Masayoshi Hyodo

Allodynia evoked by intrathecal administration of prostaglandin E2 to conscious mice

&NA; We recently reported that intrathecal (i.t.) administration of prostaglandin (PG) F2&agr;. to conscious mice induced allodynia that was elicited by non‐noxious brushing of the flanks. In the presents study, we demonstrate that i.t. administration of PGD2 and PGE2 to conscious mice also results in allodynia. Dose dependency of PGD2 for allodynia showed a skewed bell‐shaped pattern (0.1 ng‐2.5&mgr;g&ohgr;use), and the maximal allodynic effect was observed with 1.0 &mgr;g at 15 min after intrathecal injection. PGD2induced allodynia showed a time course and dose dependency similar to that induced by PGF2&agr;, but with lower scores. On the other hand, dose dependency of PGE2 for allodynia showed a bell‐shaped pattern over a wide range of dosage from 10 fg to 2.0 &mgr;g/mouse. The maximal allodynic effect was observed with 0.01–0.1 &mgr;g at 5 min after i.t. injection, and the response gradually decreased over the experimental period of 50 min. Intrathecally administered strychnine and the GABA A antagonist bicuculline also induced allodynia in conscious mice. The time courses of allodynia evoked by strychnine and bicuculline coincided with those by PGE2 and PGF2&agr;, respectively. PGE2‐induccd allodynia was dose‐dependently relieved by the strychnine‐sensitive glycine receptor agonist taurine, the NMDA receptor antagonist kctamine, and a high dose of the &agr;2‐adrenergic agonist clonidine, but not by the GABAA agonist muscimol or by the GABAB agonist baclofen. In contrast, PGF2&agr;‐induced allodynia was dramatically inhibited by clonidine and baclofen, but not by taurine, ketamine or muscimol. In addition, PGE2&agr;‐induced hyperalgesia assessed by the hot plate test was not suppressed by taurine or clonidine. These results demonstrate that the mechanism of PGE2&agr;‐induced allodynia is different from that of PGF2‐induced allodynia and from that of PGE2&agr;‐induced hyperalgesia.

Nociceptive effects induced by intrathecal administration of prostaglandin D2, E2, or F2α to conscious mice

The effects of intrathecal administration of prostaglandins on pain responses in conscious mice were evaluated by using hot plate and acetic acid writhing tests. Prostaglandin D2 (0.5-3 ng/mouse) had a hyperalgesic action on the response to a hot plate during a 3-60 min period after injection. Prostaglandin E2 showed a hyperalgesic effect at doses of 1 pg to 10 ng/mouse, but the effect lasted shorter (3-30 min) than that of prostaglandin D2. Similar results were obtained by acetic acid writhing tests. The hyperalgesic effect of prostaglandin D2 was blocked by simultaneous injection of a substance P antagonist (greater than or equal to 100 ng) but not by AH6809, a prostanoid EP1-receptor antagonist. Conversely, prostaglandin E2-induced hyperalgesia was blocked by AH6809 (greater than or equal to 500 ng) but not by the substance P antagonist. Prostaglandin F2 alpha had little effect on pain responses. These results demonstrate that both prostaglandin D2 and prostaglandin E2 exert hyperalgesia in the spinal cord, but in different ways.

Characterization of EP-receptor subtypes involved in allodynia and hyperalgesia induced by intrathecal administration of prostaglandin E2 to mice.

1 Intrathecal (i.t.) administration of prostaglandin E2 (PGE2) to conscious mice induced allodynia, a state of discomfort and pain evoked by innocuous tactile stimuli, and hyperalgesia as assessed by the hot plate test. We characterized prostaglandin E receptor subtypes (EP1–3) involved in these sensory disorders by use of 7 synthetic prostanoid analogues. 2 Sulprostone (EP1 < EP3) induced allodynia over a wide range of dosages from 50 pg to 5 μg kg−1. The maximal allodynic effect was observed at 5 min after i.t. injection, and the response gradually decreased over the experimental period of 50 min. This sulprostone‐induced allodynia showed a time course similar to that induced by PGE2. 3 17‐Phenyl‐ω‐trinor PGE2 (EP1 > EP3) and 16,16‐dimethyl PGE2 (EP1 = EP2 = EP3) were as potent as PGE2 in inducing allodynia, and more potent than sulprostone. Butaprost (EP2), 11‐deoxy PGE1 (EP2 = EP3), MB 28767 (EP3), and cicaprost (prostaglandin I2 (IP‐) receptor) induced allodynia, but with much lower scores. 13,14‐Dihydro‐15‐keto PGE2, a metabolite of PGE2, did not induce allodynia. 4 16,16‐Dimethyl PGE2 as well as PGE2 induced hyperalgesia over a wide range of dosages (16,16‐dimethyl PGE2: 5 pg–0.5 μg kg−1 PGE2: 50 pg–0.5 μg kg−1) with two apparent peaks at 0.5 ng kg−1 and 0.5 μg kg−1. Sulprostone (EP1 < EP3) and 17‐phenyl‐ω‐trinor PGE2 (EP1 > EP3) showed a bell‐shaped hyperalgesia at lower doses of 5 pg–5 ng kg−1 and 50 pg–50 ng kg−1, respectively. MB 28767 (EP3) showed a monophasic hyperalgesic action over a wide range of dosages at 50 pg–5 μg kg−1. Butaprost (EP2) induced hyperalgesia at doses higher than 50 ng kg−1. 5 These results demonstrate that PGE2 may exert allodynia through the EP1‐receptor and hyperalgesia through EP2‐ and EP3‐receptors in the mouse spinal cord.

