Intracerebroventricular injection of prostaglandin E2 induces thermal hyperalgesia in rats: the possible involvement of EP3 receptors

BRAIN RESEARCH ELSEVIER

Brain Research 663 (1994) 287-292

Research report

Intracerebroventricular injection of prostaglandin E 2 induces thermal hyperalgesia in rats: the possible involvement of E P 3 receptors Takakazu Oka *, Shuji Aou, Tetsuro Hori Department of Physiology, Faculty of Medicine, Kyushu University 60, Fukuoka 812, Japan Accepted 16 August 1994

Abstract

To determine what types of prostanoid receptors are involved in the central effect of prostaglandin E 2 (PGE 2) on nociception, we administered PGE z and its agonists, i.e., 17-phenyl-w-trinor PGE 2 (an EP 1 receptor agonist), butaprost (an EP 2 receptor agonist), ll-deoxy PGE 1 (an EP2/EP 3 receptor agonist, EP 2 >> EP 3) and M&B28767 (an EP 3 receptor agonist) into the lateral cerebroventricle (LCV) of rats and observed the changes of paw-withdrawal latency on a hot plate. The LCV injection of PGE 2 (10 pg/kg-10 ng/kg), ll-deoxy PGE 1 (100 pg/kg-10 ng/kg) and M&B28767 (1 pg/kg-100 pg/kg) produced a significant reduction in the paw-withdrawal latency. The maximal reduction was observed 15 min after the LCV injection of these drugs. Neither 17-phenyl-w-trinor PGE 2 (1 pg/kg-1 /xg/kg) nor butaprost (1 pg/kg-100 p.g/kg) induced any significant changes in the paw-withdrawal latency. The LCV injection of PGE 2 (1 /xg/kg) and 17-phenyl-w-trinor PGE 2 (50 /xg/kg) increased the latency only 5 min after LCV injection. These findings indicate that the LCV injection of PGE 2 induces thermal hyperalgesia through EP 3 receptors and analgesia through EP 1 receptors by its central action in rats.

Keywords: Prostaglandin E2; Thermal hyperaigesia; Analgesia; Pain; M&B28767; 17-Phenyl-w-trinor PGE2; Central nervous system

1. Introduction

Prostaglandin E 2 ( P G E 2) is widely distributed in the brain [20] and exerts a variety of neurophysiological actions, i.e. fever [10,18], luteinizing hormone releasing-hormone release [22], sympathetic modulation [6] and wakefulness [15,16]. The binding sites of P G E 2 are also widely distributed in the brain [17,34,35]. In rats, either moderate or high bindings of P G E 2 are found in the nociceptive afferent pathways (superficial layers of the dorsal horn, i.e., laminae 1 and 2, caudal part of the spinal trigeminal nucleus, dorsal parabrachial nucleus and thalamic nuclei) and in the descending inhibitory pathways of nociception (central gray and dorsal raphe nuclei) [17]. This suggests the possibility that P G E 2 is involved, at least in part, in pain modulation by central mechanisms as well as the peripheral sensitizing effects on nociceptors [5,30,31].

* Corresponding author. Fax: (81) (92) 632-2373 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 1 0 0 9 - 9

However, previous reports on the central effects of P G E 2 on nociception are controversial. It has been shown that the intrathecal (i.t.) administration of P G E 2 induces hyperalgesia [29,32], whereas the intracerebroventricular (i.c.v.) or intracisternal (i.cist.) administration of P G E 2 has been reported to produce either hyperalgesia [21], hypoalgesia [26], bimodal (i.e. hyperalgesia at low doses and analgesia at high doses) [11] or no effect at all [28]. One possible explanation for these discrepancies is that multiple sites in the central nervous system (CNS) are involved in the PGE2-induced modulation of nociception in different ways. Recent studies have revealed that P G E 2 receptors can be pharmacologically classified into three subtypes, i.e. EP t, EP 2 and EP 3 receptors [4,8]. We thus hypothesized that each type of EP-receptor has a different effect on nociception, and that this might be another explanation for the different results regarding the effects of centrally administered P G E 2 on nociception. Therefore, in order to determine what types of prostanoid receptors are involved in the PGEz-induced

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changes in nociception, we administered P G E 2 and its agonists, i.e., 17-phenyl-w-trinor P G E 2 (an EP 1 receptor agonist), butaprost (a selective EP 2 receptor agonist) and ll-deoxy P G E l (an E P 2 / E P 3 receptor agonist, EP 2 >> EP 3) and M&B28767 (an EP 3 receptor agonist) into the lateral cerebroventricle (LCV) of rats and then observed the changes in the nociception as assessed by a hot-plate test. Part of these results has been previously reported in abstract form [24].

