Complete inhibition of purinoceptor agonist-induced nociception by spinorphin, but not by morphine

Peptides 21 (2000) 1215–1221

Complete inhibition of purinoceptor agonist-induced nociception by spinorphin, but not by morphine Hiroshi Uedaa,*, Shinobu Matsunagaa, Makoto Inouea, Yukio Yamamotob, Tadahiko Hazatob a

Department of Molecular Pharmacology and Neuroscience, Nagasaki University School of Pharmaceutical Sciences, 1–14 Bunkyo-machi, Nagasaki 852-8521, Japan b Molecular Oncology, The Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan Received 23 February 2000; accepted 2 May 2000

Abstract We found that spinorphin, a novel neuropeptide showed analgesia in a different manner compared with morphine. By measuring flexor responses induced by the intraplanter injection of substances, the presence of three different types of sensory neurons were demonstrated. Although spinorphin completely blocked 2-metylthioadenosine (2-MeS ATP, a P2X3 agonist)-induced responses, morphine did not. On the other hand, morphine-induced blockade of bradykinin (BK, a B2-receptor agonist)-responses was attenuated by pertussis toxin (PTX) treatment, whereas that of spinorphin was not. Thus it is suggested that spinorphin has a spectrum of analgesia which covers the blockade of nociception insensitive to morphine. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Spinorphin; P2X3; Bradykinin; 2-MeS ATP; PGI2 agonist; Gi

1. Introduction Spinorphin has been discovered as an endogenous factor that inhibits enkephalin-degrading enzymes from bovine spinal cords [5,15,16]. Structural analysis revealed that the factor is Leu-Val-Val-Tyr-Pro-Trp-Thr, and it inhibits a neural endopeptidase (EC 3.4.24.11; NEP) as well as aminopeptidase (EC 3.4.1; AP), dipeptidyl aminopeptidase (EC 3.4.14.1; DPP), and angiotensin-conversing enzyme (EC 3.4.15.1; ACE) from monkey brain [15]. Like LVV-hemorphin-6 [3] and valorphin [2], spinorphin is related to hemoglobin beta chain. Human polymorphonuclear neutrophil (PMN) induces some inflammatory responses, such as chemotaxis, O2⫺ generation, and exocytosis stimulated by Nformylmethionyl-leucyl-phenylalanine (FMLP) [1,12]. Spinorphin also inhibits PMN function by suppressing FMLP binding to its receptor on PMNs [21]. In addition, spinorphin when administered intracerebroventricularly (i.c.v.) shows analgesic activity in the tail-pinch method [17], possibly through an inhibition of brain enkephalin degrading enzymes. Recently, we developed a series of new strategies

* Corresponding author. Tel.: ⫹81-95-844-4248; fax: ⫹81-95-8444248.

to characterize sensory neurons stimulated by pain-producing substances given into the intraplantar (i.pl.) space of hind paw of mouse [7–10,18]. Here we further attempted to characterize nociceptive sensory fibers into three types, and examined the mode of action of spinorphin-induced analgesia, in comparison to morphine-induced one.

2. Methods 2.1. Animals Male ddY mice weighing 20 to 22 g were used in all experiments. Procedures were approved by Nagasaki University Animal Care Committee and complied with the recommendations of International Association for the Study of Pain [22]. 2.2. Drugs The following drugs were used: bradykinin (BK; Sigma, St. Louis, MO, USA), 2-metylthioadenosine triphosphate tetrasodium (2-MeS ATP; Research Biochemicals International, Wayland, MA, USA), morphine hydrochloride (Takeda Pharmac. Co. Ltd., Japan), pertussis toxin (PTX;

0196-9781/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 6 - 9 7 8 1 ( 0 0 ) 0 0 2 6 2 - X

