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Effects induced by cysteamine on chemically-induced nociception in mice
Life Sciences, Vol. 54, No. 15, pp. 1091-1099, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved o024-3205/94 $6.0o + .0o
Pergamon
EFFECTS INDUCED BY CYSTEAMINE ON CHEMICALLY-INDUCED NOCICEPTION IN MICE
Stefano Pieretti I , Amalia Di Giannuario I , Anna Capasso 2, Ludovico Sorrentino 2 and Alberto Loizzo 1
llstituto Superiore di Sanita', V.Le Regina Elena 299, 00161, Rome and 2 School of Pharmacy, University of Salerno, P.a V. Emanuele 9, 88084, Penta di Fisciano, Fisciano, Salerno, Italy ( R e c e i v e d in final f o r m J a n u a r y 20, 1994)
Summary The effects were investigated of cysteamine - a well known somatostatin depletor - on the pain induced by chemical stimuli in mice. Cysteamine injected intraperitoneally 4 h before the test at doses of 50 and 100 mg/kg reduced the second phase of the licking response which was induced by formalin injected into the hind paw. Furthermore, cysteamine administered at the doses of 10, 50 and 100 mg/kg reduced the writhing induced by acetic acid. Naloxone, yohimbine and CGP 35348 administered in cysteamine-pretreated animals were not able to change the cysteamine antinociceptive effects in the formalin test. Intrathecally injected somatostatin was able to revert the cysteamine antinociceptive effects in the second phase of the formalin test and in the writhing test, whereas intracerebroventricularly injected somatostatin reduced the antinociceptive effects induced by cysteamine in the second phase of the formalin test. Intrathecally injected cyclo(7-aminoheptanoyI-Phe-D-Trp-LysThr[Bzl]) - a reported somatostatin antagonist increased cystearnine antinociceptive effects in the second phase of the formalin test and in the writhing test. These results suggest that somatostatin is involved in the effects of cysteamine on the nociceptive threshold. Cysteamine (beta-mercaptoethylamine) is a thiol compound that depletes somatostatin in the brain (1,2) and spinal cord (3). This depletion is observed within minutes after administration; after 4 h it reaches maximal values, and within 24-78 h it is absent (4-8). The lack of cysteamine effects on other brain peptides (9-11) has suggested that cysteamine may be a potentially useful pharmacological tool for understanding the role of somatostatin in the central nervous system. Few data are reported on the effects induced by cysteamine on the nociceptive threshold. In this report Correspondence to: Stefano Pieretti, Istituto Superiore di Sanita', v.le Regina Elena 299, 00161, Rome, Italy. Phone (1139)-6-4990; fax (1139)-6-4440053
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we investigate the effects induced by cysteamine administered systemically on the licking response induced by formalin injected subcutaneously into the hind paw. For comparative purposes the cysteamine effects on the writhing induced by acetic acid injected intraperitoneally in mice was also investigated. Taking into cosideration the previously reported time-dependent somatostatin depletion caused by cysteamine (4-8), in the first series of experiments we investigated whether cysteamine injected 1, 4 and 24 h before the experimental sessions might be able to change the response to nociceptive stimuli. Since drugs acting on opioid (12) as well as alfa-2 (13) or GABA B (14) receptors are able to induce antinociception, we investigated whether naloxone, yohimbine or CGP 35348 - a reported GABA B antagonist (15) - might be able to change cysteamineinduced antinociceptive effects. Furthermore, the effects induced by somatostatin and cyclo(7-aminoheptanoyI-Phe-D-Trp-Lys-Thr[Bzl]) - a reported somatostatin antagonist (16) - on cysteamine-induced antinociception were also investigated in cysteamine pretreated animals. Since cysteamine may decrease locomotor activity (5) and drugs that impair locomotor activity may produce false positive results in the nociceptive test (17), we performed some experiments before the nociceptive test with the aim of evaluating the cysteamine effects on this behavior. Materials and Methods Male CD-1 mice (Charles River, Italy) weighing 25-30 gr were used in the experiments. The animals were housed in colony cages (4 mice each) with free access to food and water prior to the experiments. They were maintained in a climate and light controlled room (23 + 1° C, 12/12 h dark/light cycle with light on at 07:00) for at least 7 days before testing. Testing took place during the light phase, in the climatized room. Each animal was used only in one experimental session. In all experiments attention was paid to the ethical guidelines for investigations of experimental pain in conscious animals (18). In the formalin test, 20 ul of a 1% solution of formalin in saline was injected subcutaneously (s.c.) into the dorsal surface of the right hind paw of the mouse using a microsyringe with a 27-gauge needle. The formalin injection produced a distinct biphasic response consisting in licking or biting the paw. The first phase occurred from 0 to 5 min after formalin injection and the second phase, from 15 to 30 min afterwards. After the formalin injection the mouse was put into a plexiglass cage (30 cm x 14 cm x 12 cm) which served as an observation chamber, and the total amount of time the animal spent licking or biting the paw after the formalin injection was recorded every 5 min in both the first phase and second phase (19). In the writhing test, mice were given an intraperitoneal (i.p.) injection of 0.6% v/v acetic acid in a volume of 10 ml/kg. Acetic acid induces a series of writhings, consisting in abdominal contraction and hindlimb extension and these were recorded over a 10 min period beginning 5 min after acetic acid injection (20). Locomotor activity was measured as previously reported (21). Briefly, mice were randomly assigned to one experimental group and tested in groups of four. The animals were placed in the activity cage (Ugo Basile, Italy) for at least 30 min before receiving the injection of drugs. Temperature, sound and light conditions were maintaJned uniform during the course of the experiments. Measurements were carried out at ten-