2. Materials and methods 2.1. Subjects Male Wistar rats (Kyudo, Tosu, Japan), weighing between 300 and 350 g, were used in all experiments. They were housed two per cage in a room maintained at an ambient temperature of 23_ I°C on a 12-h light/12-h dark cycle with lights on at 08.00 h. Food and water were given ad libitum.

2.2. Cannula implantation Under pentobarbitone Na anesthesia (50 m g / k g , i.p.), a 23-gauge stainless steel cannula containing a 30-gauge stainless steel wire as a stylet was implanted stereotaxically into the LCV. After confirming the correct placement of the cannula in the LCV by the rise of cerebrospinal fluid in the cannula, the cannula was fixed to the skull with acrylic dental cement. Thereafter, animals were administered sulfamethoxide (100 m g / r a t , i.p.) and then returned to the colony and housed individually. During a recovery period of at least 7 days, the animals were transported to the experimental room and placed on an unheated (25+_0.1°C) aluminum plate which was used for the hot-plate test for about 5 min daily to familialize them with the experimental procedures.

2.3. Drugs PGE 2 was purchased from Sigma, St Louis, MO. 17-Phenyl-w-trinor PGE 2 and 11-deoxy PGE l were obtained from Cayman Chemicals, USA. The following compounds were gifts which we would like to gratefully acknowledge: SC19220 from Dr. G. Fleet, Searle, USA; butaprost from Dr. P.J. Gardiner, Bayer, UK Ltd., UK; M&B28767 from Dr. L. Webb, Rhone-Poulenc, Ltd., UK. PGE 2 was dissolved in saline. M&B28767 was dissolved in 1% NaHCO 3 in saline. Butaprost, ll-deoxy PGE~ and 17-phenyl-to-trinor PGE 2 were dissolved in 99.5% ethanol. They were stored at 80°C and diluted with saline before use. Each drug was injected in a volume of 5/zl into the LCV of rats. The same saline dilution of ethanol or NaHCO 3 as the maximal dose of the test solutions was used as the control solution of each drug. SC19220 was dissolved in dimethyl sulfoxide (DMSO) and injected in a volume of 3/xl into the LCV. All solutions were passed through a 0.22-~m 'millipore filter (Millipore Lab., Tokyo, Japan)' before injection. All glassware, syringes and injection needles were autoclaved before use. -

2. 4. Experimental procedures The effects of the LCV injections of PGE 2 and its related substances on nociception were determined using the hot-plate test. On the experimental day, each rat was placed on an aluminum hot

(50+0.1°C) plate and the time until the animal showed the first avoidance response (i.e. withdrawing, jumping or licking the hindpaw) was recorded. The paw-withdrawal latency was determined three times at 10 rain intervals before the LCV injection and the average of three values was taken as the baseline latency. Only rats which consistently exhibited latencies between 11 and 29 s (three values did not differ from each other by 25%) were used in the experiments. The paw-withdrawal latency was measured 5, 15, 30 and 60 min after the injection of drugs or their vehicles. The rats were divided into different groups (n = 7-10/group). The control animals received an LCV injection of vehicle (5 ~zl). The PGE 2- and its agonists-treated groups were given different doses (between 0.1 p g / k g and 100 ~ g / k g ) of each drug. Both the control and PGE2-treated groups were subdivided into groups which were pretreated either with SC19220 or DMSO which were given into the LCV 10 min before injections of PGE 2 or saline. For the LCV injection, the guide cannula was opened and a 30-gauge injection needle, which was connected to a 10-/zl microsyringe, was inserted into the cannula 0.5 mm beyond its tip. The injection rate was 1 /xl/min. After injection, the injection needle was replaced by the stylet and then the animal was returned to its home cage until it was used for further testing at the scheduled times. All the experiments were performed between 10.00 and 16.00 in a laboratory room kept at 23_+1°C. Each rat was used for three experiments with different drugs on different days at least 5 days apart.

