Pii: s0014-2999(01)009

Antinociceptive effects of intracerebroventricularly administered Masato Fukui, Takayuki Nakagawa, Masabumi Minami, Masamichi Satoh) Department of Molecular Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto UniÕersity, Kyoto 606-8501, Japan Received 4 January 2001; received in revised form 21 March 2001; accepted 28 March 2001 Abstract
We examined the effects of adenosine 5 -triphosphate Ž ATP and its analogues administered intracerebroventricularly on nociceptive thresholds in rats. Intracerebroventricular Ži.c.v. administration of ATP Ž10 and 100 nmolr rat dose-dependently elevated the mechanical nociceptive threshold in the paw pressure test. These antinociceptive effects were rapid and short-lasting, peaking at 5 min and disappearing by 20 min after theadministration. However, i.c.v. administration of b,g-methylene-ATP Ž1–30 nmolr significant effects on the mechanical nociceptive threshold. In other tests, i.c.v. administration of a ,b-methylene-ATP Ž10 and 30nmolr rat prolonged the thermal nociceptive latency in the hot plate test, but only a higher dose Ž30 nmolr prolonged the latency in the tail flick test. a ,b-Methylene-ATP produced no motor deficit in the inclined plane test. These results suggestthat P2X purinoceptors play an inhibitory role in nociception at the supraspinal level. q 2001 Elsevier Science B.V. All rights reserved.
Keywords: ATP; a ,b-Methylene-ATP; Antinociception; Purinoceptor 1. Introduction
ported that mRNA of the P2X purinoceptor in the dorsal In addition to diverse intracellular roles, extracellular root ganglia is selectively expressed in capsaicin-sensitive, small diameter afferent neurons, which are probably asso- neurotransmitter or neuromodulater in both the peripheral 1997 demonstrated that nociceptive, but not non-nocicep- systems ŽEdwards et al., 1992; Ueno et al., 1992; Jo and tive, sensory neurons had P2X immunoreactivity in their 1999 . ATP is contained in synaptic vesicles nerve endings and cell bodies. In electrophysiological stud- and co-released with noradrenaline, acethylcholine or other ies, ATP and a ,b-methylene-ATP, a P2X receptor agonist, substances ŽSneddon et al., 1982; Stone, 1981; Jo and evoked inward currents in capsaicin-sensitive, small diam- 1999 , and then acts on specific receptors, eter dorsal root ganglion neurons ŽUeno et al., designated as P2 purinoceptors, on the cell surface. P2 spinal dorsal horn neurons ŽBardoni et al., 1997; Ping et purinoceptors are classified into two subfamilies, ionotropic 1998 . Furthermore, in vivo studies have provided a P2X receptors and metabotropic P2Y receptors, on the body of evidence of the role of P2 purinoceptors at periph- basis of their structures and signal transduction systems eral and spinal sites in nociception. Peripheral administra- tion of ATP and a ,b-methylene-ATP have been shown to of P2X receptors and five subtypes of P2Y receptors have cause nociceptive responses ŽBland-Ward and Humphrey, been cloned as P2 purinoceptors expressed in mammalian Recent studies suggest the involvement of ATP and its tration of a ,b-methylene-ATP induced thermal hyperalge- receptors in peripheral and spinal nociceptive transmission sia, which was blocked by P2 purinoceptor antagonistsŽDriessen et al., 1994; Tsuda et al., tions strongly support the idea that ATP plays a crucial ) Corresponding author. Tel.: q81-75-753-4526; fax: q81-75-753- role in facilitating pain transmission at peripheral and E-mail address: [email protected] ŽM.
spinal sites, probably via the P2X purinoceptor.
0014-2999r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 0 1 4 - 2 9 9 9 Ž 0 1 . 0 0 9 4 7 - 5 M. Fukui et al.r European Journal of Pharmacology 419 ( At supraspinal sites, several studies have shown that ATP and its analogues induced fast synaptic currents in thecultured neurons derived from the hippocampus ŽInoue et Mechanical nociceptive threshold was evaluated by the 1992 and nucleus of the solitary trace ŽUeno et al., paw pressure test using an analgesimeter ŽUgo Basile, 1992 as well as in the slices from the rat medial habenula Italy with a cuneate piston. The piston was put on the right hind paw and the pressure was loaded at a rate of it was reported that ATP enhances or inhibits the release 32 grs. The pressure, which elicited paw withdrawal of some neurotransmitters including noradrenaline ŽVon behavior, was determined as a nociceptive threshold.
For assessing thermal nociception, the hot plate test was performed by placing the rat on a plate heated to 528C and measuring the latency to licking a hindpaw or jumping.