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min intervals and cumulative counts were recorded after two h. Locomotor activity was measured 0-2, 3-5 and 23-25 h after cysteamine administation. Motor coordination of the mice was evaluated by using a rotarod apparatus (Ugo Basile, Italy) consisting of a bar with a diameter of 3.0 cm, subdivided into five compartments by a disk 24 cm in diameter. The bar rotated at a constant speed of 16 rev/min. A preliminary selection of mice was made on the day of experiment by excluding those that did not remain on the rotarod bar for two consecutive periods of 45 sec each. Afterwards, mice were tested 10 min and 1, 4 and 24 h after cysteamine administration, and the time spent on the rotating bar was recored for a maximum of 45 sec (22). On the day of testing all drugs used in the experimental sessions were dissolved in saline for administration. Drugs were injected in a volume of 5 ml/kg for i.p. administrations; for intracerebroventricular (i.c.v.) and intrathecal (i.t.) administrations, they were injected in a volume of 2.5 ul/mouse using a 10 ul Hamilton syringe with a 27 gauge needle. Cysteamine solution was neutralized to pH 7.2 using 0.1 M NaOH. The i.c.v. injection was performed using the method of Haley and McCormick (23, 24); the i.t. injection was made according to the method of Hylden and Wilcox (25). Cysteamine (Sigma Chemical, U.S.A.) was administered i.p. at doses of 10-50-100-200 mg/kg; naloxone (Sigma Chemical, U.S.A.) was administered i.p. at the dose of 0.5 mg/kg; yohimbine (a gift of Dr. T. Costa, Istituto Superiore di Sanita', Italy) was administered i.p. at the dose of 0.5 mg/kg; CGP 35348 (a gift of Dr. G. Monza, Ciba-Geigy, Italy) was administered i.p. at the dose of 1 mg/kg; somatostatin (Sigma Chemical, U.S.A.) was administered i.c.v, or i.t. at the dose of 0.1 ug/mouse; cyclo(7-aminoheptanoyI-Phe-DTrp-Lys-Thr[Bzl]) (Sigma Chemical, U.S.A.) was administered i.c.v, or i.t. at the dose of 0.1 ug/mouse. The doses were calculated as the weight of the base; the peptide purity was not checked and the concentrations were calculated according to the purity quoted from the source. All data (expressed as mean + s.e.m.) were analyzed by the analysis of variance (ANOVA) and Dunnett's procedure for multiple comparisons with a single control group. When the analysis was restricted to two means, Student's t-test (two-tailed)was used. Fisher's exact test was used to analyze the rotarod data. Significance was assumed at a 5% level. Results As previously reported (19), frequent licking of the paw was observed 0-5 min (first phase) and 15-30 min (second phase) after s.c. formalin injection. Cysteamine administered at the higher doses (50 and 100 mg/kg) only reduced the second phase of the licking due to the formalin injection. This effect appeared 1 h after administration (Fig. 1 A) and cysteamine induced the maximal antinociceptive effects 4 h after administration (Fig. 1 B). This effect was absent when cysteamine was administered 24 h (Fig. 1 C) before the test. After these experiments we investigated whether naloxone, yohimbine, CGP 35348, somatostatin, or cyclo(7-aminoheptanoyI-Phe-D-Trp-Lys-Thr[Bzl]) at a dose that by itself did not change the response to nociceptive stimuli, might be able to modify the cysteamine effects in animals treated with cysteamine at the dose of 50 or 100 mg/kg 4 h before the tests. Since the response in the first phase of the formalin test did not change after cysteamine treatment, we reported only the results obtained in the second phase of the formalin test, as a sum of the licking response recorded from 15 to 30
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Fig. 1 Effects induced by saline (5 ml/kg, i.p.), and by cysteamine (10-50-100 mg/kg, i.p.) in the formalin test performed 1 (A), 4(B) or 24 (C) h after cysteamine administration. ** is for p<0.01 versus saline. N=10.o.osaline; A--~cysteamine 10 mg/kg; H c y s t e a m i n e 50 mg/kg; Hcysteamine 100 mg/kg.
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min after formalin injection. Naloxone, yohimbine and CGP 35348 administered 10 min before the formalin test did not change the response to formalin injected into the hind paw (data not shown). In animals that had received cysteamine at the dose of 100 mg/kg 4 h before the formalin test, neither naloxone, nor yohimbine, nor CGP 35348 administered 10 min before the formalin test was able to change the cysteamine antinociceptive effects (mean + s.e.m, of the licking activity recorded from 15 to 30 min after cysteamine injection : cysteamine, 68 + 3; cysteamine plus naloxone, 72 + 5; cysteamine plus yohimbine, 71 + 4; cysteamine plus CGP35348, 69 + 3; F(3,36)=0.18, n.s.). Somatostatin or cyclo(7aminoheptanoyI-Phe-D-Trp-Lys-Thr[Bzl]) administered alone i.c.v, or i.t. at the dose of 0.1 ug/mouse did not change the response to formalin injection (data not shown). Somatostatin administered i.c.v, or i.t. at the dose of 0.1 ug/mouse reduced or reverted, respectively, the cysteamine induced effects when cysteamine was administered at the dose of 100 mg/kg 4 h before the tests (Fig. 2). Cyclo(7-aminoheptanoyI-Phe-D-TrpLys-Thr[Bzl]) administered i.c.v, at the dose of 0.1 ug/mouse in animals that were pretreated with cysteamine at the dose of 50 mg/kg 4 h before the test did not change the cysteamine antinociceptive effects. On the contrary, cyclo(7-aminoheptanoyI-Phe-DTrp-Lys-Thr[Bzl]) administered i.t. enhanced the cysteamine induced effects when cysteamine was administered at the dose of 50 mg/kg 4 h before the tests (Fig. 2). I
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Fig. 2 Effects induced by saline (SAL, 5 ml/kg, i.p.) and by somatostatin (SST, 0.1 ug/mouse i.c.v, or i.t.), in animals pretreated with cysteamine (CSH, 100 mg/kg, i.p.) and the effects induced by cyclo(7-aminoheptanoyI-Phe-D-Trp-LysThr[Bzl] (SSA, 0.1 ug/mouse i.c.v, or i.t.) in animals pretreated with cysteamine (CSH, 50 mg/kg, i.p.) in the second phase of the formalin test. The animals were treated with cysteamine 4 h before the formalin test, whereas somatostatin or cyclo(7-aminoheptanoyI-Phe-D-Trp-Lys-Thr[Bzl] was administered i.c.v. or i.t. 15 min before the test. Licking was recorded from 15 to 30 min after formalin injection. * is for p<0.05 and ** is for p<0.01. N=10. In the writhing test, cysteamine administered at the doses of 10, 50 and 100 mg/kg reduced the response to nociceptive stimuli when administered 4 h before the test (Table1). Somatostatin or cyclo(7-aminoheptanoyI-Phe-D-Trp-Lys-Thr[Bzl]) administered i.c.v, or i.t. at the dose of 0.1 ug/mouse did not change the response to acetic acid injection. Somatostatin administered i.c.v, at the dose of 0.1 ug/mouse did not change cysteamine induced antinociceptive effects, whereas somatostatin administered i.t. at the
