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The behavioural effects of intrathecally administered [d -PRO2,d -TRP7,9]-substance P, an analogue with presumed antagonist actions, in the rat
THE BEHAVIOURAL EFFECTS OF INTRATHECALLY ADMINISTERED [D-PRO’, D-TRP7,9]-SUBSTANCE P, AN ANALOGUE WITH PRESUMED ANTAGONIST ACTIONS, IN THE RAT R. E. RODKG~:EZ.* Department
of Pharmacology.
T. E. SALT.? P. M. B. CAHWA~ University
of Bristol.
Medical
and R. G. HILL
School.
Bristol.
BSX ITD. U.K.
Summary--[u-Pro’, u-Trp’.“]-substance P. an analogue of the putative nociceptive primary alkrent neurotransmitter substance P, was admimstered to rats via chronically implanted intrathecal catheters (0.5~-2.5 nmol per rat). Several ammals showed large elevations of thermal and mechanical nociceptive thresholds. but these effects were always accompanied by profound. often long-lasting. Impairments 01 motor function and thus cannot be regarded as a specific antinociceptive effect of the drug. These behavloural effects are considered to be probably due to direct spinal actions of the drug rather than antagonism of spinal substance P systems.
Wistar rats (270.-300 g) was chronically catheterised under either pentobarbital (60mg,‘kg, i.p.) or tribromoethanol (250 mgikg, i.p.) anaesthesia (Yaksh and Rudy, 1977). At least four days recovery was allowed before further testing of the animals. Injections of DPDT-SP (Peninsula Labs) were made in volumes of 5 ~11of sterile isotonic saline followed by 15 1~1 of saline to ensure that the drug reached the spinal cord. Control injections consisted of 20 ~11of saline. Solutions of DPDT-SP were freshly prepared shortly before use. Following the experimental use of animals. cannula positions were verified upon autopsy. and only animals with cannulae correctly positioned between T12 and L4 are included in the data analysis.
P (SP), an undecapeptide found in some sensory neurones (Hiikfelt, Kellerth. Nilsson and Pernow. 1975) may be a neurotransmitter of nociceptive primary afferent fibres (Nicoll, Schenker and Leeman, 1980; Henry, 1980). Recently. several synthetic analogues of substance P have been found to have actions as antagonists of substance P (Folkers. Hiirig, Rose11 and Bjiirkroth, 1981; Leander, Hgkanson. Resell, Folkers, Sundler and Tornqvist. 1981; Engberg. Svensson. Rose11 and Folkers, 1981). In particular. one such analogue. [D-Pro’. D-Trp’*‘]substance P (DPDT-SP) has been found to be antinociceptive in the rat (Resell. 1982) and mouse (Lembeck, Folkers and Donnerer, 1981) thus apparently supporting the hypothesis that substance P may be a transmitter of nociceptive primary afferent fibres. However, it has recently been found, in electrophysiological experiments, that DPDT-SP does not act as an antagonist of substance P in the rat spinal cord (Salt. De Vries, Rodriguez. Cahusac, Morris and Hill, 1982). It is thus possible that DPDT-SP produces analgesia by a novel mechanism unrelated to antagonism of substance P. It was therefore of interest to study the effects of this analogue, administered intrathecally, in various behavioural nociceptive tests in an attempt to determine its spectrum of activity. Substance
small
Behacioural
The tail immersion test (Janssen. Niemegeers and Dony. 1963) using water of 54.5 k 0.5’C and a cut-off time of 10sec (approx. twice the control response latency) was performed immersing 10 cm of tail three times at one minute intervals, with only the last reading being used in the data analysis (Irwin, Houde, Bennett, Hendershot and Seevers, 1951). The hotplate test (Woolfe and MacDonald, 1944) was used with a surface temperature of 56.5 &- 0.2”C. The time taken for the rat to display either a hind paw lick, stamp or jumping behavicur was taken as the end point and a cut-off time of 20sec (approx. twice the control response latency) was taken to avoid tissue damage. Mechanical nociceptive thresholds were determined using the paw pressure test (Randall and Selitto. 1957) on each hind paw. Pressure was steadily increased until a clear vocalisation or attempt to escape occurred. The maximum (cut-off) pressure was chosen so that normal control responses occurred at approximately half this value.
METHODS
Intrathecal
catheterisation
The lumbar
spinal
and injection
subarachnoid
space
testing
of male
Key words: substance P. [o-Pro’. D-Trp’,“]-substance P, substance P antagonists. nociception. * Permanent address: Coleglo de Medicina de Avila. University of Salamanca, Spain. t Present address for correspondence: Department of Physiology. University College. Cardiff CFI IXL. U.K. I73
I74
K. II
RooKi(illt / c’t trl.
