Proc. Natl. Acad. Sci. USAVol. 90, pp. 3564-3568, April 1993Pharmacology

Substance P markedly potentiates the antinociceptive effects ofmorphine sulfate administered at the spinal level

(pain/analgesia/tachykinin/endogenous opioids)

RICHARD M. KREAM*, TOSHIMASA KATOt, HIROYUKI SHIMONAKAt, JAMES E. MARCHAND,AND W. HEINRICH WURMAnesthesia Research, Department of Anesthesiology, Tufts University School of Medicine, New England Medical Center, 136 Harrison Avenue,Boston, MA 02111

Communicated by Susan E. Leeman, January 19, 1993

ABSTRACT The undecapeptide substance P and the al-kaloid morphine sulfate are two agents previously thought tohave opposite roles in the mediation of spinal nociceptiveprocesses. The present report, however, demonstrates that lowdoses of substance P when coadministered with marginallyeffective doses of morphine sulfate into the rat subarachnoidspace produce a markedly enhanced analgesic response, asmonitored by the tail-flick test. This pharmacological effect isblocked by prior treatment with the opioid antagonist nalox-one, indicating that the potentiated analgesic response is me-diated by opioid-responsive neurons. In addition, the putativeimmediate precursor form of substance P (i.e., substanceP-glycine) may substitute for the mature compound in thepotentiated pharmacological effect. Moreover, the describedsynergism is unaffected by transection of the spinal cord,demonstrating the lack of supraspinal modulation of the ob-served phenomenon. Based on these observations, we are nowable to dissociate opioid-potentiating and analgesic propertiesof substance P from traditional hyperalgesic effects realized atsignificantly higher concentrations. Consistent with previousbiochemical data, a likely mechanism underlying the peptide-mediated enhancement of opioid analgesia may center on theability of substance P to release endogenous opioid peptideswithin the local spinal cord environment. Finally, the phar-macological relationship of coadministered substance P andmorphine sulfate established here supports the hypothesis thatspinal tachykinin and opioid systems have a direct functionalinteraction in the modulation of local nociceptive responses.

An extensive pharmacological literature has documented theanalgesic properties of morphine and related opioid agonistswhen administered directly at the spinal level (1-3). Incontrast, the undecapeptide substance P (SP) has long beenidentified as a neurotransmitter intimately associated withthe transfer of painful or nociceptive stimuli from peripheralreceptive fields into the central nervous system (4-6). Forexample, direct application of large (microgram) doses of SPinto the lumbar spinal subarachnoid space produces hyper-algesia or increased sensitivity to pain (7-9). Concomitant orprior administration of similar large doses of SP markedlydiminishes the magnitude of the analgesic response producedby intrathecal morphine (8). Furthermore, intrathecal mor-phine blocks the hyperalgesic effects of administered SP (10,11) and inhibits the evoked release of SP from spinal cord invivo (12, 13).

In contrast to the spinal effects of SP, administration oflow(nanogram) doses of the peptide at supraspinal sites has beenshown to produce a modest analgesic response (14-16). Thiseffect is presumably due to the SP-induced excitation of

descending modulatory systems and has been operationallycharacterized as opioid in nature by virtue of its reversibilityby the antagonist naloxone (14-17). Thus, it was proposedthat supraspinal administration of SP secondarily releasesendogenous opioid peptides both in the brainstem and spinalcord, thereby producing an opioid-dependent analgesia. Thisproposed mechanism was later supported by experimentaldata demonstrating evoked release of endogenous opioids bySP (17-19).The present study attempts to evaluate whether any ex-

citatory or secretagogic effects of SP are normally in effect tomodulate pain sensation at the spinal level. For these pur-poses, we have utilized a pharmacological paradigm in whichlow doses of SP and morphine sulfate (MS) are coadminis-tered by the intrathecal route to adult male rats followed byanalgesic testing via the tail-flick test. We are thus able todemonstrate synergistic analgesic effects of SP and MS andto dissociate opioid-potentiating and analgesic properties ofSP from traditional hyperalgesic effects realized at signifi-cantly higher concentrations. This differentiation of pharma-cological effects mediated by low and high concentrations ofSP provides insight for understanding spinal analgesic pro-cesses and confirms previous biochemical data describingtachykinin-induced activation ofendogenous opioid systems.