2.5. Statistical analyses The data are presented as mean _+S.E.M. Analysis of variance (ANOVA) was used to determine statistical differences between the values of the vehicle control group and those of the drug-treated groups. When a significant overall F score was obtained, comparisons of the individual groups were carried out by Dunnett's test. Differences were considered to be significant if P < 0.05.

3. Results

3.1. The effects of an L C V injection of PGE 2 on nociception The LCV injection of P G E 2 (1 p g / k g ) did not produce any significant changes in the nociceptive responses. The LCV injection of P G E 2 (10 p g / k g - 1 0 ng/kg) reduced the paw-withdrawal latency in a Ushaped, dose-dependent manner, producing a maximal response at 1 n g / k g (Figs. 1A and 1B). At this dose, a significant reduction of the paw-withdrawal latency began to appear 5 min after the LCV injection, reached a peak (12.0_+ 1.1 s) within 15 min and then gradually subsided. The LCV injection of P G E 2 of 100 n g / k g did not produce any significant changes in the nociceptive responses. However, when P G E 2 of 1 /zg/kg was injected, the paw-withdrawal latency significantly increased (27.0 _ 2.4 s) only 5 min after injection. On the other hand, saline-treated control rats responded to the hot plate with fairly constant latencies between 18.2 _+ 1.2 and 20.3 +_ 1.5 s during the observation period, which did not differ significantly from the corresponding baseline latency (19.5 _+ 1.8 s).

T. Oka et al. / Brain Research 663 (1994) 287-292

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Fig. 1. Effect of an intracerebroventricular (LCV) injection of PGE 2 on hot plate responses as measured by the paw-withdrawal latency in rats. In A, five groups of rats (n = 10/group) were injected with PGE 2 at 1 pg/kg ( zx), 10 pg/kg ( • ), 100 pg/kg ( • ) or 1 ng/kg (e), or saline (0). In B, five groups of rats (n = 10/group) were injected with PGE 2 at 1 ng/kg (e), 10 ng/kg ( • ) , 100 ng/kg ( r, ) or 1/xg/kg (13), or saline (0). The data on the rats injected with PGE 2 of 1 ng/kg (e) and saline (©) shown in A are also illustrated in B for comparison. Each point represents the mean_+ S.E.M. The symbols adjacent to points represent the level of significance (one-way ANOVA followed by Dunnett's test) when compared with the saline-injected control. * P < 0.05; ** P < 0.01.

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Pre(0) 20 40 1~0 Time after LCV injection (min) Fig. 2. Effect of an LCV injection of 17-phenyl-w-trinor PGE 2 on the paw-withdrawal latency on a hot-plate in rats. Six groups of rats (n = 8/group) were injected with 17-phenyl-oJ-trinor PGE 2 at 1 pg/kg ( zx), 100 pg/kg ( 13), 10 ng/kg ( • ), 1 ~zg/kg ( • ) or 50 ~ g / k g (e), or vehicle (©). The same format was used as in Fig. 1.

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3.2. The effects o f an L C V injection o f EP-receptor agonists on nociception To investigate what types of prostanoid receptors a r e i n v o l v e d in t h e P G E 2 - i n d u c e d c h a n g e s in t h e p a w withdrawal latency, we injected the prostanoid receptors a g o n i s t s , i.e., 17-phenyl-o~-trinor P G E 2 (an E P t receptor agonist), butaprost (an selective EP 2 receptor agonist), l l - d e o x y P G E 1 ( a n E P 2 / E P 3 r e c e p t o r a g o nist, E P 2 >> EP3) , M & B 2 8 7 6 7 ( a n E P 3 r e c e p t o r a g o nist) a n d t h e i r v e h i c l e s a n d t h e n o b s e r v e d t h e c h a n g e s in t h e p a w - w i t h d r a w a l l a t e n c y o n t h e h o t - p l a t e . I n e a c h g r o u p , t h e v e h i c l e - t r e a t e d c o n t r o l rats r e s p o n d e d to t h e h o t - p l a t e w i t h fairly c o n s t a n t l a t e n c i e s , w h i c h w e r e not significantly different from the corresponding baseline latencies. The LCV injection of 17-phenyl-oJ-trinor P G E 2 (1 p g / k g - 1 / x g / k g ) d i d n o t p r o d u c e any signific a n t c h a n g e s in t h e p a w - w i t h d r a w a l latency• H o w e v e r , 17-phenyl-~o-trinor P G E 2 in a d o s e o f 50 / z g / k g inc r e a s e d t h e p a w - w i t h d r a w a l l a t e n c y (26.5 + 1.8 s) o n l y