Motor performance of the rats was assessed using a rotarod apparatus (Ugo Basile). The animals wcrc placed on the stationary treadmill. following which the rotation speed was gradually increased. The end point was taken as the speed at which animals were unable to remain on the treadmill. Before the test xssions. rats were trained to achieve a minimum level of proficiency (5 rpm). All behavioural tests were carried out with the operator unaware of whether an animal was drug- or vehicle-treated. In the nociceptive behavioural tests. t-eadings from animals which showed control responses greater than 75”(, of the chosen cut-off‘ value were dlscardcd. The order of testins ammats was (i) tail immersion. (ii) paw-pressure. (iii) hot-plate. (iv) rotarod. in order to reduce any errors due tO interactions between different tests. In some experimental sessions the hot-plate and rotarod tests were not used. Following determination of control scores In the beha\ioural tests. cithcr DPDT-SP or vehicle was admlnistcrcd, and the animals were re-tested 30 min later. In some experiments the animals were also tested IO min after drug or vehicle administration. Thuh. each animal served as its own control and a percentage analgesia for each rat was calculated as follows:
apparent. The behavioural effects typically consisted of complete or almost complete unresponsiveness to the noxious stimuli used. However. the proportion 01 animals so affected was greater in the group treated
Hoi
1on
I
PlatP
1
“(, Analgesia _
(Post-drug
score)-
(Control score) x ,oo
(Cut-off) - (Control score) In the case of the rotarod test. as the animals were removed from the treadmill if they achieved their predrug control scores following treatment. no cut-off value v.as used and the score v.as quantitied as: “,, Rotarod
(Post-drug score) score = ~~ ~~ x 100 (Control score)
The randomisation test (Siegel. I9561 was used in all statistical analyses of the results This non-parametric test has the advantage that it is not necessary to assume normal distributions or homogeneity of variance in the populations involved
Experiments were performed on 28 catheterised rats and. wherever possib!c. cross-over studies were performed on consecutive days. In practice. this meant that vehicle-treated animals and a small number of DPDT-SP-treated animals could be used on subsequent days (see below). Doses of 0.5, t .O and 7.5 nmol (0.75. I.5 and 3.75 /cg respectively) of DPDT-SP were admimstered, and results of the four behavioural tests performed IO and 30 min after administration of drug or vehicle are shown in Figure 1. Administration of vehicle was not found to greatly alter the scores in the various behavioural tests. With all the drug doses used. it was a general finding that behaviournl effects were either absent or readily
Fig. I. Histograms showing the mean effect i +SEM) of admlnistratlon of vehicle (open barb) or DPDT-SP (0.5 nmol. hatched bars; 1.0 nmol. cross-hatched bars; 7.5 nmol. sohd bars) in the three nociceptlve tests and rotarod test at IO mln after administration (A) and 30 mln after administration (B). Numbers of animals per group arc shown in parcnthescs. except in the case of the paw-pressure test where the number of tested paws is given. Values which arc significantly different from the xhlcle-treated groups are lndicatcd with *(P < 0.05). **(P < ().()I).
[n-Proz.
D-T~~‘.‘]-SP
with I .Onmol of DPDT-SP than in the group which received 0.5 nmol. and thus the mean nociceptlve threshold elevations were greater (Fig. 1). Nociceptive threshold elevations were also seen with doses of 2.5 nmol. but in this case the mean effects were similar to. or smaller than those seen with 1.0 nmol of DPDT-SP. As before, behavioural eflects were either absent or readily apparent. Deficits in the motor performance of the rats was also seen in many cases, and consisted of a flaccid paralysis of the tail and hind limbs and occasionally also the forelimbs. As with the nociceptive tests. animals were either completely unaffected or had paralysis as described above. and such affected animals showed low scores on the rotarod. leading to a reduction of the mean scores (Fig. I). Those animals which showed paralysis also showed analgesia and no cases of specific analgesia were seen. This correlation is illustrated in Figure 2. In all cases, the motor paralysis caused by DPDT-SP was found to be longlasting (more than 24 hr). IXXUSSION