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same dose was able to the revert the cysteamine antinociceptive effects (Table 1). Cyclo(7-aminoheptanoyI-Phe-D-Trp-Lys-Thr[Bzl]) administered i.c.v, was not able to change the cysteamine induced effects whereas cyclo(7-aminoheptanoyI-Phe-D-TrpLys-Thr[Bzl]) administered i.t. enhanced the cysteamine-induced antinociceptive effects (Table 1). The results obtained with the activity cage and in the rotarod test revealead that cysteamine administered only at the highest dose (200 mg/kg) 1 or 4 h before the experimental session significantly reduced locomotor activity (Fig. 3) and the performance in the rotarod test (Fig. 4). Discussion The results obtained in our experiments indicate that cysteamine is able to modify the nociceptive threshold in assays involving prolonged pain stimuli in mice. Indeed, when cysteamine was administered at doses that did not significantly change locomotor activity, it reduced the response to prolonged pain induced by chemical stimuli in the formalin as well as in the writhing test. Regarding the absence of effects in the first phase of the formalin test, many findings suggest that in the formalin test the first phase (licking activity recorded between 0-5 min after formalin injection) and the second phase (licking activity recorded between 15-30 min after formalin injection) may have different nociceptive Table 1. Effects induced by saline (5 ml/kg, i.p.), and by cysteamine (10-50100 mg/kg i.p.) in the writhing test performed 4 h after cysteamine administration and the effects induced by somatostatin (0.1 ug/mouse i,c.v, or i.t.) or by cyclo(7-aminoheptanoyI-Phe-D-Trp-Lys-Thr[Bzl]) (SSA, 0.1 ug/mouse i.c.v, or i.t.) in animals pretreated with cysteamine 4 h before the writhing test. Somatostatin or cyclo(7-aminoheptanoyI-Phe-D-Trp-LysThr[Bzl]) were administered i.c.v, or i.t. 15 min before the test. Treatment
number of writhings mean + s.e.m.
SAL CSH 10 CSH 50 CSH 100 SST i.c.v. SST i.t. CSH 100 + SST i.c.v. CSH 100 + SST i.t. SSA i.c.v. SSA i.t. CSH 10 + SSA i.c.v. CSH 10 + SSA i.t.
31.5 + 1.5 21.3 + 2.2** 18.9 + 1.5** 11.5 + 1.0.* 30.8 + 1.5 30.4 + 1.2 16.9+2.7.* 28.3 + 1.0§§ 29.1 + 1.4 30.0 + 1.1 18.5+ 1.5.* 11.0 + 1.3**##
Statistical analysis was performed by using Student's t-test (two-tailed) *'* is for p<0.01 v/s SAL; §§ is for p<0.01 v/s CSH 100; ## is for p<0.01 v/s CSH 10. N=10
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Fig. 3 Effects on locomotor activity induced by saline (SAL, 5 ml/kg, i.p.) or cysteamine (CSH) administered at different doses (10-50-100-200 mg/kg, i.p.) as measured between 0-2, 3-5 h and 23-25 h after administration. * is for p<0.05 and *'** is for p<0.001 versus saline. N=16. 100-
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Fig. 4 Effects on rotarod performance induced by saline (SAL, 5 ml/kg, i.p.) or cysteamine (CSH) administered at different doses (10-50-100-200 mg/kg, i.p.) as measured 10 min or 1, 4 or 24 h after administration. * is for p<0.05 and ** is for p<0.01 versus saline. N=10. mechanisms; i.e., the first phase representing an acute pain stimuli probably due to a direct effect on nociceptors, and the second phase representing a prolonged pain stimuli probably due to an inflammatory responses (26). Indeed, the first phase is sensitive to drugs that interact with the opioid system whereas the second phase can also be inhibited by antiinflammatory drugs (12). Ohkubo et al. (3) reported that cysteamine administered i.t. was not able to change the response in tests involving acute pain stimuli such as the
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tail pinch and hot plate test. The same results were reported by Dalsgaard et al. (27) who found that i.t. administration of cysteamine did not change the response to the hot plate in the rat. These findings, together with those obtained in our experiments may suggest that cysteamine exerts its effects only on the pain mechanisms involved in the response to prolonged-inflammatory stimuli. Our results also indicate that somatostatin reduced the cysteamine-induced antinociceptive effects, and that somatostatin antagonist cyclo(7-aminoheptanoyI-PheD-Trp-Lys-Thr[Bzl] enhanced them. Somatostatin involvement in cysteamine-induced antinociception has already been reported by Ohkubo et al (3) who suggest that cysteamine antinociception induced by intrathecal cysteamine administration may be related to the cysteamine capacity to reduce somatostatin levels in the spinal cord. In our experiments, the reduction or the increase of the cysteamine effects obtained in cysteamine pretreated animals in the formalin test after injection with somatostatin and cyclo(7-aminoheptanoyI-Phe-D-Trp-Lys-Thr[Bzl], respectively, may indicate that the cysteamine antinociceptive effects may be mediated by somatostatin. Furthermore, in our experiments cysteamine did not change the response to formalin injection when cysteamine was administered 24 h before the test, whereas cysteamine-induced antinociception appeared 1 h after administration, and cysteamine induced the maximal antinociceptive effects 4 h after administration. These effects may be related to the effects that cysteamine exert on somatostatin CNS content. Assays performed on CSF and on brain somatostatin levels showed that cysteamine induced a time-dependent depletion of cortical somatostatin levels - that reaches maximal values 2-4 h after cysteamine administration which was accompained by a parallel increase in CSF levels of somatostatin (1-8). The possibility that the cysteamine antinociceptive effects in our experiments are due to decreased somatostatin levels or increased somatostatin release in the CNS could be considered. In addition, cysteamine antinociceptive effects may not involve opioid, adrenergic or GABA systems since naloxone, yohimbine or CGP 35348 did not change the cysteamine antinociceptive effects. However, cysteamine also reduced pituitary prolactin levels (28, 29) and following administration of high doses, there was an inhibition of dopamine-beta-hydroxylase activity, with cortical changes in norepinephrine and dopamine levels (30). At present, we cannot exclude that cysteamine effects may be mediated also by these effects. Finally, given the evidence mentioned above, it is of interest that cysteamine is able to modify the nociceptive response induced by chemical stimuli, and that these effects may be mediated by somatostatin. References 1. 2. 3. 4. 5.