20

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5 mm after injection (Fig. 2). A m o n g t h e E P 2 r e c e p t o r a g o n i s t s , b u t a p r o s t (1 pg/kg-100 / z g / k g ) d i d n o t p r o d u c e any s i g n i f i c a n t c h a n g e s in n o c i c e p t i v e b e h a v i o r (Fig. 3), w h e r e a s l l d e o x y P G E 1 at c o n c e n t r a t i o n s b e t w e e n 100 p g / k g a n d 10 n g / k g r e d u c e d t h e p a w - w i t h d r a w a l l a t e n c y (Fig. 4). T h e m a x i m a l r e d u c t i o n w a s o b t a i n e d 15 m i n a f t e r L C V i n j e c t i o n at a d o s e o f 100 p g / k g . T h e L C V i n j e c t i o n o f l l - d e o x y - P G E 1 o f 10 p g / k g , 100 n g / k g a n d 5 0 / x g / k g did not change the nociceptive responses. T h e L C V i n j e c t i o n o f M & B 2 8 7 6 7 (1 p g / k g - 1 0 0 p g / k g ) r e d u c e d t h e p a w - w i t h d r a w a l l a t e n c y in a U s h a p e d , d o s e - d e p e n d e n t m a n n e r (Fig. 5). T h e m a x i m a l r e d u c t i o n o f t h e p a w - w i t h d r a w a l l a t e n c y was o b t a i n e d

~lS m

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Fig. 4• Effect of an LCV injection of ll-deoxy PGE t on the pawwithdrawal latency on a hot-plate test in rats. In A, three groups of rats (n = 8/group) were injected with ll-deoxy PGE 1 at 10 pg/kg (O) or 100 pg/kg (e), or vehicle (©). In B, six groups of rats (n = 8/group) were injected with ll-deoxy PGE I at 100 pg/kg (e), 1 ng/kg (A), 10 ng/kg ( • ) , 100 ng (A) or 50 tzg/kg (U), or vehicle (O). The data on the rats injected with ll-deoxy PGE 1 of 100 pg/kg (e) and vehicle (o) shown in A are also illustrated in B for comparison. The same format was used as in Fig. 1.

7". Oka et al. / B r a i n Research 663 (1994) 287-292

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Fig. 5. Effect of an LCV injection of M&B28767 on the pawwithdrawal latency on a hot-plate in rats. In A, four groups of rats (n = 8 / g r o u p ) were injected with M&B28767 at 0.1 p g / k g (z~), 1 p g / k g ( • ) or 10 p g / k g (e), or vehicle (©). In B, six groups of rats (n = 8 / g r o u p ) were injected with M&B28767 at 10 p g / k g (e), 100 p g / k g ( • ), 1 n g / k g ( A ), 1 /zg/kg ( [] ) or 2 0 / z g / k g ( • ), or vehicle (o). The data on the rats injected with M&B28767 of 10 p g / k g (e) and vehicle ( o ) shown in A are also illustrated in B for comparison. The same format was used as in Fig. 1.

15 min after injection at a dose of 10 p g / k g (12.2 _+ 1.2 s). When the amount of M&B28767 was increased to more than 1 n g / k g (up to 20 /xg/kg) or decreased to 0.1 pg/kg, the paw-withdrawal latency did not change significantly. 3.3. The effect of EP 1 receptor antagonist on the PGE 2induced changes in nociception To further confirm that the EP l receptor is involved in the PGE2-induced prolongation of paw-withdrawal latency, SC19220 (300/xg/kg) or its vehicle was administered to the LCV of the rats 10 • i n before the LCV injection of either PGE 2 (1 /xg/kg) or saline and the changes in the paw-withdrawal latency on the hot plate were then measured over a 60 min period. The rats 30 v >, O C

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Fig. 6. Effects of pretreatment with SC19220 on PGE2-induced increase in the paw-withdrawal latency in rats. The rats were injected (LCV) with SC19220 (300 Izg/kg) or vehicle 10 min before PGE 2 (1 /zg/kg) or saline was given (LCV). [] SC19220/saline; o vehicle/saline; • vehicle/PGE2; • SC19220/PGE2. n = 7, respectively. The symbols adjacent to points represent the level of significance when compared with the vehicle/saline-treated rats. * P < 0.05, ** P < 0.01.