METHODSCannula Implantation. All experimental procedures em-

ployed in the present study were approved by the TuftsUniversity Animal Research Committee, protocol 226-90.Adult male Sprague-Dawley rats, weighing 275-300 g, wereanesthetized by intramuscular administration of ketamine(100 mg/kg)/xylazine (10 mg/kg) and implanted with chronicin-dwelling intrathecal catheters by a modification of theprocedure of Yaksh and Rudy (20). Briefly, after opening thedura at the atlanto-occipitaljunction, a catheter was insertedfor a distance of 8.5 cm, thereby positioning the tip near theL1-L2 lumbar spinal level. The catheter consisted of Silastictubing (o.d., 0.625 mm; i.d., 0.30 mm; 602-105, Dow) with adead volume of 10 ,.d into which a stylet (fused silica, 062442,Scientifi'c Glass Engineering, Austin, TX) was inserted towithin 0.5 cm of the catheter tip to stiffen the Silastic tubingand to facilitate threading of the catheter through the intra-thecal space. After placement, the stylet was removed andthe catheter was secured by dental cement to stainless steelscrews implanted in the skull. The catheter was then pluggedwith a stainless steel insert and threaded through the skin viaa separate small opening lateral to the main skin incision

Abbreviations: MS, morphine sulfate; SP, substance P; SP-G, sub-stance P-glycine.*To whom reprint requests should be addressed.tPresent address: Department of Anesthesiology, Gifu UniversitySchool of Medicine, Gifu City 500, Japan.

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Proc. Natl. Acad. Sci. USA 90 (1993) 3565

leaving a 3-cm external length. After surgery, animals wereindividually housed to ensure the patency of the catheter ina temperature- and light-controlled environment, with freeaccess to food and water. Animals exhibiting any sign ofneurological or motor deficit were excluded from the study.After completion of drug testing, verification of catheterposition was performed in each animal by postmortem ex-amination of the spinal cord.

Analgesic Testing. Animals were given 1 week in which torecover from surgery, during which time they were habitu-ated daily to the laboratory environment and analgesic testingapparatus. For assessment of thermal nociceptive response,a custom-made tail-flick apparatus (Department of MedicalEngineering, New England Medical Center) consisted of avariable-intensity 300-W quartz projector bulb focused -2cm from the underside of the tail and a photodetector-automatic timer sensitive to 0.01-sec intervals. Latency torespond after the onset ofthe radiant heat stimulus applied tothe tail was monitored via a visible twitch or flick, which inturn activated the photodetector. Light intensity wasadjusted to yield a mean baseline latency of 3.5-4.5 sec, andthe automatic cutoff was set at 10 sec to avoid damage to thetail. Before each drug trial, a series of three pre-drug-administration latency measurements were made to establisha stable baseline. Typically, these values varied by <10o.Post-drug latency measurements were performed at 5, 15, 30,45, 60, 75, and 90 min. The respective differences betweenthe baseline and post-drug latencies constituted the time-dependent analgesic response profile. Difference values werefurther normalized as percent maximum possible effect(MPE) units, by the following formula: % MPE = 100 x [TF- BL]/[10.0 - BL], where TF is the post-drug tail-flicklatency, BL is the baseline latency, and 10.0 is the cutoff time.Data from the treatment groups were evaluated usingANOVA followed by post-hoc testing using the Bonferronimethod via the GRAPHPAD INSTAT program.

