Involvement of S-nitrosylation of actin in inhibition of neurotransmitter release by nitric oxide

<p>Abstract</p> <p>Background</p> <p>The role of the diffusible messenger nitric oxide (NO) in the regulation of pain transmission is still a debate of matter, pro-nociceptive and/or anti-nociceptive. <it>S</it>-Nitrosylation, the reversible post-translational modification of selective cysteine residues in proteins, has emerged as an important mechanism by which NO acts as a signaling molecule. The occurrence of <it>S</it>-nitrosylation in the spinal cord and its targets that may modulate pain transmission remain unclarified. The "biotin-switch" method and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were employed for identifying <it>S</it>-nitrosylated proteins.</p> <p>Results</p> <p>Here we show that actin was a major protein <it>S</it>-nitrosylated in the spinal cord by the NO donor, <it>S</it>-nitroso-<it>N</it>-acetyl-DL-penicillamine (SNAP). Interestingly, actin was <it>S</it>-nitrosylated, more in the S2 fraction than in the P2 fraction of the spinal homogenate. Treatment of PC12 cells with SNAP caused rapid <it>S</it>-nitrosylation of actin and inhibited dopamine release from the cells. Just like cytochalasin B, which depolymerizes actin, SNAP decreased the amount of filamentous actin cytoskeleton just beneath the membrane. The inhibition of dopamine release was not attenuated by inhibitors of soluble guanylyl cyclase and cGMP-dependent protein kinase.</p> <p>Conclusion</p> <p>The present study demonstrates that actin is a major <it>S</it>-nitrosylated protein in the spinal cord and suggests that NO directly regulates neurotransmitter release by <it>S</it>-nitrosylation in addition to the well-known phosphorylation by cGMP-dependent protein kinase.</p

Anti-nociceptive responses produced by human putative counterpart of nocistatin

b-nocistatin is a heptadecapeptide produced from bovine prepronociceptin and blocks the induction of hyperalgesia and touch-evoked pain (allodynia) by intrathecal administration of nociceptin or prostaglandin E(2) (PGE(2)). Human prepronociceptin may generate a 30-amino acid peptide different in length from b-nocistatin. Here, we examine whether the human putative counterpart of nocistatin (h-nocistatin) possessed the same biological activities as b-nocistatin. Simultaneous intrathecal injection of h-nocistatin in mice blocked the induction of allodynia by nociceptin and PGE(2) in a dose-dependent manner with ID(50) values of 329 pg kg(−1) and 16.6 ng kg(−1), respectively. h-nocistatin was about 10 times less potent than b-nocistatin. h-nocistatin also attenuated the nociceptin- and PGE(2)-induced hyperalgesia. These results demonstrate that h-nocistatin is biologically active and may be involved in the processing of pain at the spinal level in humans

Inhibition of nociceptin-induced allodynia in conscious mice by prostaglandin D(2)

1. We recently showed that intrathecal administration of nociceptin induced allodynia by innocuous tactile stimuli and hyperalgesia by noxious thermal stimuli in conscious mice. In the present study, we examined the effect of prostaglandins on nociceptin-induced allodynia and hyperalgesia. 2. Prostaglandin D(2) (PGD(2)) blocked the allodynia induced by nociceptin in a dose-dependent manner with an IC(50) of 26 ng kg(−1), but did not affect the nociceptin-induced hyperalgesia at doses up to 500 ng kg(−1). BW 245C (an agonist for PGD (DP) receptor) blocked the allodynia with an IC(50) of 83 ng kg(−1). 3. The blockade of nociceptin-induced allodynia by PGD(2) was reversed by the potent and selective DP-receptor antagonist BW A868C in a dose-dependent manner with an ED(50) of 42.8 ng kg(−1). 4. Glycine (500 ng kg(−1)) almost completely blocked the nociceptin-induced allodynia. A synergistic effect on the inhibition of nociceptin-evoked allodynia was observed between glycine and PGD(2) at below effective doses. 5. Dibutyryl cyclic AMP, but not dibutyryl cyclic GMP, blocked the nociceptin-induced allodynia with an IC(50) of 2.9 μg kg(−1). 6. PGE(2), PGF(2α), butaprost (an EP(2) agonist) and cicaprost (a PGI receptor agonist) did not affect the nociceptin-induced allodynia. 7. These results demonstrate that PGD(2) inhibits the nociceptin-evoked allodynia through DP receptors in the spinal cord and that glycine may be involved in this inhibition

Characterization of nociceptin hyperalgesia and allodynia in conscious mice

1. Intrathecal (i.t.) administration of nociceptin and high doses of morphine induced allodynia in response to innocuous tactile stimuli, and i.t. nociceptin evoked hyperalgesia in response to noxious thermal stimuli in conscious mice. Here we have characterized the nociceptin-induced allodynia and compared it with the morphine-induced allodynia and the nociceptin-evoked hyperalgesia. 2. Nociceptin-induced allodynia was evoked by the first stimulus 5 min after i.t. injection, reached a maximum at 10 min, and continued for a 50 min experimental period. Dose-dependency of the allodynia showed a bell-shaped pattern from 50 pg to 5 ng kg(−1), and the maximum effect was observed at 2.5 ng kg(−1). 3. Morphine-induced allodynia reached the maximum effect at 15 min and declined progressively until cessation by 40–50 min. The dose-response curve showed a bell-shaped pattern, similar to that induced by nociceptin, with a maximum effect at 0.5 mg kg(−1), five orders of magnitude higher than that of nociceptin. 4. The allodynia evoked by nociceptin and morphine were dose-dependently blocked by glycine, D(−)-2-amino-5-phosphonovaleric acid (D-AP5, an N-methyl-D-aspartate (NMDA) receptor antagonist), γ-D-glutamylaminomethyl sulphonic acid (GAMS, a non-NMDA receptor antagonist) and methylene blue (a soluble guanylate cyclase inhibitor), but were not affected by muscimol (a γ-aminobutyric acid(A) (GABA(A)) receptor agonist) and baclofen (a GABA(B) receptor agonist). 5. Morphine did not inhibit forskolin-stimulated cyclicAMP formation in cultured cells expressing the nociceptin receptor. 6. Nociceptin-induced hyperalgesia was evoked 10–15 min after i.t. injection. Nociceptin produced a monophasic hyperalgesic action over a wide range of doses from 5 fg to 50 ng kg(−1). The nociceptin-induced hyperalgesia was blocked by glycine only among the agents examined. 7. None of the pain responses evoked by nociceptin and morphine were blocked by naloxone. 8. These results demonstrate that, whereas the mechanisms of the nociceptin-induced allodynia and hyperalgesia are evidently distinct, they involve a common neurochemical event beginning with the disinhibition of the inhibitory glycinergic response. Morphine may induce allodynia through a pathway common to nociceptin, but the nociceptin receptor does not mediate the action of high doses of morphine