which received an LCV injection of PGE 2 at 1 /xg/kg following the vehicle treatment showed a significant prolongation in the paw-withdrawal latency only 5 min after injection (Fig. 6), which was similar to that of the rats which received 1 /zg/kg of P G E 2 alone (Fig. 1A) in terms of its magnitude and time course. Pretreatment with SC19220 (300 /zg/kg) completely blocked the PGE2-induced prolongation in the paw-withdrawal latency, whereas SC19220 (300 /zg/kg) followed by a saline injection had no effect on the nociceptive behavior.

4. Discussion

The changes in the paw-withdrawal latency of rats after the LCV injection of P G E 2 and its agonists are not considered to be secondarily caused by possible changes in either the body temperature or the level of vigilance. In our previous study [25], no significant changes in the colonic temperature were observed in rats after the LCV injection of P G E 2 (10 p g / k g - 1 0 ng/kg), 11-deoxy P G E 1 (100 p g / k g - 1 0 ng/kg) or M & B28767 (1 p g / k g - 1 0 0 pg/kg), which were found to shorten the paw-withdrawal latency in the present study. The awaking action of PGE z is also unlikely to cause the decrease in the paw-withdrawal latency, because it was shown that reduction of sleep during intracerebral infusion of PGE 2 was accompanied by an elevation of the body temperature [15,16] whereas only non-pyrogenic doses of PGE 2 decreased the pawwithdrawal latency in the present study. On the other hand, the increase in the paw-withdrawal latency observed after LCV injection of P G E 2 (1 /xg/kg) or 17-phenyMo-trinor PGE 2 (50 ~ g / k g ) may be accompanied by hyperthermia according to our previous study [25]. However, our electrophysiological study has revealed that the firing rate responses of nociceptive neurons in the trigeminal nucleus caudalis to noxious stimuli were suppressed by an LCV injection of P G E 2 (1 /~g/kg) or 17-phenyl-o~-trinor P G E 2 ( 5 0 / x g / k g ) and enhanced by P G E 2 (1 ng/kg), ll-deoxy P G E 1 (100 pg/kg) or M&B28767 (10 pg/kg) (unpublished data). These findings, taken together, indicate that the decrease and increase of the paw-withdrawal latency after LCV injections of PGE 2 and its analogues reflect hyperalgesia and analgesia, respectively. PGs in the brain have been suggested to be involved in the modulation of nociception. There are many lines of evidence that non-steroidal anti-inflammatory drugs (NSAIDs), which are known to inhibit PG synthesis [7,33], induce analgesia by their central actions. For instance, the i.c.v, injection of NSAIDs abolishes the reperfusion hyperalgesia of the rat's tail at lower doses than those necessary when administered systemically [9]. A microinjection of NSAIDs into the preoptic