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Research Biochemicals International), capsaicin (Nacalai Tesque, Japan), MK-801 (Research Biochemical International), CNQX (Research Biochemicals International), leuhistin (Sigma). CP-99994 and CP-100263 were generously provided by Pfizer Pharmaceuticals (New York, NY, USA). ONO-54918-07 (a PGI2 agonist) was gift from Ono Pharmaceutical Co., Ltd. (Osaka, Japan). Spinorphin (Leu-ValVal-Tyr-Pro-Trp-Thr) was synthesized by the solid-phase method using Fmoc-chemistry with a peptide synthesizer (model 431A; Applied Biosystems, Foster City, CA, USA). After the synthesis, the peptide was deprotected and cleaved from the resin with 95% trifluoroacetic acid. The structure of the synthesized peptide was verified by amino acid analysis and molecular mass analysis. The lyophilized crude peptide was purified by preparative reverse-phase HPLC on a C-18 column [21]. All drugs except for capsaicin and CNQX were dissolved in physiological saline. Capsaicin was dissolved in 10% ethanol plus 10% Tween 80 in physiological saline, while CNQX in 25% dimethyl sulfoxide. Drugs were given by i.pl. injection in a volume of 2 ␮l. 2.3. Evaluation of analgesia on nociceptive flexor responses All experiments were performed in compliance with the relevant laws and institutional guidelines. Experiments were performed, as described earlier [7–10,18]. Briefly, mice were lightly anesthetized with ether and held in a cloth sling with their 4 limbs hanging free through holes. The sling was suspended on a metal bar. All limbs were tied with strings, and three were fixed to the floor, whereas the other one was connected to an isotonic transducer and recorder. Two polyethylene cannulae (0.61 mm in outer diameter) were inserted into right hind-limb planta, filled with drug solution, and connected to a microsyringe. One cannula was filled with BK, 2-MeS ATP, PGI2 agonist or physiological saline, and the other with spinorphin or morphine. As we used light and soft polyethylene cannulae, they did not fall off the paw during the experiments. All experiments were started after complete recovery (20 –30 min) from the light ether anesthesia and i.pl. injection of saline did not show any significant flexor responses. BK, 2-MeS ATP, or PGI2 agonist were given at 10 and 5 min before and 5, 10, 20, and 30 min after spinorphin- or morphine-injection. As the intensity of the flexor responses differs from one mouse to another, we used the largest one of 10 to 20 spontaneous flexor response occurring immediately after cannulation (maximal reflex), to normalize the test drug-induced responses. Therefore, the nociceptive activity was expressed as the % of maximal reflex observed before drug challenges at the beginning of each experiment. In these experiments, the pain-producing compound was contained in a tandem manner with an air space in the cannula. Nociceptive responses were measured every 5 min unless otherwise stated. The effects of test drugs were expressed as the ratio of the response observed

over the average of twice repeated control BK, 2-MeS ATP, or PGI2 agonist-induced responses obtained in the beginning of experiments. Test drugs affecting each agonist responses were given through another cannula immediately after the second control response was measured. PTX was given through another cannula at 5 min before the second control of BK response. All animals were used for only one experiment by the observer who did not know what kind of pretreatments had been given. The area under the curve (AUC) was evaluated by plotting antinociception (%) on the ordinate and time after antagonist (i.pl.) administration (min) on the abscissa. In this case, antinociception was assessed by % of the maximal AUC (2500% 䡠 min) which represents the analgesia when pain-producing compoundresponse is completely inhibited during periods from 5 to 30 min after drug injection. The median antinociceptive dose (AD50) was calculated from the linear regression curve of the percentage of maximal AUC against log dose of antagonist. We determined the single AD50 from separate experiments using three different doses of antagonist on the same day, and carried out similar trials six times (days) to calculate SEM of AD50. 2.4. Capsaicin-treatment Capsaicin was injected subcutaneously (s.c.) into the back of newborn (P4) ddY mice in a dose of 50 mg/kg. This treatment is known to cause a degeneration of small-diameter afferent sensory neurons [4,6], and we confirmed the loss of substance P immunoreactivity substance P in the dorsal horn of spinal cord [9]. For control mice, vehicle (10% ethanol and 10% Tween 80 in physiological saline) was treated. Nociception tests were carried out with weighing 20 to 22 g thus treated mice. 2.5. Statistical analysis Statistical evaluations were performed using the Student’s t-test, after one-way ANOVA at each time (5, 10, 20, or 30 min) after antagonist-treatment. In the experiment using different doses of antagonist, statistical evaluations were performed using the Dunnett test for multiple comparisons, after one way ANOVA. In the experiment evaluating effect of PTX on spinorphin- or morphine-analgesia, statistical evaluations were performed using the Scheffe test for multiple comparisons, after one way ANOVA. Data were expressed as mean ⫾ SEM. Significance was established at *P ⬍ 0.05. 3. Results 3.1. Pharmacological identification of three different kinds of nociceptive sensory neurons The local application of BK at 2 pmol into the planta of hind limb (i.pl.) produced a nociceptive flexor response, and