M.PALKOVITS, M.J. BROWNSTEIN, L.E. EIDEN, M.C. BEINFELD, J. RUSSEL, A. ARIMURA and S. SZABO, Brain Res. 240 178-180 (1982). S.M. SAGAR, D.L. LANDRY, W.J. MILLARD, T.M. BADGEM,A. ARNOLD and J.B. MARTIN, (1982) J. Neurosci. _2225-231. T. OHKUBO, M. SHIBATA, H. TAKAHASKI and R. INOKI, J. Pharmacol. Exp. Ther.252 1261-1268 (1990). V.HAROUTUNIAN, R. MANTIN, G.A. CAMPBELL, G.K. TSUBOYAMA and K.L. DAVIS, Brain Res. 403 234-242 (1987). L. VECZEI, R. EKMAN, C. ALLING and E. WlDERLOV, J. Neural. Transm [GenSect]
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7_88209-220 (1989). 6. Y.C. PATEL and I. PIERZCHALA, Endocrinology 166 1699-1702 (1985) 7. R. WlDMANN, D. MAAS and G. SPERK, J. Neurochem. 50 1682-1686 (1988). 8. C.B. SRIKANT and Y.C. PATEL, Endocrinology 115 990-995 (1984). 9. C.J. HELKE and J.H. SELSKY, Peptides 4 699-672 (1983). 10. W.J. MILLARD, S.M. SAGAR, T.M. BADGER, D.B. CARR, M.A. ARNOLD, E. SPINDEL, N.W. KASTING, J.B. MARTIN, Endocrinology 112 518-525 (1983). 11. G.K. CHA'I-I-A and M.F. BEAL, Brain Res. 4..O0359-364 (1987). 12. S. HUNSKAAR and K. HOLE, Pain 3._0103-114 (1987) 13. M. SKINGE, A.G. HAYES and M.B. TYERS, Life Sciences 3.~11123-1132 (1982) 14. J. SAWYNOK, Pharmacol. Biochem. Behav. 2_.66463-474 (1987) 15. M. MALCANGIO, C. GHELARDINI, A. GIOI-I-I, P. MALMBERG-AIELLO and A. BARTOLINI, Br. J. Pharmacol. 103 1303-1308 (1991). 16. J.L. FRIES, W.A. MURPHY, J. SUEIRAS-DIAS and D.H. COY, Peptides 3 811814 (1982) 17. S.M. CARTMELL, L. GELGOR and D. MITCHELL, J. Pharmacot. Meth. 2..66149-159 (1991). 18. M. ZIMMERMMAN, Pain 1._6_6109-110(1983). 19. S. HUNSKAAR, O.B. FASMER and K. HOLE, J. Neurosci. Meth. 1,$ 69-76 (1985). 20. R. KOSTER, M. ANDERSON and E.J. DE BEER, Fed. Proc. 18 412 (1959) 21. A. CAPASSO, A. DI GIANNUARIO, A. LOIZZO, S. PIERE-I-I-I and L, SORRENTINO, Life Sciences 4.991411 - 1418 (1991). 22. J.H. ROSLAND, S. HUNSKAAR and K. HOLE, Pharmacol. Toxicol. 66 382-386 (1990). 23. T.J. HALEY and W.G. MCCORMICK, Br. J. Pharmacol. 1_2212-15 (1957) 24. S. PIEREI-FI, A. DI GIANNUARIO, A. LOIZZO, Gen. Pharmacol. 2,$ 83-88 (1993) 25. J.L.K. HYLDEN and G. L. WILCOX, Eur. J. Pharmacol. 67 313-316 (1980). 26. S. HUNSKAAR, O.-G. BERGE and K. HOLE, Pain 25 125-132 (1986) 27. C.-J. DALSGAARD, S.R. VINCENT, T. HOKFELT, Z. WlESENFELD-HALLIN, L. GUSTAFSSON, R. ELDE and G.J. DOCKRAY, Eur. J. Pharmacol.104 295-301 (1984). 28. W.J. MILLARD, S.M. SAGAR, D.M.D. LANDIS and J.B. MARTIN, Science 217 452454 (1982) 29. M.Y. LORENSON and L.S. JACOBS, Endocrinology 115 1492-1495 (1984) 30. L.VECSEI, L. KOVACS, M. FALUDI, I. BOLLOK and G. TELEGDY, Acta Physiol. Hung. 6._66213-217(1984)
Pergamon
EFFECTS INDUCED BY CYSTEAMINE ON CHEMICALLY-INDUCED NOCICEPTION IN MICE
Stefano Pieretti I , Amalia Di Giannuario I , Anna Capasso 2, Ludovico Sorrentino 2 and Alberto Loizzo 1
llstituto Superiore di Sanita', V.Le Regina Elena 299, 00161, Rome and 2 School of Pharmacy, University of Salerno, P.a V. Emanuele 9, 88084, Penta di Fisciano, Fisciano, Salerno, Italy ( R e c e i v e d in final f o r m J a n u a r y 20, 1994)
Summary The effects were investigated of cysteamine - a well known somatostatin depletor - on the pain induced by chemical stimuli in mice. Cysteamine injected intraperitoneally 4 h before the test at doses of 50 and 100 mg/kg reduced the second phase of the licking response which was induced by formalin injected into the hind paw. Furthermore, cysteamine administered at the doses of 10, 50 and 100 mg/kg reduced the writhing induced by acetic acid. Naloxone, yohimbine and CGP 35348 administered in cysteamine-pretreated animals were not able to change the cysteamine antinociceptive effects in the formalin test. Intrathecally injected somatostatin was able to revert the cysteamine antinociceptive effects in the second phase of the formalin test and in the writhing test, whereas intracerebroventricularly injected somatostatin reduced the antinociceptive effects induced by cysteamine in the second phase of the formalin test. Intrathecally injected cyclo(7-aminoheptanoyI-Phe-D-Trp-LysThr[Bzl]) - a reported somatostatin antagonist increased cystearnine antinociceptive effects in the second phase of the formalin test and in the writhing test. These results suggest that somatostatin is involved in the effects of cysteamine on the nociceptive threshold. Cysteamine (beta-mercaptoethylamine) is a thiol compound that depletes somatostatin in the brain (1,2) and spinal cord (3). This depletion is observed within minutes after administration; after 4 h it reaches maximal values, and within 24-78 h it is absent (4-8). The lack of cysteamine effects on other brain peptides (9-11) has suggested that cysteamine may be a potentially useful pharmacological tool for understanding the role of somatostatin in the central nervous system. Few data are reported on the effects induced by cysteamine on the nociceptive threshold. In this report Correspondence to: Stefano Pieretti, Istituto Superiore di Sanita', v.le Regina Elena 299, 00161, Rome, Italy. Phone (1139)-6-4990; fax (1139)-6-4440053