Transection of the Spinal Cord. To assess whether theeffects ofthe combined drug regimen were spinally mediated,a separate group of rats was surgically treated to achieve anacute transection of the spinal cord. These animals had beenpreviously implanted with in-dwelling cannulae and werefully habituated to the testing apparatus (above). Briefly,animals were anesthetized with 1.5% (vol/vol) halothane,and a laminectomy was performed at the level of the secondthoracic vertebrae. After opening of the dura, the spinal cordwas completely transected without affecting the patency ofthe intrathecal cannula. The wound was closed in layers andanimals were allowed to recover from the anesthesia for =2h before administration of drugs. Immediately after comple-tion of drug testing, animals with spinal cord transectionswere sacrificed by lethal injection of Nembutal. For eachanimal, verification of the completeness of transection wasachieved by postmortem examination of the spinal cord.Drugs. Tested compounds were dissolved in artificial ce-

rebrospinal fluid (pH 7.33), prepared as described by Crid-land and Henry (9) and injected in a volume of 10 ,ul followedby 10 ,ul of artificial cerebrospinal fluid to flush the catheter.Drug administrations were completed within 1 min and werefollowed by analgesic testing beginning at the 5-min point. SPwas obtained from Peninsula Laboratories, and was furtherpurified to homogeneity by HPLC, as described by Kream etal. (21). SP-glycine (SP-G) was enzymatically generated fromsynthetic SP-Gly-Lys (Peninsula Laboratories) and purifiedto homogeneity by HPLC (21). Stock concentrations ofHPLC-purified peptides were determined by amino acidanalysis and comparative chromatogram peak height ratios.Working solutions ofMS and the opioid antagonist naloxonewere prepared as indicated (22).

RESULTSCoadministration of 0.1 pg ofMS (equivalent to 300 pmol ofmorphine base) and 10 pmol ofSP into the rat central nervoussystem by the intrathecal route produced a maximal analgesicresponse, as reflected in tail-flick latency reaching cutoff atthe 5-, 15-, and 30-min points, with return to baseline after 60min (Fig. 1A). The magnitude of the analgesia was consid-

** ** **A100 A A A A

MS + SP80

60 -\

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20

-20~~ ~ ~ ~ ~~05 15 30 45 60

Time after administration (min)

FIG. 1. (A) Time-dependent analgesic responses after intrathecaladministration of a relatively low concentration of MS (0.1 pg; opentriangles) or the same concentration ofMS in combination with SP (0.1pg and 10 pmol, respectively; solid triangles). By comparison, ad-ministration of SP alone (10 pmol) or artificial cerebrospinal fluid(open and solid circles, respectively) produced insignificant changes intail-flick latencies. Ordinal values represent tail-flick latency measure-ments normalized as percent maximum possible effect (means ±SEM, n = 10 for each ofthe four groups). The absence ofan error barindicates that the value of the SEM is smaller than the size of thespecified symbol. Whereas MS alone produced a modest analgesicresponse peaking at -30%o maximum possible effect, the combinationofMS and SP produced maximal responses at 5, 15, and 30 min. Theanalgesic response produced by MS in combination with SP wasessentially eliminated by pretreatment with intrathecally administerednaloxone at 1.0 M.g (n = 6; open squares, dotted trace). Naloxone wasgiven 10 min prior to administration of MS and SP. *, P < 0.01; **,P < 0.001 (significantly different from SP alone and artificial cere-brospinal fluid treatment groups). The MS and MS plus SP treatmentgroups were significantly different from each other (**, P < 0.001) at5, 15, 30, and 45 min. (B) Time-dependent analgesic responses afterintrathecal administration of MS (0.1 ,ug; open squares) or MS incombination with the unamidated precursor SP-G (0.1 ,ug and 10 pmol,respectively; solid squares). Administration of SP-G alone (10 pmol)and artificial cerebrospinal fluid are represented by open and solidcircles, respectively. Ordinal values are as defined above (n = 6 foreach of the four groups). For comparative purposes, an additionalgroup of six animals was treated with 0.1 ,ug ofMS plus 10 pmol ofSP(broken trace), yielding an analgesic response curve that was indis-tinguishable from that produced by the combination ofMS and SP-G.*, P <0.01; **, P <0.001 (significantly different from SP-G alone andartificial cerebrospinal fluid treatment groups). The MS and MS plusSP-G treatment groups were significantly different from each other. *,P < 0.01, at 45 min; **, P < 0.001, at 5, 15, and 30 min.