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Fig. 1. Type classification of flexor-responses induced by BK, 2-MeS ATP and PGI2 agonist. A, C, and E: BK (2 fmol-2 pmol), 2-MeS ATP, a specific agonist for ATP P2X3 receptor, (1 pmol-1 nmol) and PGI2 agonist (10 fmol-100 pmol) were i.pl. given. Capsaicin (50 mg/kg) or vehicle was injected into the back of newborn (P4) ddY mice. B, D, and F: Differential blockade by various neurotransmitter antagonists. CP-99994 (a selective NK1 antagonist), CP-100263 (an inactive isomer), MK-801 (NMDA receptor antagonist), or CNQX (AMPA/kainate receptor antagonist) was administrated intrathecally at 20 min before the first challenge of BK, 2-MeS ATP or PGI2 agonist. Data represent the mean ⫾ SEM from separate four through nine experiments. *P ⬍ 0.05, compared with the value of vehicle treated group.

there were stable responses in amplitude upon repeated applications every 5 min, as previously reported [8,18]. The mean ⫾ SEM of BK responses (2 pmol) corresponds to the force of 6.86 ⫾ 0.25 g (n ⫽ 50). BK in ranges of 0.002 to 2 pmol (i.pl.) showed such responses in a dose-dependent manner (Fig. 1A), and the average of ND50, nociceptive dose ( ⫾ SEM) showing 50% of maximal reflex, was 0.71 ⫾ 0.09 pmol (n ⫽ 5). To characterize the sensory neuron involved in BK-responses, mice were pretreated

with capsaicin that did not cause any gross behavioral changes, compared to the case with vehicle. BK-responses in this dose range were completely abolished by capsaicintreatment at dose of 2 fmol, F(1,6) ⫽ 0.81, P ⫽ 0.40, 20 fmol, F(1,6) ⫽ 17.38, P ⬍ 0.01, 200 fmol, F(1,6) ⫽ 12.35, P ⫽ 0.01, 2 pmol, F(1,5) ⫽ 19.63, P ⬍ 0.01 (Fig. 1A). When CP-99994, an NK1 antagonist was intrathecally (i.t.) pretreated in a dose of 100 pmol at 20 min before BK (2 pmol, i.pl.)-challenge, BK-responses were markedly