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we investigate the effects induced by cysteamine administered systemically on the licking response induced by formalin injected subcutaneously into the hind paw. For comparative purposes the cysteamine effects on the writhing induced by acetic acid injected intraperitoneally in mice was also investigated. Taking into cosideration the previously reported time-dependent somatostatin depletion caused by cysteamine (4-8), in the first series of experiments we investigated whether cysteamine injected 1, 4 and 24 h before the experimental sessions might be able to change the response to nociceptive stimuli. Since drugs acting on opioid (12) as well as alfa-2 (13) or GABA B (14) receptors are able to induce antinociception, we investigated whether naloxone, yohimbine or CGP 35348 - a reported GABA B antagonist (15) - might be able to change cysteamineinduced antinociceptive effects. Furthermore, the effects induced by somatostatin and cyclo(7-aminoheptanoyI-Phe-D-Trp-Lys-Thr[Bzl]) - a reported somatostatin antagonist (16) - on cysteamine-induced antinociception were also investigated in cysteamine pretreated animals. Since cysteamine may decrease locomotor activity (5) and drugs that impair locomotor activity may produce false positive results in the nociceptive test (17), we performed some experiments before the nociceptive test with the aim of evaluating the cysteamine effects on this behavior. Materials and Methods Male CD-1 mice (Charles River, Italy) weighing 25-30 gr were used in the experiments. The animals were housed in colony cages (4 mice each) with free access to food and water prior to the experiments. They were maintained in a climate and light controlled room (23 + 1° C, 12/12 h dark/light cycle with light on at 07:00) for at least 7 days before testing. Testing took place during the light phase, in the climatized room. Each animal was used only in one experimental session. In all experiments attention was paid to the ethical guidelines for investigations of experimental pain in conscious animals (18). In the formalin test, 20 ul of a 1% solution of formalin in saline was injected subcutaneously (s.c.) into the dorsal surface of the right hind paw of the mouse using a microsyringe with a 27-gauge needle. The formalin injection produced a distinct biphasic response consisting in licking or biting the paw. The first phase occurred from 0 to 5 min after formalin injection and the second phase, from 15 to 30 min afterwards. After the formalin injection the mouse was put into a plexiglass cage (30 cm x 14 cm x 12 cm) which served as an observation chamber, and the total amount of time the animal spent licking or biting the paw after the formalin injection was recorded every 5 min in both the first phase and second phase (19). In the writhing test, mice were given an intraperitoneal (i.p.) injection of 0.6% v/v acetic acid in a volume of 10 ml/kg. Acetic acid induces a series of writhings, consisting in abdominal contraction and hindlimb extension and these were recorded over a 10 min period beginning 5 min after acetic acid injection (20). Locomotor activity was measured as previously reported (21). Briefly, mice were randomly assigned to one experimental group and tested in groups of four. The animals were placed in the activity cage (Ugo Basile, Italy) for at least 30 min before receiving the injection of drugs. Temperature, sound and light conditions were maintaJned uniform during the course of the experiments. Measurements were carried out at ten-
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min intervals and cumulative counts were recorded after two h. Locomotor activity was measured 0-2, 3-5 and 23-25 h after cysteamine administation. Motor coordination of the mice was evaluated by using a rotarod apparatus (Ugo Basile, Italy) consisting of a bar with a diameter of 3.0 cm, subdivided into five compartments by a disk 24 cm in diameter. The bar rotated at a constant speed of 16 rev/min. A preliminary selection of mice was made on the day of experiment by excluding those that did not remain on the rotarod bar for two consecutive periods of 45 sec each. Afterwards, mice were tested 10 min and 1, 4 and 24 h after cysteamine administration, and the time spent on the rotating bar was recored for a maximum of 45 sec (22). On the day of testing all drugs used in the experimental sessions were dissolved in saline for administration. Drugs were injected in a volume of 5 ml/kg for i.p. administrations; for intracerebroventricular (i.c.v.) and intrathecal (i.t.) administrations, they were injected in a volume of 2.5 ul/mouse using a 10 ul Hamilton syringe with a 27 gauge needle. Cysteamine solution was neutralized to pH 7.2 using 0.1 M NaOH. The i.c.v. injection was performed using the method of Haley and McCormick (23, 24); the i.t. injection was made according to the method of Hylden and Wilcox (25). Cysteamine (Sigma Chemical, U.S.A.) was administered i.p. at doses of 10-50-100-200 mg/kg; naloxone (Sigma Chemical, U.S.A.) was administered i.p. at the dose of 0.5 mg/kg; yohimbine (a gift of Dr. T. Costa, Istituto Superiore di Sanita', Italy) was administered i.p. at the dose of 0.5 mg/kg; CGP 35348 (a gift of Dr. G. Monza, Ciba-Geigy, Italy) was administered i.p. at the dose of 1 mg/kg; somatostatin (Sigma Chemical, U.S.A.) was administered i.c.v, or i.t. at the dose of 0.1 ug/mouse; cyclo(7-aminoheptanoyI-Phe-DTrp-Lys-Thr[Bzl]) (Sigma Chemical, U.S.A.) was administered i.c.v, or i.t. at the dose of 0.1 ug/mouse. The doses were calculated as the weight of the base; the peptide purity was not checked and the concentrations were calculated according to the purity quoted from the source. All data (expressed as mean + s.e.m.) were analyzed by the analysis of variance (ANOVA) and Dunnett's procedure for multiple comparisons with a single control group. When the analysis was restricted to two means, Student's t-test (two-tailed)was used. Fisher's exact test was used to analyze the rotarod data. Significance was assumed at a 5% level. Results As previously reported (19), frequent licking of the paw was observed 