The present results show that DPDT-SP, administered intrathecally. is capable of affecting neural function in the spinal cord to the extent of producing considerable changes in nociceptive thresholds and motor performance in rats. These results are consistent with the thermal analgesia reported by Akerman. Rose11 and Folkers (1982) (1.0 nmol DPDT-SP, given intrathecelly in rats) and Lembeck et ul. (1981) (3.3 nmol DPDT-SP. given intraspinally in mice) but also suggest that the elevations of nociceptive thresholds are not specific, as little or no dissociation between the “analgesia” and motor impairment caused by the drug was found (Fig. 2). This lack of specificity appears to be at variance with the findings of Akerman pt a/. (1982) and Lembeck et ul. (I981 ). In a few cases it did appear that it was possible for rats to have slight elevations of nociceptive threshold with little effect on the rotarod score. However, certain vehicle-treated rats also showed similar effects, and thus such effects are likely to be within the limits of experimental variability (Fig. 2). It must also be noted that it was possible for animals to achieve a score of 50”, on the rotarod using only their fore-limbs, which would clearly be much less affected than hind-limbs by lumbar administration of DPDT-SP and thus rotarod scores for treated animals are likely to be artificially elevated. As substance P is present within the ventral horn of the spinal cord (Ljungdahl, HBkfelt and Nilsson, 1978) and as this peptide will depolarize motoneurones (Zieglgansberger and Tuiloch, 1979), it is possible that the effects on motor performance produced by DPDT-SP, are due to antagonism of the effects of endogenous substance P in the ventral horn. However, this does not seem likely in view of the previous finding that DPDT-SP did not antagonise the de-
I75
and nociception
polarisations of motoneurones evoked by substance P in the isolated rat spinal cord (Salt et al., 1982). Similarly, an antagonist action of DPDT-SP in substance P systems of the dorsal horn ~H~kfelt et al., 1975) also seems unlikely (Salt rt nl., 1982). Thus. a direct effect of DPDT-SP in the spinal cord appears to be the likely cause of the effects seen and this is supported by the finding that this analogue will depolarise motoneurones in rim (Salt et (11..1982). In this respect, it is of interest that substance P itself can have the same eKects as DPDT-SP, although of shorter duration, when administered intrathecally (15 nmol; J. Brown and R. G. Hill, unpublished observations). Thus. the prolonged effects seen in some cases with DPDT-SP may be due to “neurotoxic” actions of this analogue (Hiikfelt, Vincent, Hellsten. Resell. Folkers, Markey, Goldstein and Cuello, 1981). Other workers have found that an analogue of subP, structurally similar to DPDT-SP, stance [a-Pro’. D-Phe’, D-Trp9]-SP. had analgesic actions in thermal and chemical nociceptive tests when administered to mice (3.3-6.5 nmol) (Lembeck rt pi.. 1981; Pierce);. Schroeder. Folkers, Xu and Hiirig, 1981). Furthermore, Piercey er ai. (1981) found that larger doses of their analogue (20nmol) produced motor deficits, and these results were interpreted in terms of antagonism of substance P. Similar findings have recently been reported by Akerman et al. (1982) using DPDT-SP (IO nmol) in the rat. The structural and
0
20
LO
60
80
% Analgesm
Fig. 2. Plot of percentage rotarod score against percentage analgesia values for individual rats treated with either vehicle. 0.5 nmol DPDT-SP or 1.0nmol DPDT-SP. The percentage analgesia value is a mean value computed from results obtained in all three nociceptive tests, except in the case of one animal. in which the results from the hot-plate were not included in any of the analyses (see Methods section). Note that animals which showed clear analgesia also showed clear motor impajrment. Both at 10 and 30 min after administration this correlation was significant (Spearman rank correlation co-efficient ?s = --0.618 and -0.482. respectively. giving P -c0.01and P < 0.05, respectively). Plots of percentage analgesia for each noclceptive test against percentage rotarod score (not shown) did not reveal any selective effect of DPDT-SP in any of the three nociceptive tests used (see also Fig. I).
100
pharmacological similarities between these two analogues of substance P (Folkers et ul.. 1981; Leander rt al., 1981: Lembeck et ul.. 1981). together with the fact that neither has been shown to be an antagonist at spinal substance P receptors and that both have agonist actions in certain tissues (Hawcock. Hayes and Tyers. 1987; Salt QT al.. 1983) suggests that the be]~~~vioLlralresults obtained may. at least in part. be due to direct effects rather than ~~l~t~~~otiislnof substance P. Thus. results from behavioural experiments must be interpreted cautiously. The existence of multiple receptor sub-types for substance P (Lee, Iversen. Han& and Sandberg, 1982) further indicates the possibility that central substance P receptors may have a different specificity- to the peripheral receptors at which these analogues function as antagomsts. It IS thus evident that further information is required to complete the central pharmacological profiles of those analopues of substance P which have peripheral ~II~~~~oI~IS~ properties. However. it appears likely that the be~i~~~~ioLlr~~1 effects of DPDT-SP are not due to antagonism of substance P (Salt c’t trl.. 1987: HZikfelt c’t ui.. 19X1)and thus do not provide evidence for a role of substance P in nociceptive primary
afferent
neurotransmission.
Furthermore.
DPDT-SP does not have any specific antinociceptive properties, as motor impairment always accompanied such efftcts. il~~kr~~~~t/er/yc~n~r~~~r.sWe wish to thank Dr M. Skmgle for dernorrstrating the intrathecal catheterisation technique and Dr M. Tyers foi- the loan of the rotarod appararus. This work was supported by grants from the MRC and SRC.