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blocked, F(1,7) ⫽ 21.19, P ⬍ 0.01, (Fig. 1B). However, 100 pmol of CP-100263, an inactive derivative of CP-99994 did not affect it, F(1,7) ⫽ 0.04, P ⫽ 0.85. Neither MK-801 (100 pmol, i.t.), an NMDA receptor antagonist, nor CNQX (1 nmol, i.t.), an AMPA/kainate receptor antagonist, showed any significant attenuation (MK-801, F(1,6) ⫽ 0.33, P ⫽ 0.59, CNQX, F(1,8) ⫽ 0.33, P ⫽ 0.58). The validity of the dose for MK-801 or CNQX has been confirmed in preliminary studies, where the tail flick responses were not significantly inhibited by either treatment (data not shown). 2-MeS ATP, a specific agonist for ATP P2X3 receptor [4], also dose-dependently induced flexor responses in the range of 1 pmol to 1 nmol (i.pl.) (Fig. 1C). These responses were also blocked by capsaicin pretreatment at dose of 1 pmol, F(1,10) ⫽ 3.82, P ⫽ 0.08, 10 pmol, F(1,10) ⫽ 12.40, P ⬍ 0.01, 100 pmol, F(1,10) ⫽ 7.09, P ⫽ 0.02, 1 nmol, F(1,10) ⫽ 11.83, P ⬍ 0.01, whereas some activities with higher doses were insensitive (Fig. 1C). The intrathecal pretreatment with CP-99994, however, did not affect these 2-MeS ATP-responses, F(1,11) ⫽ 0.00041, P ⫽ 0.98, (Fig. 1D). Interestingly, MK-801 but not CNQX significantly inhibited these responses (MK-801, F(1,11) ⫽ 22.65, P ⬍ 0.01, CNQX, F(1,11) ⫽ 0.08, P ⫽ 0.78). Different profiles of nociceptive responses were observed with PGI2 agonist, ONO-54918-07. This compound also induced nociceptive flexor responses in the range of 10 fmol to 100 pmol (i.pl.), as shown in Fig. 1E. However, capsaicin pretreatment had no effect on these responses at dose of 100 fmol, F(1,11) ⫽ 0.000090, P ⫽ 0.99, 1 pmol, F(1,11) ⫽ 0.05, P ⫽ 0.82, 10 pmol, F(1,8) ⫽ 0.34, P ⫽ 0.58, 100 pmol, F(1,8) ⫽ 0.03, P ⫽ 0.88. As in the case with 2-MeS ATP, PGI2 agonist– responses were inhibited by MK-801 (F(1,6) ⫽ 7.33, P ⫽ 0.04), but not CP-99994 (F(1,8) ⫽ 0.02, P ⫽ 0.89) or CNQX (F(1,8) ⫽ 0.40, P ⫽ 0.55) (Fig. 1F). 3.2. Analgesic effects of spinorphin on three different primary afferent neurons In all experiments for spinorphin-induced analgesia, leuhistin (1 nmol, i.pl.) was injected with spinorphin to protect this peptide from the degradation by aminopeptidase N [17,20]. When 3 nmol of spinorphin was injected by i.pl. immediately after the second BK (2 pmol)-challenge, the following BK-induced nociceptive responses were rapidly attenuated and abolished completely at 20 min after the spinorphin challenge (Fig. 2A). Similar blockade was also observed when a 1 nmol of morphine was given (Fig. 2B). Analgesic effects of both materials were maximal at 20 min after treatments and they lasted at least for 30 min (Fig. 2C). The nociception induced by 2 pmol of BK was blocked by 3 nmol of spinorphin, at 5 min, F(1,12) ⫽ 7.43, P ⫽ 0.02, 10 min, F(1,12) ⫽ 33.46, P ⬍ 0.01, 20 min, F(1,12) ⫽ 37.91, P ⬍ 0.01, 30 min, F(1,12) ⫽ 158.32, P ⬍ 0.01 (Fig. 2C). Morphine blocked the nociception induced by 2 pmol of BK at the dose of 1 nmol at 5 min, F(1,13) ⫽ 12.55, P ⬍ 0.01, 10 min, F(1,13) ⫽ 34.38, P ⬍ 0.01, 20 min, F(1,13) ⫽

35.18, P ⬍ 0.01 30 min, F(1,13) ⫽ 102.23, P ⬍ 0.01 (Fig. 2C). The dose-dependency was also observed in both cases when the analgesia was expressed as percent of maximal AUC for 30 min (Fig. 2D). However the slope for spinorphin was less steep than that for morphine. The median analgesic dose, AD50, was 42.8 ⫾ 8.7 pmol (n ⫽ 6) for spinorphin, whereas was 274 ⫾ 23 pmol for morphine. Spinorphin significantly inhibited 2-MeS ATP (1 nmol)– induced responses at a dose of 3 nmol (i.pl.) at 5 min, F(1,5) ⫽ 2.91, P ⫽ 0.15, 10 min, F(1,5) ⫽ 1.14, P ⫽ 0.33, 20 min, F(1,5) ⫽ 3.78, P ⫽ 0.11, 30 min, F(1,5) ⫽ 8.72, P ⫽ 0.03 as shown in Fig. 3A. Complete inhibition was observed with 10 nmol (i.pl.) of this peptide at 5 min, F(1,7) ⫽ 3.53, P ⫽ 0.10, 10 min, F(1,7) ⫽ 5.02, P ⫽ 0.06, 20 min, F(1,7) ⫽ 31.66, P ⬍ 0.01, 30 min, F(1,7) ⫽ 65.33, P ⬍ 0.01. When morphine was given, on the other hand, 2-MeS ATP-responses were significantly inhibited by 10 nmol (i.pl.) of morphine (at 5 min, F(1,7) ⫽ 1.42, P ⫽ 0.27, 10 min, F(1,7) ⫽ 3.81, P ⫽ 0.09, 20 min, F(1,7) ⫽ 2.64, P ⫽ 0.15, 30 min, F(1,7) ⫽ 9.64, P ⫽ 0.02), whereas no dose-dependency was observed between cases with 3 and 10 nmol (Fig. 3B). Marked difference from the case with spinorphin was observed in the fact that morphine-analgesia against 2-MeS ATP-response was not complete. As shown in Fig. 4, no significant analgesia against PGI2 agonist (100 pmol, i.pl.)-responses was observed with each 10 nmol of spinorphin (at 5 min, F(1,6) ⫽ 1.30, P ⫽ 0.30, 10 min, F(1,6) ⫽ 0.31, P ⫽ 0.60, 20 min, F(1,6) ⫽ 1.39, P ⫽ 0.28, 30 min, F(1,5) ⫽ 0.06, P ⫽ 0.81) or morphine (at 5 min, F(1,7) ⫽ 1.54, P ⫽ 0.25, 10 min, F(1,8) ⫽ 0.23, P ⫽ 0.64, 20 min, F(1,8) ⫽ 1.08, P ⫽ 0.33, 30 min, F(1,5) ⫽ 0.12, P ⫽ 0.74) coinjection. 3.3. PTX-insensitive spinorphin-analgesia against BKnociception To characterize the mechanism of spinorphin-analgesia against BK-induced nociceptive responses, pertussis toxin (PTX) was treated after the second BK-challenge. As shown in Fig. 5A, there was no significant attenuation of spinorphin-analgesia by PTX (10 ng, i.pl.) (at 5 min, F(2,23) ⫽ 2.27, P ⫽ 0.13, 10 min, F(2,23) ⫽ 8.79, P ⬍ 0.01, 20 min, F(2,23) ⫽ 25.13, P ⬍ 0.01, 30 min, F(2,22) ⫽ 42.89, P ⬍ 0.01). In contrast, morphine-analgesia was significantly blocked by this treatment (at 5 min, F(2,16) ⫽ 7.50, P ⬍ 0.01, 10 min, F(2,16) ⫽ 19.79, P ⬍ 0.01, 20 min, F(2,16) ⫽ 21.57, P ⬍ 0.01, 30 min, F(2,16) ⫽ 51.44, P ⬍ 0.01), whereas the PTX-treatment itself did not affect BK-responses (data not shown).