0-5 min (first phase) and 15-30 min (second phase) after s.c. formalin injection. Cysteamine administered at the higher doses (50 and 100 mg/kg) only reduced the second phase of the licking due to the formalin injection. This effect appeared 1 h after administration (Fig. 1 A) and cysteamine induced the maximal antinociceptive effects 4 h after administration (Fig. 1 B). This effect was absent when cysteamine was administered 24 h (Fig. 1 C) before the test. After these experiments we investigated whether naloxone, yohimbine, CGP 35348, somatostatin, or cyclo(7-aminoheptanoyI-Phe-D-Trp-Lys-Thr[Bzl]) at a dose that by itself did not change the response to nociceptive stimuli, might be able to modify the cysteamine effects in animals treated with cysteamine at the dose of 50 or 100 mg/kg 4 h before the tests. Since the response in the first phase of the formalin test did not change after cysteamine treatment, we reported only the results obtained in the second phase of the formalin test, as a sum of the licking response recorded from 15 to 30
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Somatostatin on Chemically-induced Pain
A
80-
60.
40,
20-
D
120-
f
i
B
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80
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20-
i
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,
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Fig. 1 Effects induced by saline (5 ml/kg, i.p.), and by cysteamine (10-50-100 mg/kg, i.p.) in the formalin test performed 1 (A), 4(B) or 24 (C) h after cysteamine administration. ** is for p<0.01 versus saline. N=10.o.osaline; A--~cysteamine 10 mg/kg; H c y s t e a m i n e 50 mg/kg; Hcysteamine 100 mg/kg.
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min after formalin injection. Naloxone, yohimbine and CGP 35348 administered 10 min before the formalin test did not change the response to formalin injected into the hind paw (data not shown). In animals that had received cysteamine at the dose of 100 mg/kg 4 h before the formalin test, neither naloxone, nor yohimbine, nor CGP 35348 administered 10 min before the formalin test was able to change the cysteamine antinociceptive effects (mean + s.e.m, of the licking activity recorded from 15 to 30 min after cysteamine injection : cysteamine, 68 + 3; cysteamine plus naloxone, 72 + 5; cysteamine plus yohimbine, 71 + 4; cysteamine plus CGP35348, 69 + 3; F(3,36)=0.18, n.s.). Somatostatin or cyclo(7aminoheptanoyI-Phe-D-Trp-Lys-Thr[Bzl]) administered alone i.c.v, or i.t. at the dose of 0.1 ug/mouse did not change the response to formalin injection (data not shown). Somatostatin administered i.c.v, or i.t. at the dose of 0.1 ug/mouse reduced or reverted, respectively, the cysteamine induced effects when cysteamine was administered at the dose of 100 mg/kg 4 h before the tests (Fig. 2). Cyclo(7-aminoheptanoyI-Phe-D-TrpLys-Thr[Bzl]) administered i.c.v, at the dose of 0.1 ug/mouse in animals that were pretreated with cysteamine at the dose of 50 mg/kg 4 h before the test did not change the cysteamine antinociceptive effects. On the contrary, cyclo(7-aminoheptanoyI-Phe-DTrp-Lys-Thr[Bzl]) administered i.t. enhanced the cysteamine induced effects when cysteamine was administered at the dose of 50 mg/kg 4 h before the tests (Fig. 2). I
**'
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Fig. 2 Effects induced by saline (SAL, 5 ml/kg, i.p.) and by somatostatin (SST, 0.1 ug/mouse i.c.v, or i.t.), in animals pretreated with cysteamine (CSH, 100 mg/kg, i.p.) and the effects induced by cyclo(7-aminoheptanoyI-Phe-D-Trp-LysThr[Bzl] (SSA, 0.1 ug/mouse i.c.v, or i.t.) in animals pretreated with cysteamine (CSH, 50 mg/kg, i.p.) in the second phase of the formalin test. The animals were treated with cysteamine 4 h before the formalin test, whereas somatostatin or cyclo(7-aminoheptanoyI-Phe-D-Trp-Lys-Thr[Bzl] was administered i.c.v. or i.t. 15 min before the test. Licking was recorded from 15 to 30 min after formalin injection. * is for p<0.05 and ** is for p<0.01. N=10. In the writhing test, cysteamine administered at the doses of 10, 50 and 100 mg/kg reduced the response to nociceptive stimuli when administered 4 h before the test (Table1). Somatostatin or cyclo(7-aminoheptanoyI-Phe-D-Trp-Lys-Thr[Bzl]) administered i.c.v, or i.t. at the dose of 0.1 ug/mouse did not change the response to acetic acid injection. Somatostatin administered i.c.v, at the dose of 0.1 ug/mouse did not change cysteamine induced antinociceptive effects, whereas somatostatin administered i.t. at the
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same dose was able to the revert the cysteamine antinociceptive effects (Table 1). Cyclo(7-aminoheptanoyI-Phe-D-Trp-Lys-Thr[Bzl]) administered i.c.v, was not able to change the cysteamine induced effects whereas cyclo(7-aminoheptanoyI-Phe-D-TrpLys-Thr[Bzl]) administered i.t. enhanced the cysteamine-induced antinociceptive effects (Table 1). The results obtained with the activity cage and in the rotarod test revealead that cysteamine administered only at the highest dose (200 mg/kg) 1 or 4 h before the experimental session significantly reduced locomotor activity (Fig. 3) and the performance in the rotarod test (Fig. 4). Discussion The results obtained in our experiments indicate that cysteamine is able to modify the nociceptive threshold in assays involving prolonged pain stimuli in mice. Indeed, when cysteamine was administered at doses that did not significantly change locomotor activity, it reduced the response to prolonged pain induced by chemical stimuli in the formalin as well as