REFERENCES
Akerman. B.. Resell. S. and Folkers, K. (1982). Intrathecal (n-Pro”. D-Trp”“]-SP elicits hypoalgesia and motor blockade in the rat and antagonizes noxious responses induced by substance P. Acru ph~siol. stand. 114: 631.-633. Engberg, G., Svensson, T. H.. Resell, S. and Folkers, K. (1981). A synthetic peptide as an antagonist of substance P. Narure, tend. 293: 222-223. Folkers. K.. Harig. J.. Resell, S. and Bjiirkroth. U, (1981). Chemical design of antagonists to substance P. Arrcr ph?siol. .scmd. 111: 505-506. Hancock, A. B., Hayes. A. G. and Tyers. M. B. (1982). Agonist properties of [o-Pro* D-Phe’. u-Trp”] substance P i17rirrcl-----evidence for more than one type of substance P receptor. Br. J. Pl~armac. 75: 113P. Henry. J. L. (1980). Substance P and pain: an updating. Trenlls Nemmi. 3: 95-97.
HBkfelt, T.. Kellerth, J. 0.. Nilsson. G. and Pcrnow. B. (1975). Experimental lmmunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurons. Brairl Re,s. 100: 235%252. Hiikfelt. T.. Vincent, S., Hellsten, L., Rosell. S.. Folkers, K.. Markey. K.. Goldstein. M. and Cuello, C. (1981I. Immunohistochemical evidence for a “neurotoxic” action of [n-Pro’. D-Trp’,“i-substance P. an analogue with substance P ~~nta~ollistic ;ictivity. ;Icrtr ~)~~~.\jo~..wtifiLi. 113: 571 573. Irwin. S.. Houde. R. W.. Bennett. D. R.. Hcndcrshot. L. C‘. and Ssevers, M. H. (1951).The effects of morphine. methadone and meperidine on some rcllc\ r-e\ponw\ of spinal animals to nociceptive stimulation. .I. P/wrwrt,. L’V,‘.7110. 101: 132 143. Janssen. P. A. J.. Niemegeers. c‘. .I. F.. and Don!. J. (i. H (lY63). The inhibltory effects of fentanyl and other mom-phinc-like analgcbich on the harm-induced tall w1111drawal reHex in rats. ~r3rlc~in,ittr,/-Ff~~s~/i. 13: 501 507. Leander. S.. HBkanson. R., Roscll. S.. Folkcrs. K.. Sundlcr. F. and Tornqvist. K. (IYXI). A specific suhstancc P alitagonist blocks smooth muscle contractions induced h> i~o~l-choliner~ic, non-adrcncrglc nerve ~tinllil~~ti(~~~. ~Vlirittrw,Lonti. 294: 467 46’). Lee. C. M.. Iversen. L. L.., Hanlev. M. R. and Sand&w
Lembeck. F.. Folkcrs, K. and Donnercr. J. (IYXI).Analgeslc effects of antagonists of substance P. Biwhcw. hoerplow Rcs. C‘omnwt. 103: I3 IX 132I. Ljungdahl. A.. Htikfelt. T. and Nilsson. G. (1978). Dlstribution of substance P-like immunoreactivit> in the central nervous system of the rat I. Cell bodies and nerve tcrminals. iVr~irosc,irnc,e 3: Hh I --94.3. Nicoll. R. A.. Schenker. c‘. and Leeman. S. E. (1YXO). Substance P as a transmitter candidate. -1. Rer. .X’curo.w. 3: __ 777-268. Piercey, M. F., Schroeder. L. A.. Folkers. K.. Xu, J.-c‘. and Hiirig. 3. (1981). Sensory and motor fllnctions of spinal cord substance P. Scirwr~ 214: 1361~ 1363. Randall. L. 0. and Seiitto. 3. J. (19.57). A method for measurement of analgesic activity on inflamed tissue. Archs rnt. Pharrmxodyn. Ther. 111 : 409 -4 19. Resell. S. (1981). Development of novel substance P antagonists. In: Cihtr Fowlduriwl Symposium ,Yo. 9/: Suhatirrlcc* P in t/w ~~mvus s!‘sttw. Pitman. London (In Press). Salt. T. E.. De Vrles. G. J.. Rodriguez. R. E.. Cahusac. P. M. B.. Morris, R. and Hill. R. G. (1982). Evaluation of [~-Pro’. D-Trp’.“]-substance P as an antagonist of substance P responses in the rat central nervous system. ,V~~rosci. Lat. 30: 29 l-295. Siegel. S. (1956). .~u~~p~~~}~~~tr;~ Sfufistics :lbr fhr Brhiormi Scirnces. pp. 152- 156.McGrawHdl. Woolfe. G. and MacDonald, A. D. (1944). The evaluation of the analgesic action of pethldine llydrocliloride (Demerol). f. Piwwrac. c’rn Tlier. 80: 30# 307. Yaksh. T. L. and Rudy. T A. (1977). Studies on the direct spinal actlon of narcotics in the production of analgesia In the rat. .I. Phcrrrm~. c’vp. T/xv. 202: 41 I 42X ZieglgBnsberger. W. and Tulloch. 1. F. 119701. Effects 01 sugstancc p on neuroncs in the dorsal horn of the spinal cord of the cat. Rrcco~ Rc\. 166: 173 2x2.