The cut-off time was 60 s to prevent tissue damage. The considered to play various physiological roles at supraspinal tail flick test was performed with the use of a light beam sites. However, little is known about the involvement of supraspinal ATP and its receptors in nociception. The goal that the radiant heat was focused onto the dorsal surface of of the present study was to determine the effects of ATP the tail at 5–6 cm proximal to the tip. The latency to analogues administered i.c.v. on mechanical and thermal withdrawing the tail was recorded. The cut-off time was 15 nociception and to assess whether the effects observed can be ascribed to a particular type or subtype of P2 purinocep- The inclined plane test was carried out using a sliding tors through the use of type- or subtype-selective agonists.
tions. Each rat was placed on the stainless steel plate 2. Materials and methods
inclined at 308, and the angle of the plate was increased ata rate of 28rs. The angle at which the animal began to slip 2.1. Animals and surgical procedures to slip down at 46.0 " 0.88 Ž n s All experiments using male Sprague–Dawley rats each animals was calculated in accordance with the follow- weighing 220–280 g followed the ethical guidelines for investigations of experimental pain in conscious animals r Žslope angle at which the animal began to slip down cycle with free access to food and water. Under pento- The procedures for behavioral testing were carried out barbital Ž50 mgrkg, i.p. anesthesia, a stainless steel guide three times per day for habituation. After 2 days of habitu- ation, the threshold or latency was measured following two 2.0. implanted on the right side according to the atlast of additional habituation procedures, and the value was taken as a control. Soon after measuring the control value, the were returned to cages and housed individually. They were drugs were administered intracerebroventricularly and the allowed to recover for 5 to 7 days until the following threshold or latency was measured at 5, 10, 20, 30 and 60 min after the intracerebroventricular Ži.c.v. administration.
2.2. Drugs and drug administration ATP, a ,b-methylene-ATP ŽP2X - and P2X -selective Statistical analyses were performed by the Dunnett multiple comparisons test following one-way analysis of agonist , b,g-methylene-ATP ŽP2X -selec- ANOVA or Mann–Whitney U-test. Differences agonist and uridine 5 -triphosphate ŽUTP, P2Y-selec- at P - 0.05 were considered significant.
agonist were purchased from Sigma ŽSt. Louis, MO, USA . These drugs were dissolved in phosphate-bufferedsaline Ž PBS and administered via the injection cannula, 3. Results
which reached the right lateral ventricle ŽP 0.8, L 1.5, H4.0. when attached to the guide cannula. The i.c.v. injec- 3.1. Effects of i.c.Õ. administration of ATP and its ana- tion was carried out in a volume of 5 ml at a constant rate logues on mechanical nociceptiÕe threshold in the paw and administered intraperitoneally. The experimenter wasnot informed of the names and doses of drugs until the measurements of nociceptive threshold or latency and anal- but significantly, elevated the mechanical nociceptive yses of the data had been completed.
threshold to the paw-pressure stimulation ŽFig.
M. Fukui et al.r European Journal of Pharmacology 419 ( Fig. 1. The effects of i.c.v. administration of ATP Ž .
nociceptive threshold in the paw pressure test. Drugs were administered intracerebroventricularly at time 0. The nociceptive threshold of each animalbefore the i.c.v. administration served as the control value Ž 100% . The values are presented as the means of the % of controls " S.E.M Ž n s 6– P - 0.001 compared with the vehicle-treated group Žmultiple comparison by Dunnett multiple comparisons M. Fukui et al.r European Journal of Pharmacology 419 ( In contrast, i.c.v. administration of b,g-methylene-ATPŽ1–30 nmolr significantly elevate the mechanical nociceptive thresholdŽFig. 1D,E.
3.2. Effect of i.c.Õ. administration of a ,b-methylene-ATPon thermal nociceptiÕe latency in the hot plate test and thetail flick test In the hot plate test, i.c.v. administration of a ,b-meth- short-lasting prolongation of thermal nociceptive latencyŽFig.
2A . A significant effect was observed at 5 min after Fig. 2. The effects of i.c.v. administration of a ,b-methylene-ATP on thethermal nociceptive latency in the hot plate test Ž .
Drugs were administered intracerebroventricularly at time 0. The values are presented as the means"S.E.M. of the latency Ž .
P - 0.01 compared with the vehicle-treated group Žmultiple comparison by Dunnett multiple comparisons administration of a ,b-methylene-ATP Ž1–30 nmolr produced a dose-dependent elevation of the mechanicalnociceptive threshold ŽFig.
the threshold was observed at 5 min after i.c.v. administra-tion of a ,b-methylene-ATP at doses of 10 and 30 nmolrratŽ156 " 12% and 173 " 12% of control, at 10 min at a dose of 30 nmolrrat Ž135 " 13% of compared with the group administered with vehicle Ž102 "3% and 97 " 3% of control, rapid and short-lasting, which peaked at 5 min and disap-peared by 20 min after i.c.v. administration. Similarly,Bz-ATP Ž1–30 nmolr

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