4. Discussion The major findings in the present study have two issues. Firstly, peripherally stimulated nociceptive or sensory stimulations could be divided into three types from pharmaco-

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Fig. 2. Blockade of BK-induced nociceptive responses by spinorphin (SPN) and morphine (MOR). A, B: A representative trace for SPN (A)- or MOR (B)-induced blockade of BK-response. BK (2 pmol) was given by i.pl. as indicated by the arrow. SPN (3 nmol) or MOR (1 nmol) was given by i.pl. through another cannula immediately after the second control BK-challenge. C: Antagonism of BK-responses by SPN or MOR. Results represent the percentage of BK-response after SPN or MOR injection to the average of twice preceding control responses. D: Dose-dependency of SPN or MOR -induced analgesia. The results were expressed with % of maximal AUC, as described in the text. The maximal AUC was represented the analgesia when BK-response is completely inhibited during periods from 5 to 30 min. Data represent the mean ⫾ SEM from separate 6 through 10 experiments. *P ⬍ 0.05, compared with the value of vehicle treated group at the corresponding time.

logical characterizations. Type I fibers are stimulated by BK, type II by 2-MeS ATP and type III by PGI2 agonist. The response induced by BK was completely abolished by capsaicin treatment that had been carried out to neonatal mice. As it is well accepted that capsaicin treatment degenerates primary afferent unmyelinated C fibers [4,6], the BK-response is thought to be nociceptive. Here we attempted to characterize the neurotransmitter to be released from such primary afferent fibers to the dorsal root of spinal cord by use of intrathecal injection of antagonists for NK1, NMDA and AMPA/kainate receptors, because these receptors and related neurotransmitters (substance P or glutamate) are representative candidates in primary pain transmission. As the BK-response was abolished by intrathecal injection of NK1-antagonist, but not by NMDA or AMPA/ kainate receptor antagonist, it is evident that BK selectively stimulates polymodal substance P -containing C-fibers, because substance P is predominantly found in small dorsal

root ganglion (DRG) neurons (C-fiber neuron) containing immunoreactive substance P [9]. Similar pharmacological characterizations were also carried out in the case with 2-MeS ATP, a P2X agonist that has a relative selectivity to P2X3 type receptor [4]. The 2-MeS ATP-induced response was mostly, but not completely sensitive to capsaicin treatment. As the response was not blocked by intrathecal injection of NK1-antagonist, but by NMDA antagonist (Fig. 1D), it is evident that BK- and 2-MeS ATP-induced responses are attributed to different nociceptive sensory neurons, though both neurons are sensitive to capsaicin. However it remains to be determined whether small parts of capsaicin-insensitive responses are nociceptive. There is a report that ATP-activated currents in small-sized DRG neurons (C fiber neurons) expressing only P2X3 are sensitive to capsaicin, whereas those in mediumsized neurons (probably A␦-fiber neurons) expressing both P2X2 and P2X3 are not [19]. Although A␦-neurons are also