of Pharmacology.
T. E. SALT.? P. M. B. CAHWA~ University
of Bristol.
Medical
and R. G. HILL
School.
Bristol.
BSX ITD. U.K.
Summary--[u-Pro’, u-Trp’.“]-substance P. an analogue of the putative nociceptive primary alkrent neurotransmitter substance P, was admimstered to rats via chronically implanted intrathecal catheters (0.5~-2.5 nmol per rat). Several ammals showed large elevations of thermal and mechanical nociceptive thresholds. but these effects were always accompanied by profound. often long-lasting. Impairments 01 motor function and thus cannot be regarded as a specific antinociceptive effect of the drug. These behavloural effects are considered to be probably due to direct spinal actions of the drug rather than antagonism of spinal substance P systems.
Wistar rats (270.-300 g) was chronically catheterised under either pentobarbital (60mg,‘kg, i.p.) or tribromoethanol (250 mgikg, i.p.) anaesthesia (Yaksh and Rudy, 1977). At least four days recovery was allowed before further testing of the animals. Injections of DPDT-SP (Peninsula Labs) were made in volumes of 5 ~11of sterile isotonic saline followed by 15 1~1 of saline to ensure that the drug reached the spinal cord. Control injections consisted of 20 ~11of saline. Solutions of DPDT-SP were freshly prepared shortly before use. Following the experimental use of animals. cannula positions were verified upon autopsy. and only animals with cannulae correctly positioned between T12 and L4 are included in the data analysis.
P (SP), an undecapeptide found in some sensory neurones (Hiikfelt, Kellerth. Nilsson and Pernow. 1975) may be a neurotransmitter of nociceptive primary afferent fibres (Nicoll, Schenker and Leeman, 1980; Henry, 1980). Recently. several synthetic analogues of substance P have been found to have actions as antagonists of substance P (Folkers. Hiirig, Rose11 and Bjiirkroth, 1981; Leander, Hgkanson. Resell, Folkers, Sundler and Tornqvist. 1981; Engberg. Svensson. Rose11 and Folkers, 1981). In particular. one such analogue. [D-Pro’. D-Trp’*‘]substance P (DPDT-SP) has been found to be antinociceptive in the rat (Resell. 1982) and mouse (Lembeck, Folkers and Donnerer, 1981) thus apparently supporting the hypothesis that substance P may be a transmitter of nociceptive primary afferent fibres. However, it has recently been found, in electrophysiological experiments, that DPDT-SP does not act as an antagonist of substance P in the rat spinal cord (Salt. De Vries, Rodriguez. Cahusac, Morris and Hill, 1982). It is thus possible that DPDT-SP produces analgesia by a novel mechanism unrelated to antagonism of substance P. It was therefore of interest to study the effects of this analogue, administered intrathecally, in various behavioural nociceptive tests in an attempt to determine its spectrum of activity. Substance
small
Behacioural
The tail immersion test (Janssen. Niemegeers and Dony. 1963) using water of 54.5 k 0.5’C and a cut-off time of 10sec (approx. twice the control response latency) was performed immersing 10 cm of tail three times at one minute intervals, with only the last reading being used in the data analysis (Irwin, Houde, Bennett, Hendershot and Seevers, 1951). The hotplate test (Woolfe and MacDonald, 1944) was used with a surface temperature of 56.5 &- 0.2”C. The time taken for the rat to display either a hind paw lick, stamp or jumping behavicur was taken as the end point and a cut-off time of 20sec (approx. twice the control response latency) was taken to avoid tissue damage. Mechanical nociceptive thresholds were determined using the paw pressure test (Randall and Selitto. 1957) on each hind paw. Pressure was steadily increased until a clear vocalisation or attempt to escape occurred. The maximum (cut-off) pressure was chosen so that normal control responses occurred at approximately half this value.