Pharmacology: Kream et al.

3566 Pharmacology: Kream et al.

erably greater in a statistically significant manner than thatproduced by an equivalent intrathecal administration of MSalone at 0.1 ,ug through the 45-min point. Integration of therespective response curves between 5 and 60 min demon-strated that the combination of agents provided an -4-foldincrease in analgesic potency, as compared to MS alone.Additionally, a very similar analgesic response was observedafter coadministration of 0.1 ,g of MS and 10 pmol of theunamidated putative precursor to SP [i.e., the dodecapeptideSP-G (21, 23)], where the intensity and duration of the effectwere indistinguishable from that produced by the combina-tion of MS and SP (Fig. 1B). By comparison, control injec-tions ofSP or SP-G alone at 10 pmol, or vehicle, produced nomeasurable analgesic response at these same time points.Finally, the enhanced analgesic response of MS in combina-tion with SP was essentially eliminated by pretreatment withintrathecally administered naloxone at 1.0 ,ug (Fig. 1A),reflecting the opioid nature of the effect.The dose dependency of the described phenomenon was

further investigated (Fig. 2). At a set concentration of 0.1 ,ugof MS, the peptide-mediated potentiation of opioid analgesiawas maximal at 10-100 pmol of coinjected SP, with marginalefficacy at 1 and 1000 pmol. Thus, the SP dose-responsecurve appeared to be of a bell-shaped or inverted-U config-uration, consistent with previous work describing analgesicproperties of supraspinally administered SP (15). In ourstudy, the apparent lack of potentiation by 1000 pmol of SPmay result from the statistically significant hyperalgesiceffect produced by administration of this relatively high dose.We further assessed the analgesic responses elicited by low

and high concentrations of MS in combination with a setconcentration of 10 pmol of SP. At a relatively ineffectivedose of 0.02 ,g of MS, a strong SP-mediated enhancement ofopioid analgesia was observed (Fig. 3A). Here the potentia-tion of the analgesic response was -6-fold, as determined

4-)

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- - 1 10 100 1000 SP(pmol)

FIG. 2. Dose dependency of the SP-mediated potentiation ofMS-induced analgesia. Normalized changes in tail-flick latenciesrepresented as percent maximum possible effect are monitored 15min after administration of increasing doses of SP in combinationwith a set concentration of 0.1 ,ug of MS (open bars). The effects ofincreasing doses of SP alone on tail-flick latencies are represented bythe solid bars. Ordinal values are as defined above (n = 6) for all ofthe treatment groups. The maximal analgesic effects of SP either at10 or 100 pmol in combination with 0.1 ug of MS were significantlydifferent (**, P < 0.001) from those observed in all of the othertreatment groups. The hyperalgesic effect produced by administra-tion of 1000 pmol of SP alone (represented as a -25% change in themaximum possible effect) was significantly different (**, P < 0.001)from those observed in the respective treatment groups receiving 1,10, and 100 pmol of SP alone.

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Time after administration (min)

FIG. 3. (A) Time-dependent analgesic responses after intrathecaladministration of a very low concentration of MS (0.02 ug; opentriangles) or the same concentration of MS in combination with 10pmol of SP (solid triangles). Whereas administration of MS aloneproduced a weak response, addition of SP resulted in a strong 6-foldenhancement of opioid analgesia, as quantified by integration of therespective response curves. (B) Time-dependent analgesic responsesafter intrathecal administration ofa high concentration ofMS (0.5 ,ug;open triangles) or the same concentration ofMS in combination with10 pmol of SP (solid triangles). Administration of 0.5 ,ug ofMS aloneelicited a maximal analgesic response between 5 and 30 min, whichdeclined to baseline at 60 min (open triangles). The same concen-tration of MS in combination with 10 pmol of SP resulted in a 2-foldenhancement in ahalgesic potency, as compared to MS alone. ForAand B, ordinal values are as defined above (n = 6) for all of thetreatment groups. **, P < 0.001 (significantly different from the MStreatment group).