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Fig. 3. Differential effects of SPN and MOR on 2-MeS ATP-induced responses. Doses for each drug are indicated in the figure. Other details are given in the legend of Fig. 2. Data represent the mean ⫾ SEM from separate four through six experiments. *P ⬍ 0.05, compared with the value of vehicle-treated group at the corresponding time.

known to be nociceptive ones, the possibility that capsaicininsensitive responses are attenuated to the stimulation of innocuous neurons can not be excluded. Regarding this observation there were reports that 2-MeS ATP also has weak agonist actions on P2Y1 receptor which couples to Gq-phospholipase C activation [11], and that P2Y1 receptors exist in sensory neurons that cause touch-induced impulse generation [14]. Thus the identification of such capsaicininsensitive neurons should require more experiments. The presence of type III fiber was observed when PGI2 agonist was given. The PGI2 agonist-induced response showed no sensitivity to capsaicin treatment, whereas the response was blocked by NMDA-antagonist. Although it is difficult to characterize the PGI2 agonist-induced response, it is evident that the response is derived from non-poly-

modal C fibers. As it is reported that PGI2 play a painstimulatory role in vivo from mutant mice lacking this receptor [13], the sensory neurons sensitive to PGI2 agonist should be more likely A␦-fibers than A␤-ones. Under these conditions, the inhibitory action of spinorphin is unique, because this peptide completely inhibited the 2-MeS ATP-induced response as well as BK-response, whereas the complete inhibition by morphine was only observed with the latter response. In addition to it, the mode of inhibition of BK-response by morphine was different from the case with spinorphin, because the former action was sensitive to PTX, whereas the latter was not. Although it is likely that the 2-MeS ATP-induced response includes unidentified mechanisms as well as the stimulation of capsaicin-sensitive nociceptor endings, the contribution of capsaicin-sensitive mechanism seems to be much higher than the other one (Fig. 1C). In the present study, we demonstrated that nociceptive primary afferent neurons could be classified into three-types from pharmacological studies including the different sensitivity to capsaicin and neurotransmitter antagonists. As such a strategy has not been reported elsewhere, the present finding should be new and valuable, though we understand that the present characterization of 3-types of responses through three different sensory stimulants is not perfect. Furthermore, we demonstrated that spinorphin may be a unique peptide to inhibit the nociception resistant to morphine. It would be interesting to determine whether spinorphin or more active related compounds inhibit neuropathic pain, known to be insensitive to morphine.

Fig. 4. Differential effects of SPN and MOR on PGI2 agonist -induced responses. Doses for each drug are indicated in the figure. Other details are given in the legend of Fig. 2. Data represent the mean ⫾ SEM from separate four through six experiments. *P ⬍ 0.05, compared with the value of vehicle-treated group on the corresponding time.

Acknowledgments Parts of this work were supported by Grants-in-Aid from the Ministry of Education, Science, Culture and Sports of

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Fig. 5. Differential effects of PTX on SPN- and MOR-induced analgesia. PTX (10 ng, i.pl.) or physiological saline (veh) was treated at 20 min before SPN (A: 3 nmol, i.pl.) or MOR (B: 1 nmol, i.pl.) injection. Results represent the data obtained 5 min after the toxin or vehicle-challenge as the mean ⫾ SEM from separate 4 through 12 experiments. *P ⬍ 0.05, compared with the value of vehicle treated group on the corresponding time.

Japan, by research grants from The Naito Foundation and Ono Medical Research Foundation.