METHODS
Intrathecal
catheterisation
The lumbar
spinal
and injection
subarachnoid
space
testing
of male
Key words: substance P. [o-Pro’. D-Trp’,“]-substance P, substance P antagonists. nociception. * Permanent address: Coleglo de Medicina de Avila. University of Salamanca, Spain. t Present address for correspondence: Department of Physiology. University College. Cardiff CFI IXL. U.K. I73
I74
K. II
RooKi(illt / c’t trl.
Motor performance of the rats was assessed using a rotarod apparatus (Ugo Basile). The animals wcrc placed on the stationary treadmill. following which the rotation speed was gradually increased. The end point was taken as the speed at which animals were unable to remain on the treadmill. Before the test xssions. rats were trained to achieve a minimum level of proficiency (5 rpm). All behavioural tests were carried out with the operator unaware of whether an animal was drug- or vehicle-treated. In the nociceptive behavioural tests. t-eadings from animals which showed control responses greater than 75”(, of the chosen cut-off‘ value were dlscardcd. The order of testins ammats was (i) tail immersion. (ii) paw-pressure. (iii) hot-plate. (iv) rotarod. in order to reduce any errors due tO interactions between different tests. In some experimental sessions the hot-plate and rotarod tests were not used. Following determination of control scores In the beha\ioural tests. cithcr DPDT-SP or vehicle was admlnistcrcd, and the animals were re-tested 30 min later. In some experiments the animals were also tested IO min after drug or vehicle administration. Thuh. each animal served as its own control and a percentage analgesia for each rat was calculated as follows:
apparent. The behavioural effects typically consisted of complete or almost complete unresponsiveness to the noxious stimuli used. However. the proportion 01 animals so affected was greater in the group treated
Hoi
1on
I
PlatP
1
“(, Analgesia _
(Post-drug
score)-
(Control score) x ,oo
(Cut-off) - (Control score) In the case of the rotarod test. as the animals were removed from the treadmill if they achieved their predrug control scores following treatment. no cut-off value v.as used and the score v.as quantitied as: “,, Rotarod
(Post-drug score) score = ~~ ~~ x 100 (Control score)
The randomisation test (Siegel. I9561 was used in all statistical analyses of the results This non-parametric test has the advantage that it is not necessary to assume normal distributions or homogeneity of variance in the populations involved
Experiments were performed on 28 catheterised rats and. wherever possib!c. cross-over studies were performed on consecutive days. In practice. this meant that vehicle-treated animals and a small number of DPDT-SP-treated animals could be used on subsequent days (see below). Doses of 0.5, t .O and 7.5 nmol (0.75. I.5 and 3.75 /cg respectively) of DPDT-SP were admimstered, and results of the four behavioural tests performed IO and 30 min after administration of drug or vehicle are shown in Figure 1. Administration of vehicle was not found to greatly alter the scores in the various behavioural tests. With all the drug doses used. it was a general finding that behaviournl effects were either absent or readily
Fig. I. Histograms showing the mean effect i +SEM) of admlnistratlon of vehicle (open barb) or DPDT-SP (0.5 nmol. hatched bars; 1.0 nmol. cross-hatched bars; 7.5 nmol. sohd bars) in the three nociceptlve tests and rotarod test at IO mln after administration (A) and 30 mln after administration (B). Numbers of animals per group arc shown in parcnthescs. except in the case of the paw-pressure test where the number of tested paws is given. Values which arc significantly different from the xhlcle-treated groups are lndicatcd with *(P < 0.05). **(P < ().()I).
[n-Proz.