in the writhing test. Regarding the absence of effects in the first phase of the formalin test, many findings suggest that in the formalin test the first phase (licking activity recorded between 0-5 min after formalin injection) and the second phase (licking activity recorded between 15-30 min after formalin injection) may have different nociceptive Table 1. Effects induced by saline (5 ml/kg, i.p.), and by cysteamine (10-50100 mg/kg i.p.) in the writhing test performed 4 h after cysteamine administration and the effects induced by somatostatin (0.1 ug/mouse i,c.v, or i.t.) or by cyclo(7-aminoheptanoyI-Phe-D-Trp-Lys-Thr[Bzl]) (SSA, 0.1 ug/mouse i.c.v, or i.t.) in animals pretreated with cysteamine 4 h before the writhing test. Somatostatin or cyclo(7-aminoheptanoyI-Phe-D-Trp-LysThr[Bzl]) were administered i.c.v, or i.t. 15 min before the test. Treatment
number of writhings mean + s.e.m.
SAL CSH 10 CSH 50 CSH 100 SST i.c.v. SST i.t. CSH 100 + SST i.c.v. CSH 100 + SST i.t. SSA i.c.v. SSA i.t. CSH 10 + SSA i.c.v. CSH 10 + SSA i.t.
31.5 + 1.5 21.3 + 2.2** 18.9 + 1.5** 11.5 + 1.0.* 30.8 + 1.5 30.4 + 1.2 16.9+2.7.* 28.3 + 1.0§§ 29.1 + 1.4 30.0 + 1.1 18.5+ 1.5.* 11.0 + 1.3**##
Statistical analysis was performed by using Student's t-test (two-tailed) *'* is for p<0.01 v/s SAL; §§ is for p<0.01 v/s CSH 100; ## is for p<0.01 v/s CSH 10. N=10
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Somatostatin on Chemically-induced Pain
0-2h
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1097
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Fig. 3 Effects on locomotor activity induced by saline (SAL, 5 ml/kg, i.p.) or cysteamine (CSH) administered at different doses (10-50-100-200 mg/kg, i.p.) as measured between 0-2, 3-5 h and 23-25 h after administration. * is for p<0.05 and *'** is for p<0.001 versus saline. N=16. 100-
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Fig. 4 Effects on rotarod performance induced by saline (SAL, 5 ml/kg, i.p.) or cysteamine (CSH) administered at different doses (10-50-100-200 mg/kg, i.p.) as measured 10 min or 1, 4 or 24 h after administration. * is for p<0.05 and ** is for p<0.01 versus saline. N=10. mechanisms; i.e., the first phase representing an acute pain stimuli probably due to a direct effect on nociceptors, and the second phase representing a prolonged pain stimuli probably due to an inflammatory responses (26). Indeed, the first phase is sensitive to drugs that interact with the opioid system whereas the second phase can also be inhibited by antiinflammatory drugs (12). Ohkubo et al. (3) reported that cysteamine administered i.t. was not able to change the response in tests involving acute pain stimuli such as the
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tail pinch and hot plate test. The same results were reported by Dalsgaard et al. (27) who found that i.t. administration of cysteamine did not change the response to the hot plate in the rat. These findings, together with those obtained in our experiments may suggest that cysteamine exerts its effects only on the pain mechanisms involved in the response to prolonged-inflammatory stimuli. Our results also indicate that somatostatin reduced the cysteamine-induced antinociceptive effects, and that somatostatin antagonist cyclo(7-aminoheptanoyI-PheD-Trp-Lys-Thr[Bzl] enhanced them. Somatostatin involvement in cysteamine-induced antinociception has already been reported by Ohkubo et al (3) who suggest that cysteamine antinociception induced by intrathecal cysteamine administration may be related to the cysteamine capacity to reduce somatostatin levels in the spinal cord. In our experiments, the reduction or the increase of the cysteamine effects obtained in cysteamine pretreated animals in the formalin test after injection with somatostatin and cyclo(7-aminoheptanoyI-Phe-D-Trp-Lys-Thr[Bzl], respectively, may indicate that the cysteamine antinociceptive effects may be mediated by somatostatin. Furthermore, in our experiments cysteamine did not change the response to formalin injection when cysteamine was administered 24 h before the test, whereas cysteamine-induced antinociception appeared 1 h after administration, and cysteamine induced the maximal antinociceptive effects 4 h after administration. These effects may be related to the effects that cysteamine exert on somatostatin CNS content. Assays performed on CSF and on brain somatostatin levels showed that cysteamine induced a time-dependent depletion of cortical somatostatin levels - that reaches maximal values 2-4 h after cysteamine administration which was accompained by a parallel increase in CSF levels of somatostatin (1-8). The possibility that the cysteamine antinociceptive effects in our experiments are due to decreased somatostatin levels or increased somatostatin release in the CNS could be considered. In addition, cysteamine antinociceptive effects may not involve opioid, adrenergic or GABA systems since naloxone, yohimbine or CGP 35348 did not change the cysteamine antinociceptive effects. However, cysteamine also reduced pituitary prolactin levels (28, 29) and following administration of high doses, there was an inhibition of dopamine-beta-hydroxylase activity, with cortical changes in norepinephrine and dopamine levels (30). At present, we cannot exclude that cysteamine effects may be mediated also by these effects. Finally, given the evidence mentioned above, it is of interest that cysteamine is able to modify the nociceptive response induced by chemical stimuli, and that these effects may be mediated by somatostatin. References 1. 2. 3. 4. 5.