from the integration values for the respective responsecurves. In contrast, a 0.5-,ug dose of MS alone elicited amaximal analgesic response between 5 and 30 min, whichdeclined to baseline at 60 min. This same concentration ofMSin combination with SP resulted in an =2-fold increase inanalgesic potency, as described by extended duration of themaximal response and a prolongation of the time of effect outto 90 min (Fig. 3B). Overall, the peptide-mediated potentia-tion of opioid analgesia was more effectively realized at thelower doses of administered MS (0.02 and 0.1 ,ug vs. 0.5 ,Ag),thereby indicating a synergistic relationship between the twoagents in producing a biological response at the spinal level.

Finally, to assess the role of opioid-responsive descendingspinal systems (24) in the development of the potentiatedanalgesic response, a comparable pharmacological experi-ment was performed on animals with acute spinal cordtransections (Fig. 4). Here it was necessary to administer a2-fold higher concentration of MS to achieve a modestanalgesic response equivalent to that observed in the intactanimal (0.2 pg vs. 0.1 ug, respectively; compare with Fig.1A). In the spinally transected rat, however, the SP-mediatedpotentiation of opioid analgesia was not only preserved but

Proc. Natl. Acad. Sci. USA 90 (1993)

Proc. Natl. Acad. Sci. USA 90 (1993) 3567

? 80 MS + SP

60 -V)~~~~~~~~~*U,

° 40 - **E Mx 20

-205 15 30 45 60 75 90

Time after administration (min)

FIG. 4. Time-dependent analgesic responses after intrathecaladministration of MS (0.2 gg; open triangles) or MS in combinationwith SP (0.2 i&g and 10 pmol, respectively; solid triangles) to animalswith acutely transected spinal cord. In the spinally transected rat, theSP-mediated potentiation of opioid analgesia was significantly am-plified compared to the normal rat (compare with Fig. 1). Thecombination of agents produced a maximal response with extendedduration from 5 to 45 min and prolonged time of effect. Integrationof the respective response curves indicated that MS in combinationwith SP produced a 6-fold enhancement in analgesic potency, ascompared to MS alone. Ordinal values are as defined above (n = 6for all of the treatment groups). **, P < 0.001 (significantly differentfrom the MS treatment group).

also significantly amplified. The duration of the maximalresponse extended from 5 to 45 min and the time of the effectwas prolonged for an additional 45 min. Integration of therespective response curves indicated that the combination ofagents provided an -6-fold enhancement in analgesic po-tency, as compared to MS alone.

DISCUSSIONIn the present report, we have described a pharmacologicalrelationship in which picomole amounts of SP markedlypotentiate the analgesic potencies of modest and even rela-tively ineffective doses of MS when administered by theintrathecal route. The ability of the opioid antagonist nalox-one to effectively block the potentiated analgesic responsestrongly indicates a convergence of pharmacological effectsthrough opioid-responsive neurons. In addition, we havedetermined that the SP-mediated potentiation of opioid an-algesia is unaffected by transection of the spinal cord,thereby indicating the lack of descending supraspinal mod-ulation (24) of the observed phenomenon.From a historical perspective, it is worth comparing these

present findings with previous information relating to theanalgesic properties of SP. The potentiating properties of SPlie within a relatively narrow concentration window of 10-100 pmol of coadministered peptide (Fig. 2). Previously,administration of low (nanogram) doses of SP alone by theintracerebroventricular route was sufficient to engendernaloxone-reversible analgesia (14-16). The similarities be-tween these reported values indicate that the "nontradi-tional" antinociceptive properties of the peptide are realizedat lower doses. Since many previous pharmacological studiesemploying the intrathecal model have not observed any directanalgesic effects of SP when administered alone (7-11), it islikely that any localized excitatory or secretatogogic effectsof SP in operation at the spinal level are not solely sufficientto promote a significant analgesic response. Since data fromdrug testing of rats with acutely transected spinal cordsindicate that the potentiating effects of SP are realized in thelocal spinal cord environment (Fig. 4), the antinociceptive

role of spinally released SP may be to initiate an amplificationmechanism for opioid action subsequent to painful stimuli.