D-T~~‘.‘]-SP
with I .Onmol of DPDT-SP than in the group which received 0.5 nmol. and thus the mean nociceptlve threshold elevations were greater (Fig. 1). Nociceptive threshold elevations were also seen with doses of 2.5 nmol. but in this case the mean effects were similar to. or smaller than those seen with 1.0 nmol of DPDT-SP. As before, behavioural eflects were either absent or readily apparent. Deficits in the motor performance of the rats was also seen in many cases, and consisted of a flaccid paralysis of the tail and hind limbs and occasionally also the forelimbs. As with the nociceptive tests. animals were either completely unaffected or had paralysis as described above. and such affected animals showed low scores on the rotarod. leading to a reduction of the mean scores (Fig. I). Those animals which showed paralysis also showed analgesia and no cases of specific analgesia were seen. This correlation is illustrated in Figure 2. In all cases, the motor paralysis caused by DPDT-SP was found to be longlasting (more than 24 hr). IXXUSSION
The present results show that DPDT-SP, administered intrathecally. is capable of affecting neural function in the spinal cord to the extent of producing considerable changes in nociceptive thresholds and motor performance in rats. These results are consistent with the thermal analgesia reported by Akerman. Rose11 and Folkers (1982) (1.0 nmol DPDT-SP, given intrathecelly in rats) and Lembeck et ul. (1981) (3.3 nmol DPDT-SP. given intraspinally in mice) but also suggest that the elevations of nociceptive thresholds are not specific, as little or no dissociation between the “analgesia” and motor impairment caused by the drug was found (Fig. 2). This lack of specificity appears to be at variance with the findings of Akerman pt a/. (1982) and Lembeck et ul. (I981 ). In a few cases it did appear that it was possible for rats to have slight elevations of nociceptive threshold with little effect on the rotarod score. However, certain vehicle-treated rats also showed similar effects, and thus such effects are likely to be within the limits of experimental variability (Fig. 2). It must also be noted that it was possible for animals to achieve a score of 50”, on the rotarod using only their fore-limbs, which would clearly be much less affected than hind-limbs by lumbar administration of DPDT-SP and thus rotarod scores for treated animals are likely to be artificially elevated. As substance P is present within the ventral horn of the spinal cord (Ljungdahl, HBkfelt and Nilsson, 1978) and as this peptide will depolarize motoneurones (Zieglgansberger and Tuiloch, 1979), it is possible that the effects on motor performance produced by DPDT-SP, are due to antagonism of the effects of endogenous substance P in the ventral horn. However, this does not seem likely in view of the previous finding that DPDT-SP did not antagonise the de-
I75
and nociception
polarisations of motoneurones evoked by substance P in the isolated rat spinal cord (Salt et al., 1982). Similarly, an antagonist action of DPDT-SP in substance P systems of the dorsal horn ~H~kfelt et al., 1975) also seems unlikely (Salt rt nl., 1982). Thus. a direct effect of DPDT-SP in the spinal cord appears to be the likely cause of the effects seen and this is supported by the finding that this analogue will depolarise motoneurones in rim (Salt et (11..1982). In this respect, it is of interest that substance P itself can have the same eKects as DPDT-SP, although of shorter duration, when administered intrathecally (15 nmol; J. Brown and R. G. Hill, unpublished observations). Thus. the prolonged effects seen in some cases with DPDT-SP may be due to “neurotoxic” actions of this analogue (Hiikfelt, Vincent, Hellsten. Resell. Folkers, Markey, Goldstein and Cuello, 1981). Other workers have found that an analogue of subP, structurally similar to DPDT-SP, stance [a-Pro’. D-Phe’, D-Trp9]-SP. had analgesic actions in thermal and chemical nociceptive tests when administered to mice (3.3-6.5 nmol) (Lembeck rt pi.. 1981; Pierce);. Schroeder. Folkers, Xu and Hiirig, 1981). Furthermore, Piercey er ai. (1981) found that larger doses of their analogue (20nmol) produced motor deficits, and these results were interpreted in terms of antagonism of substance P. Similar findings have recently been reported by Akerman et al. (1982) using DPDT-SP (IO nmol) in the rat. The structural and
0
20
LO
60
80
% Analgesm
Fig. 2. Plot of percentage rotarod score against percentage analgesia values for individual rats treated with either vehicle. 0.5 nmol DPDT-SP or 1.0nmol DPDT-SP. The percentage analgesia value is a mean value computed from results obtained in all three nociceptive tests, except in the case of one animal. in which the results from the hot-plate were not included in any of the analyses (see Methods section). Note that animals which showed clear analgesia also showed clear motor impajrment. Both at 10 and 30 min after administration this correlation was significant (Spearman rank correlation co-efficient ?s = --0.618 and -0.482. respectively. giving P -c0.01and P < 0.05, respectively). Plots of percentage analgesia for each noclceptive test against percentage rotarod score (not shown) did not reveal any selective effect of DPDT-SP in any of the three nociceptive tests used (see also Fig. I).
100
pharmacological similarities between these two analogues of substance P (Folkers et ul.. 1981; Leander rt al., 1981: Lembeck et ul.. 1981). together with the fact that neither has been shown to be an antagonist at spinal substance P receptors and that both have agonist actions in certain tissues (Hawcock. Hayes and Tyers. 1987; Salt QT al.. 1983) suggests that the be]~~~vioLlralresults obtained may. at least in part. be due to direct effects rather than ~~l~t~~~otiislnof substance P. Thus. results from behavioural experiments must be interpreted cautiously. The existence of multiple receptor sub-types for substance P (Lee, Iversen. Han& and Sandberg, 1982) further indicates the possibility that central substance P receptors may have a different specificity- to the peripheral receptors at which these analogues function as antagomsts. It IS thus evident that further information is required to complete the central pharmacological profiles of those analopues of substance P which have peripheral ~II~~~~oI~IS~ properties. However. it appears likely that the be~i~~~~ioLlr~~1 effects of DPDT-SP are not due to antagonism of substance P (Salt c’t trl.. 1987: HZikfelt c’t ui.. 19X1)and thus do not provide evidence for a role of substance P in nociceptive primary
afferent
neurotransmission.