M.PALKOVITS, M.J. BROWNSTEIN, L.E. EIDEN, M.C. BEINFELD, J. RUSSEL, A. ARIMURA and S. SZABO, Brain Res. 240 178-180 (1982). S.M. SAGAR, D.L. LANDRY, W.J. MILLARD, T.M. BADGEM,A. ARNOLD and J.B. MARTIN, (1982) J. Neurosci. _2225-231. T. OHKUBO, M. SHIBATA, H. TAKAHASKI and R. INOKI, J. Pharmacol. Exp. Ther.252 1261-1268 (1990). V.HAROUTUNIAN, R. MANTIN, G.A. CAMPBELL, G.K. TSUBOYAMA and K.L. DAVIS, Brain Res. 403 234-242 (1987). L. VECZEI, R. EKMAN, C. ALLING and E. WlDERLOV, J. Neural. Transm [GenSect]
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7_88209-220 (1989). 6. Y.C. PATEL and I. PIERZCHALA, Endocrinology 166 1699-1702 (1985) 7. R. WlDMANN, D. MAAS and G. SPERK, J. Neurochem. 50 1682-1686 (1988). 8. C.B. SRIKANT and Y.C. PATEL, Endocrinology 115 990-995 (1984). 9. C.J. HELKE and J.H. SELSKY, Peptides 4 699-672 (1983). 10. W.J. MILLARD, S.M. SAGAR, T.M. BADGER, D.B. CARR, M.A. ARNOLD, E. SPINDEL, N.W. KASTING, J.B. MARTIN, Endocrinology 112 518-525 (1983). 11. G.K. CHA'I-I-A and M.F. BEAL, Brain Res. 4..O0359-364 (1987). 12. S. HUNSKAAR and K. HOLE, Pain 3._0103-114 (1987) 13. M. SKINGE, A.G. HAYES and M.B. TYERS, Life Sciences 3.~11123-1132 (1982) 14. J. SAWYNOK, Pharmacol. Biochem. Behav. 2_.66463-474 (1987) 15. M. MALCANGIO, C. GHELARDINI, A. GIOI-I-I, P. MALMBERG-AIELLO and A. BARTOLINI, Br. J. Pharmacol. 103 1303-1308 (1991). 16. J.L. FRIES, W.A. MURPHY, J. SUEIRAS-DIAS and D.H. COY, Peptides 3 811814 (1982) 17. S.M. CARTMELL, L. GELGOR and D. MITCHELL, J. Pharmacot. Meth. 2..66149-159 (1991). 18. M. ZIMMERMMAN, Pain 1._6_6109-110(1983). 19. S. HUNSKAAR, O.B. FASMER and K. HOLE, J. Neurosci. Meth. 1,$ 69-76 (1985). 20. R. KOSTER, M. ANDERSON and E.J. DE BEER, Fed. Proc. 18 412 (1959) 21. A. CAPASSO, A. DI GIANNUARIO, A. LOIZZO, S. PIERE-I-I-I and L, SORRENTINO, Life Sciences 4.991411 - 1418 (1991). 22. J.H. ROSLAND, S. HUNSKAAR and K. HOLE, Pharmacol. Toxicol. 66 382-386 (1990). 23. T.J. HALEY and W.G. MCCORMICK, Br. J. Pharmacol. 1_2212-15 (1957) 24. S. PIEREI-FI, A. DI GIANNUARIO, A. LOIZZO, Gen. Pharmacol. 2,$ 83-88 (1993) 25. J.L.K. HYLDEN and G. L. WILCOX, Eur. J. Pharmacol. 67 313-316 (1980). 26. S. HUNSKAAR, O.-G. BERGE and K. HOLE, Pain 25 125-132 (1986) 27. C.-J. DALSGAARD, S.R. VINCENT, T. HOKFELT, Z. WlESENFELD-HALLIN, L. GUSTAFSSON, R. ELDE and G.J. DOCKRAY, Eur. J. Pharmacol.104 295-301 (1984). 28. W.J. MILLARD, S.M. SAGAR, D.M.D. LANDIS and J.B. MARTIN, Science 217 452454 (1982) 29. M.Y. LORENSON and L.S. JACOBS, Endocrinology 115 1492-1495 (1984) 30. L.VECSEI, L. KOVACS, M. FALUDI, I. BOLLOK and G. TELEGDY, Acta Physiol. Hung. 6._66213-217(1984)