for different days;2. To investigate the influence on the development of morphine tolerance as well as P2X3 activation in DRGs after intervening P2X3 activity by intrathecally treating A-317491 a selective inhibitor of P2X3;3. To explore possible mechanisms involved in participation of P2X3 in morphine tolerance. Methods56 Male Sprague-Dawley rats were divided into 8 groups :1. Group M3, M5, M7 were treated intrathecally with morphine(15μg/10μl) for 3, 5, and 7 days;2. Group M+A received morphine(15μg/10μl) plus A-317491(15μg/10μl) for 7 days;3. Group A was treated with A-317491(15μg/10μl) for 7 days;4. Group M+epsilon-V1-2 was treated with morphine(15μg/10μl) plus the PKCε inhibitor epsilon-V1-2(10μg/10μl) for 7 days;5. Group epsilon-V1-2 received epsilon-V1-2(10μg/10μl) for 7 days;6. Group S was treated with saline for 7 days.All drugs were given once a day. The paw withdrawal test and the tail flick test were employed as behavior tests. The expression of P2X3 and kinase C-epsilon(PKCε) in the dorsal root ganglia(DRG) was investigated with Western Blotting and/or immunofluorescence. Results1.Both A-317491 and epsilon-V1-2 attenuated the development of tolerance to morphine-induced anti-nociception.2.Western blotting and immunofluorescence showed a morphine-induced increase of P2X3 receptor expression in the DRG, and A-317491 reduced this change.3.Up-regulated expression of PKCε protein levels in the DRG after chronic morphine exposure was inhibited by A-317491. ConclusionsInhibition of the P2X3 receptor might reduce PKCε activity during chronic morphine exposure and result in reduced development of morphine-induced anti-nociceptive tolerance. So, the P2X3 receptor might contribute to the development of morphine-induced anti-nociceptive tolerance and be a new therapeutic target in the prevention of tolerance to morphine-induced anti-nociception.