Furthermore.
DPDT-SP does not have any specific antinociceptive properties, as motor impairment always accompanied such efftcts. il~~kr~~~~t/er/yc~n~r~~~r.sWe wish to thank Dr M. Skmgle for dernorrstrating the intrathecal catheterisation technique and Dr M. Tyers foi- the loan of the rotarod appararus. This work was supported by grants from the MRC and SRC.
REFERENCES
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HBkfelt, T.. Kellerth, J. 0.. Nilsson. G. and Pcrnow. B. (1975). Experimental lmmunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurons. Brairl Re,s. 100: 235%252. Hiikfelt. T.. Vincent, S., Hellsten, L., Rosell. S.. Folkers, K.. Markey. K.. Goldstein. M. and Cuello, C. (1981I. Immunohistochemical evidence for a “neurotoxic” action of [n-Pro’. D-Trp’,“i-substance P. an analogue with substance P ~~nta~ollistic ;ictivity. ;Icrtr ~)~~~.\jo~..wtifiLi. 113: 571 573. Irwin. S.. Houde. R. W.. Bennett. D. R.. Hcndcrshot. L. C‘. and Ssevers, M. H. (1951).The effects of morphine. methadone and meperidine on some rcllc\ r-e\ponw\ of spinal animals to nociceptive stimulation. .I. P/wrwrt,. L’V,‘.7110. 101: 132 143. Janssen. P. A. J.. Niemegeers. c‘. .I. F.. and Don!. J. (i. H (lY63). The inhibltory effects of fentanyl and other mom-phinc-like analgcbich on the harm-induced tall w1111drawal reHex in rats. ~r3rlc~in,ittr,/-Ff~~s~/i. 13: 501 507. Leander. S.. HBkanson. R., Roscll. S.. Folkcrs. K.. Sundlcr. F. and Tornqvist. K. (IYXI). A specific suhstancc P alitagonist blocks smooth muscle contractions induced h> i~o~l-choliner~ic, non-adrcncrglc nerve ~tinllil~~ti(~~~. ~Vlirittrw,Lonti. 294: 467 46’). Lee. C. M.. Iversen. L. L.., Hanlev. M. R. and Sand&w
Lembeck. F.. Folkcrs, K. and Donnercr. J. (IYXI).Analgeslc effects of antagonists of substance P. Biwhcw. hoerplow Rcs. C‘omnwt. 103: I3 IX 132I. Ljungdahl. A.. Htikfelt. T. and Nilsson. G. (1978). Dlstribution of substance P-like immunoreactivit> in the central nervous system of the rat I. Cell bodies and nerve tcrminals. iVr~irosc,irnc,e 3: Hh I --94.3. Nicoll. R. A.. Schenker. c‘. and Leeman. S. E. (1YXO). Substance P as a transmitter candidate. -1. Rer. .X’curo.w. 3: __ 777-268. Piercey, M. F., Schroeder. L. A.. Folkers. K.. Xu, J.-c‘. and Hiirig. 3. (1981). Sensory and motor fllnctions of spinal cord substance P. Scirwr~ 214: 1361~ 1363. Randall. L. 0. and Seiitto. 3. J. (19.57). A method for measurement of analgesic activity on inflamed tissue. Archs rnt. Pharrmxodyn. Ther. 111 : 409 -4 19. Resell. S. (1981). Development of novel substance P antagonists. In: Cihtr Fowlduriwl Symposium ,Yo. 9/: Suhatirrlcc* P in t/w ~~mvus s!‘sttw. Pitman. London (In Press). Salt. T. E.. De Vrles. G. J.. Rodriguez. R. E.. Cahusac. P. M. B.. Morris, R. and Hill. R. G. (1982). Evaluation of [~-Pro’. D-Trp’.“]-substance P as an antagonist of substance P responses in the rat central nervous system. ,V~~rosci. Lat. 30: 29 l-295. Siegel. S. (1956). .~u~~p~~~}~~~tr;~ Sfufistics :lbr fhr Brhiormi Scirnces. pp. 152- 156.McGrawHdl. Woolfe. G. and MacDonald, A. D. (1944). The evaluation of the analgesic action of pethldine llydrocliloride (Demerol). f. Piwwrac. c’rn Tlier. 80: 30# 307. Yaksh. T. L. and Rudy. T A. (1977). Studies on the direct spinal actlon of narcotics in the production of analgesia In the rat. .I. Phcrrrm~. c’vp. T/xv. 202: 41 I 42X ZieglgBnsberger. W. and Tulloch. 1. F. 119701. Effects 01 sugstancc p on neuroncs in the dorsal horn of the spinal cord of the cat. Rrcco~ Rc\. 166: 173 2x2.