References [1] Christiansen NO. Pertussis toxin inhibits the FMLP-induced membrane association of protein kinase C in human neutrophils. J Leukoc Biol 1990;47:60 –3. [2] Erchegyi J, Kastin AJ, Zadina JE, Qiu XD. Isolation of a heptapeptide Val-Val-Tyr-Pro-Trp-Thr-Gln (valorphin) with some opiate activity. Int J Pept Protein Res 1992;39:477– 84. [3] Glamsta EL, Marklund A, Hellman U, Wernstedt C, Terenius L, Nyberg F. Isolation and characterization of a hemoglobin-derived opioid peptide from the human pituitary gland. Regul Pept 1991;34: 169 –79. [4] Hamilton SG, Wade A, McMahon SB. The effects of inflammation and inflammatory mediators on nociceptive behaviour induced by ATP analogues in the rat. Br J Pharmacol 1999;126:326 –32. [5] Hazato T. [Enkephalin, and endogenous enkephalinase inhibitor]. Seikagaku 1994;66:57– 60. [6] Hiura A, Ishizuka H. Changes in features of degenerating primary sensory neurons with time after capsaicin treatment. Acta Neuropathol. (Berl) 1989;78:35– 46. [7] Inoue M, Kobayashi M, Kozaki S, Zimmer A, Ueda H. Nociceptin/ orphanin FQ-induced nociceptive responses through substance P release from peripheral nerve endings in mice. Proc Natl Acad Sci USA 1998;95:10949 –53. [8] Inoue M, Nakayamada H, Tokuyama S, Ueda H. Peripheral nonopioid analgesic effects of kyotorphin in mice. Neurosci Lett 1997; 236:60 –2. [9] Inoue M, Shimohira I, Yoshida A, Zimmer A, Takeshima H, Ueda H. Dose-related opposite modulation by nociceptin/orphanin FQ of substance P nociception in the nociceptors and spinal cord. J Pharmacol Exp Ther 1999;291:308 –13. [10] Inoue M, Tokuyama S, Nakayamada H, Ueda H. In vivo signal transduction of tetrodotoxin-sensitive nociceptive responses by substance P given into the planta of mouse hind limb. Cell Mol Neurobiol 1998;18:555– 61.

[11] Kunapuli SP, Daniel JL. P2 receptor subtypes in the cardiovascular system. Biochem J 1998;336:513–23. [12] Liang SL, Woodlock TJ, Whitin JC, Lichtman MA, Segel GB. Signal transduction in N-formyl-methionyl-leucyl-phenylalanine and concanavalin A stimulated human neutrophils: superoxide production without a rise in intracellular free calcium. J Cell Physiol 1990;145: 295–302. [13] Murata T, Ushikubi F, Matsuoka T, et al. Altered pain perception and inflammatory response in mice lacking prostacyclin receptor. Nature 1997;388:678 – 82. [14] Nakamura F, Strittmatter SM. P2Y1 purinergic receptors in sensory neurons: contribution to touch-induced impulse generation. Proc Natl Acad Sci USA 1996;93:10465–70. [15] Nishimura K, Hazato T. Isolation and identification of an endogenous inhibitor of enkephalin-degrading enzymes from bovine spinal cord. Biochem Biophys Res Commun 1993;194:713–9. [16] Nishimura K, Hazato T. [Spinorphin, a new inhibitor of enkephalindegrading enzymes derived from the bovine spinal cord]. Masui 1993;42:1497–503. [17] Nishimura K, Ueki M, Kaneto H, Hazato T. [Study of a new endogenous inhibitor of enkephalin-degrading enzymes; pharmacological function and metabolism of spinorphin]. Masui 1993;42:1663–70. [18] Ueda H. In vivo molecular signal transduction of peripheral mechanisms of pain. Jpn J Pharmacol 1999;79:263– 8. [19] Ueno S, Tsuda M, Iwanaga T, Inoue K. Cell type-specific ATPactivated responses in rat dorsal root ganglion neurons. Br J Pharmacol 1999;126:429 –36. [20] Yamamoto Y, Kanazawa H, Shimamura M, Ueki M, Hazato T. Inhibitory action of spinorphin, an endogenous regulator of enkephalin-degrading enzymes, on carrageenan-induced polymorphonuclear neutrophil accumulation in mouse air-pouches. Life Sci 1998;62: 1767–73. [21] Yamamoto Y, Kanazawa T, Shimamura M, Ueki M, Hazato T. Inhibitory effects of spinorphin, a novel endogenous regulator, on chemotaxis, O2- generation, and exocytosis by N-formylmethionylleucyl-phenylalanine (FMLP)-stimulated neutrophils. Biochem Pharmacol 1997;54:695–701. [22] Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 1983;16:109 –10.