Involvement of glutamate receptors in allodynia induced by prostaglandins E2 and F2α injected into conscious mice

&NA; In order to investigate the involvement of glutamate receptor systems in allodynia induced by prostaglandin (PG) E2 or F2&agr;, we co‐administered antagonists for N‐methyl‐d‐aspartate (NMDA). non‐NMDA, or metabotropic glutamate receptors intrathecally with PGE2 or PGF2&agr; and examined their effects on the allodynia evoked in conscious mice by non‐noxious brushing of the flanks. MK‐801, a non‐competitive NMDA receptor channel blocker, and d‐AP‐5, a selective NMDA receptor antagonist, dose‐dependently blocked PGH2‐induced allodynia with an IC50 of 1.60 and 0.52 &mgr;g/mouse, respectively. A glycine binding‐site antagonist for the NMDA receptor, 7‐CI‐KYNA, did not influence it. None of these NMDA receptor antagonists inhibited PGF2&agr;‐evoked allodynia. Non‐NMDA receptor antagonists GAMS and CNQX inhibited both PGE2‐ and PGF2&agr;‐induced allodynia. On the other hand, l‐AP‐3 and l‐AP‐4, putative metabotropic glutamate receptor antagonists, dose‐dependently antagonized the allodynia induced by PGF2&agr; with an IC50 of 0.92 and 3.26 ng/mouse, respectively, but not that induced by PGE2. Intrathecal administration of l‐glutamate produced allodynia over a wide range of low doses from 0.1 pg to 0.1 &mgr;g/mouse, and the maximal effect was observed at 1 ng. Similar to allodynia induced by prostaglandins, the response lasted over a 50‐min experimental period. These results demonstrate that both PGE2‐ and PGF2&agr;‐evoked allodynia are mediated through a pathway that includes the glutamate receptor system but that subtypes of glutamate receptors involved and sites of action in the spinal cord may he different between them.

Alterations in nociception after intracisternal administration of prostaglandin D2, E2 or F2α to conscious mice

The effect of intracisternal administration of three major prostaglandins on nociceptive responses was evaluated in mice. Prostaglandin D2 had biphasic effects on pain thresholds (hot plate and acetic acid writhing tests) when given in a dosage range of 5 ng to 5 micrograms per mouse. Lower doses of prostaglandin D2 (less than or equal to 15 ng) increased the sensitivity to pain stimulation. Higher doses (greater than or equal to 50 ng) caused hypoalgesia, which was completely blocked by intracisternal injection of 500 pg of naloxone. Prostaglandin E2 (5 ng-5 micrograms) also had a biphasic effect on pain thresholds, similar to the effect of prostaglandin D2. However, the hypoalgesia caused by a higher dose of prostaglandin E2 (5 micrograms) was not blocked at all by naloxone doses of up to 500 ng. Prostaglandin F2 alpha had little effect on pain thresholds. These results indicate that each prostaglandin has a specific effect on the modulation of nociception.

Nitric oxide mediates allodynia induced by intrathecal administration of prostaglandin E2 or prostaglandin F2α in conscious mice

&NA; We recently reported that intrathecal (i.t.) administration of prostaglandin (PG) E2 or PGF2&agr; in conscious mice induced allodynia through a pathway that includes the glutamate receptor system. Allodynia induced by PGE2 and PGF2&agr; was blocked by antagonists for NMDA and metabotropic glutamate receptor subtypes, respectively. In the present study, we examined the possibility for the involvement of nitric oxide (NO) in the PG‐evoked allodynia. Allodynia was assessed once every 5 min by light stroking of the flank of mice with a paintbrush. Intrathecal administration of L‐arginine, a substrate of nitric oxide synthase (NOS), in conscious mice resulted in allodynia. Dose dependency of L‐arginine for allodynia showed a bell‐shaped pattern (1–10 &mgr;g/mouse). The maximal allodynic effect was observed with 5.0 &mgr;g at 10–15 min after i.t. injection, similar in time course and magnitude to that induced by L‐glutamate. L‐Arginine‐induced allodynia was dose‐dependently reduced by the NOS inhibitor N&ohgr;‐nitro‐L‐arginine methyl ester (L‐NAME) and the soluble guanylate cyclase inhibitor methylene blue with IC50 values of 7.68 and 8.70 pg/mouse, respectively. PGE2 induced allodynia was also dose‐dependently inhibited by L‐NAME and methylene blue with IC150. values of 94.7 and 74.9 pg/mouse. PGF2&agr;‐induced allodynia was inhibited by methylene blue with an IC50. value of 40.6 pg/mouse, but not by L‐NAME at doses up to 1.0 ng. These results demonstrate that PGEZ‐induced allodynia is mediated through the NO‐generating system and that PGF2&agr;‐induced allodynia may be mediated by interactions with the NO system at a site different from the NO‐generating site in the spinal cord.