T. Oka et al. / Brain Research 663 (1994) 287-292

anterior hypothalamic area [27] or the periaqueductal gray matter [2] induces analgesia. The dorsomedial part of the ventral nucleus (VDM) of the thalamus is also known to be involved in the central antinociceptive effect of NSAIDs [12]. We recently observed that an LCV injection of non-pyrogenic doses of recombinant human interleukin-1/3 induced hyperalgesia, which was blocked by a cyclooxygenase inhibitor (Na salicylate) in rats [23]. Although these findings do not elucidate what types of PGs are involved in the central effects of NSAIDs, P G E 2 may be one of the candidates because P G E 2 is widely distributed in the brain [20] and the binding sites of P G E 2 are found in the nociceptive afferent pathways and in the descending inhibitory pathways of nociception [17,34,35]. The present study has revealed that the central effects of P G E 2 on nociception differ in relation to its concentration, i.e., there was no effect at 1 p g / k g , a hyperalgesic effect at lower doses (10 p g / k g - 1 0 ng/kg), no effect at 100 n g / k g and an analgesic effect at a higher dose (1 /zg/kg). In addition, hyperalgesia was observed after the LCV injection of M&B28767 (an EP 3 agonist) at doses 10-100 times less than those necessary for P G E 2 to cause an equivalent degree of hyperalgesia, l l - D e o x y PGE~ (100 p g / k g - 1 0 ng/kg), an E P 2 / E P 3 agonist, also produced a reduction in the paw-withdrawal latency. This hyperalgesic action of 11-deoxy P G E l may be due to its EP 3 receptor agonistic activity [13] because butaprost, a selective EP 2 agonist, had no effect on nociception at any doses tested. On the other hand, 17-phenyl-to-trinor P G E 1 (50 /xg/kg), an EP~ receptor agonist, induced a transient analgesia which was similar in terms of the magnitude and time course to that observed after injection of P G E 2 (1 /xg/kg). These results, taken together, suggest that two types of EP receptors are involved in the pain modulating actions of P G E 2 in rats, i.e., a hyperalgesic action is mediated by EP 3 receptors while an analgesic action is mediated by EP 1 receptors. If neurons in the different sites of the brain have different EP receptors or a different affinity for each EP receptor, it would not be strange that contradictory conclusions could be drawn from previous studies on nociception after the i.c.v, or i.cist, injection of P G E 2 [11,21,26,28]. At present it is unclear as to why the hyperalgesic effects of M&B28767 and 11-deoxy PGEj disappeared at higher doses and exhibited a U-shaped, dose-response curve. One explanation might be that both drugs have a very weak EP t receptor agonistic activity [13] and the hyperalgesic effects thus may be obscured by possible analgesic effects at higher doses which are mediated by EP~ receptors. Another possibility may be that these drugs at higher doses might desensitize EP 3 receptors since a repeated application of P G E 2 to hypothalamic neurons results in tachyphylaxis [14].

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The stimulation of each type of EP-receptor is known to activate or inhibit the different types of second messengers. The stimulation of EP 1 receptors has been demonstrated to activate the inositol triphosphate (IP 3) metabolism in cultured bovine adrenal chromaffin cells [36]. On the other hand, the activation of EP 2 and EP 3 receptors results in the increase and decrease of intracellular cyclic AMP (cAMP), respectively [1,3,19]. Therefore, we can hypothesize that the decrease of cAMP might be involved in the central PGE2-induced thermal hyperalgesia, while the activation of IP 3 metabolism might be involved in the central PGEe-induced analgesia. In conclusion, an LCV injection of P G E 2 induces thermal hyperalgesia at low doses and analgesia at high doses. The hyperalgesic effect of P G E 2 may be mediated by EP 3 receptors which are known to decrease the intracellular cAMP while the analgesic effect of P G E 2 may be mediated by EP 1 receptors which is thought to activate the IP 3 pathway.

Acknowledgements The following compounds were gifts which the authors gratefully acknowledge: SC19220 from Dr. G. Fleet, Searle, USA; butaprost from Dr. P.J. Gardiner, Bayer, UK; M&B28767 from Dr. L. Webb, RhonePoulenc, UK. We also express our gratitude to Drs. Y. Watanabe (Osaka Biochemical Institute), S. Narumiya (Kyoto University School of Medicine) and K. Mizumura (Institute of Environmental Medicine, Nagoya University) for providing information about EP receptors agonists and antagonists. We are grateful to B.C. Quinn, Kyushu University, for reading the manuscript. This work was performed through Special Coordination Funds of the Science and Technology Agency of the Japanese Government (to T. Hori) and was also supported in part by Grant-in-Aid for Scientific Research on Priority Areas 'Pain' (03260102 to T. Hori) and Grant-in-Aid for Scientific Research (06454153 and 06557006 to T. Hori) from the Ministry of Education, Science, and Culture, Japan.

References [1] Brunton, L.L., Wiklund, R.A., Van Arsdale, P.M. and Gilman, A.G., Binding of [3H]prostaglandin E 1 to putative receptors linked to adenylate cyclase of cultured cell clones, J. Biol. Chem., 251 (1976) 3037-3044. [2] Carlsson, K-H., Helmreich, J. and Jurna, I., Activationof inhibition from the periaqueductal grey matter mediates central analgesic effect of metamizole (Dipyrone), Pain, 27 (1986) 373-390. [3] Chen, M.C.Y., Amirian, D.A., Toomey, M., Sanders, M.J. and Soll, A.H., Prostanoid inhibition of canine parietal cells: mediation by the inhibitoryguanosine triphosphate-binding protein of adenyl cyclase, Gastroenterology, 94 (1988) 1121-1129.

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