This proposed mechanism is consistent with previousbiochemical studies demonstrating evoked release of endog-enous opioid peptides by SP (17-19). In support of thisassertion, anatomical studies at the ultrastructural level havedescribed primary afferent terminals making synaptic contactwith second-order opioid-expressing neurons in the superfi-cial dorsal horn (25-27). In addition, these observations arecomplemented by recent physiological studies demonstratingthat noxious stimulation produces slow excitatory postsyn-aptic potentials in wide-dynamic-range dorsal horn neuronsspecifically through activation of SP receptors (28, 29). Sinceboth enkephalinergic and dynorphinergic phenotypes havebeen identified in this class of spinal cord neuron (30, 31), thenoxiously evoked secondary release ofendogenous opioids isconsistent with the proposed secretagogic and ultimatelyanalgesic effects of pharmacologically administered SP. Fi-nally, an immunohistochemical study has shown extensivecolocalization of SP and a proenkephalin-derived opioidpeptide within intraspinal neurons innervating the superficialdorsal horn and has also suggested a functional role forcoreleased SP and endogenous opioids in spinal analgesicprocesses (32). This morphological observation providesadditional support for a functional interrelationship of spinaltachykinin and opioid systems by outlining a local analgesiccircuit converging through opioid-responsive neurons withinthe dorsal horn.

In conclusion, the combined data presented here provideevidence that spinal tachykinin and opioid systems have adirect functional interaction in the modulation of local noci-ceptive responses. A likely mechanism underlying this phar-macological relationship centers on the ability ofSP to releaseendogenous opioid peptides within the local spinal cord envi-ronment. In addition, our study provides a means to function-ally discriminate between the potentiating or analgesic effectsand the direct hyperalgesic effects of SP at the spinal level.Recently, dissociation of traditional inhibitory and excitatoryeffects of spinal opioids has been described (33, 34), and asimilar differentiation of activity may apply to the spinaleffects of SP. Our data also indicate that the putative imme-diate precursor form of SP (i.e., SP-G) is fully active and maysubstitute for the mature compound in the potentiated phar-macological effect. Thus, the amidated C-terminal end ofmature SP is not required for this activity, suggesting theinvolvement of a nontraditional tachykinin receptor. We havepresented (23) biochemical and morphological evidence dem-onstrating significant steady-state levels of SP-G in primaryafferent terminals innervating the dorsal horn. The presence ofSP-G in primary afferent terminals suggests that unamidatedprecursor is packaged in peptide-containing vesicles and re-leased with SP subsequent to peripheral stimulation. WhetherSP-G fulfills a special biological role in ongoing spinal cordprocesses remains to be fully investigated. Finally, a recentstudy has observed that N-terminal fragments of SP directlymodulate opioid receptor binding (35). Since we have dem-onstrated that very low doses of peptide are required forenhancement of opioid action, as reflected in strong synergis-tic effects of coadministered SP and MS at stoichiometries of1:30 to 1:60 (Figs. 2 and 4, respectively), it is doubtful whetherthe pharmacological effects observed here are mediated di-rectly through changes in opioid receptor occupancies. Inconclusion, the synergistic analgesic relationship of MS andSP established here may initiate approaches to the investiga-tion of interactive spinal systems via combinatorial pharma-cological analyses and clinical applications for control of painand addiction.

We thank Drs. Carolyn A. Cohen and Klaus A. Miczek for advicerelating to the behavioral and statistical aspects of the experiments.

Pharmacology: Kream et al.

3568 Pharmacology: Kream et al.

The research in the present report was supported by the NationalInstitute on Drug Abuse (Grant DA04128 to R.M.K.) and by theDepartment of Anesthesiology, New England Medical Center, TuftsUniversity School of Medicine.

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Proc. Natl. Acad Sci. USA 90 (1993)