Effect of NMDA receptor antagonists on prostaglandin E2-induced hyperalgesia in conscious mice

Intrathecal (i.t.) injection of prostaglandin E2 (PGE2) to conscious mice produced a hyperalgesic action over a wide range of dosages with two apparent peaks at 100 pg and 10 ng per mouse, which may be mediated through EP3 and EP2 subtypes of the PGE receptor. In the present study, the effects of NMDA receptor antagonists on hyperalgesia induced by PGE2 were evaluated by the hot plate test at 30 min after i.t. injection. Hyperalgesia induced by a higher dose of PGE2 (10 ng/mouse) was relieved by D-AP5 (a competitive antagonist), 7-Cl-KynA (a glycine site antagonist), and ketamine and MK801 (non-competitive channel blockers). Intrathecal injection of butaprost (10 ng/mouse), an EP2 agonist, induced hyperalgesia, and this hyperalgesia was blocked by D-AP5, 7-Cl-KynA, ketamine, and MK801, similar to that induced by 10 ng of PGE2. On the other hand, hyperalgesia induced by a lower dose of PGE2 (100 pg/mouse) was blocked by D-AP5 and 7-Cl-KynA, but not by ketamine and MK801. Intrathecal injection of sulprostone (100 pg/mouse), an EP1 and EP3 agonist, induced hyperalgesia, and this hyperalgesia was blocked by D-AP5 and 7-Cl-KynA, but not by ketamine and MK801, similar to that induced by 100 pg of PGE2. These results first demonstrate that the NMDA receptor is involved in the PGE2-induced hyperalgesia and suggest that the hyperalgesic action by lower and higher doses of PGE2 may be mediated through EP3 and EP2 subtypes, respectively.

Blockade by ONO-NT-012, a unique prostanoid analogue, of prostaglandin E2-induced allodynia in conscious mice

1 Intrathecal (i.t.) administration of prostaglandin E2 (PGE2) to conscious mice was reported to induce allodynia, a state of discomfort and pain evoked by innocuous tactile stimuli through prostaglandin E receptor subtype EP1 and hyperalgesia through prostaglandin E receptor subtypes EP2 and/or EP3. In the present study, we investigated the effects of an EP1 antagonist on these sensory disorders by use of ONO‐NT‐012 or AH6809. 2 ONO‐NT‐012 dose‐dependently antagonized the PGE2‐induced allodynia but had no effect on the PGE2‐induced hyperalgesia by the hot plate test. On the other hand, AH6809 blocked the PGE2‐induced hyperalgesia at the highest dose examined (50 μ kg−1) but had no effect on the PGE2‐induced allodynia. The i.t. injection of AH6809 or ONO‐NT‐012 alone did not have any effect on the response to noxious or innocuous stimuli. 3 Increasing doses (5 pg kg−1‐500 ng kg−1) of ONO‐NT‐012 produced parallel shifts to the right of the dose‐response curves to PGE2. The Schild plot regression line was linear and the slope was close to unity. The pA2 value against PGE2 was calculated to be 9.96. 4 The present study demonstrates that i.t. administration of PGE2 exerts allodynia through EP1 in the mouse spinal cord and that ONO‐NT‐012 is a highly potent, simple competitive antagonist for the PGE2‐induced allodynia.

The effects of stimulation of ear acupuncture points on the body's pain threshold.

Six ear acupuncture points, one non-acupuncture ear point, and the body locus Ho-Ku (LI-4) were electrically stimulated in order to compare the effects of stimulation on the bodys pain threshold at selected loci on various points on the body by measurement with a radiation heat-type Pain Meter on 5 subjects. The ear points, with the exception of the non-acupuncture ear point, were found to be effective even in peripheral body regions in varying degrees. Ear stimulation did not increase the threshold as rapidly as Ho-Ku. In all cases where the pain threshold was raised, the effect persisted after electrical stimulation had stopped.

An experimental study on the enhancing effects of phenylalanine on acupuncture analgesia.

In this preliminary study we examined the enhancing effect of D-phenylalanine on acupuncture anesthesia. We made 4 different kinds of experiments with 3 volunteers. The results show that D-phenylalanine extends the analgesic effect of acupuncture analgesia remarkably, with no exception in 3 cases. According to these facts, we believe that these findings have an important meaning for those who are engaged in acupuncture treatment or research.