Pain, 31 (1987) 263-276

Elsevier

263

PA1 01117

Behavioural effects of receptor-specific substance P agonists

Dalia Papir-Kricheli * , Joseph Frey *, Ralph Laufer *, Chaim Gilon * * , Michael Chorev * * *, Zvi Selinger * and Marshall Devor *

* Life Sciences Institute, * * Institute of Chemistry, and * * * School of Pharmacy, Hebrew University of

Jerusalem, Jerusalem 91904 (Israel)

(Received 16 December 1986, revised received and accepted 30 April 1987)

Summary Septide and senktide are synthetic substance P (SP) agonists with extremely high

selectivity for 1 of the 3 known SP receptor subtypes. When injected intrathecally, they produced

dramatically different behavioural effects. Septide, the selective SP-P receptor agonist, evoked intense,

compulsive scratching, biting and licking of the hind limb, with no sign of motor flaccidity, and without

measurable effect on responses to noxious thermal or mechanical stimulation of the foot or tail. In

contrast, senktide, the selective SP-N receptor agonist, produced profound, but transient, motor flaccid-

ity, reduced response to noxious stimuli and, at low doses, ‘wet-dog shakes.’ These various symptoms, all

previously associated with SP and/or synthetic SP analogues, appear therefore to derive from activation

of distinct SP receptor subtypes.

Key words: Paralysis; Pain; Senktide; Septide; Substance P; Substance P receptor; Tachykinin;

Tachykinin receptor

Introduction

Intrathecal injection of substance P (SP) and synthetic SP analogues in rodents produces a bizarre combination of behavioral symptoms [1,3-7,9,10,18,20-22,24, 27-29,31,32,34,35,39,43]. On the one hand, they produce hypersensitivity to noxious mechanical and thermal stimuli immediately upon drug injection, and a constella- tion of positive signs such as distress vocalization, ‘wet dog shakes,’ and compulsive scratching, biting and licking of the relevant body part. These effects have been taken to indicate that the animal is feeling discomfort or pain and are therefore widely quoted as supporting the candidacy of SP as a neurotransmitter in the pain

Correspondence to: Dr. M. Devor, Life Sciences Institute, Hebrew University of Jerusalem, Jerusalem

91904, Israel.

0304-3959/87/$03.50 0 1987 Elsevier Science Publishers B.V. (Biomedical Division)

264

pathway. On the other hand, there are also frequent reports of (delayed) hypoalgesia with intrathecal injection of SP, and of motor flaccidity with injection of SP analogues, particularly putative SP antagonists. These signs do not fit well with the pain hypothesis [36] and suggest other roles for SP. One obvious way of reconciling these observations is the possibility that the drugs are acting simultaneously on several different functional systems, perhaps via different membrane receptors. Indeed, much of the variability of symptoms may result from variations in drug

concentration at the effective site of action, and the relative selectivity of different analogues at activating or blocking different receptor types.

In recent years it has become clear that SP is only one of a family of structurally related neuropeptides, the tachykinins [13]. Members of the tachykinin family share the C-terminal sequence, Phe-X-Gly-Leu-Met-NH,, and include the amphibian skin peptides, physalaemin and kassinin, and the molluscan salivary gland peptide, eledoisin, in addition to the mammalian neural peptides SP, neurokinin A (also known as substance K), and neurokinin B (also known as neuromedin K). All 3 mammalian tachykinins occur in the upper layers of the dorsal horn [25]. At present, 3 distinct tachykinin receptor subtypes have been identified and char-

acterized in the mammalian PNS, and there is substantial evidence suggesting that

TABLE 1

DEGREE OF DISCRIMINATION BETWEEN THE 3 TACHYKININ-RECEPTOR SUBTYPES BY

SOME NATURALLY OCCURRING AND SYNTHETIC TACHYKININ AGONISTS

Figures show the effective concentration (in nM) required to obtain a 50% response in the bioassays

listed below (EC,,).

Peptide Source Ref. Receptor subtype (EC,, (nM)

SP-P SP-N SP-E

SP Mammalian CNS

Neurokinin A Mammalian CNS

Neurokinin B Mammalian CNS

Eledoisin Mollusc gland

Physalaemin Amphibian skin

Kassinin Amphibian skin

Septide Synthetic

AcArg’Septide Synthetic

Senktide Synthetic

SP receptor

subtype

Bioassay

1411

[**I

[411

[**I

1::;

[411

[**I

[411

3.5

28

4.2

5.9

2.6

2.1

2.5

3

35,000

59 20.000

78 40

1.3 400

12 200

65 20.000 4.5 70

1300 > 200,000 *

1250 t 200,000 *

0.5 > 200,000 *

SP-P

SP-N

SP-E

Contraction of the guinea pig ileum in the presence of atropine (3 x 10 ’ M) [16]. Contraction of the guinea pig ileum after sensitization with SP methyl ester (3 X 70 ’

M) [161. Potentiation of electrically induced contraction of rat vas deferens [16].

* No effect at the indicated concentration.

* * R. Laufer. unpublished data.

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all three also occur in the CNS [2,11,13,14-1’7,23,30,38,41]. These are the SP-P receptor, for which SP and physalaemin are slightly more potent agonists than the other tachykinins, the SP-E (or SP-K) receptor, for which eledoisin, kassinin and neurokinin A are particularly potent agonists, and the SP-N receptor, for which neurokinin B is a particularly potent agonist. Although each receptor displays a certain selectivity for the various tachykinins (summarized in Table I), all three can be activated by each, and therefore the natural tachykinins are not well suited for determining the functional effects of activating the different SP receptor subtypes individually. It would obviously be of interest to investigate the behavioral effects of intrathecally applied substances that have highly selective actions on one or another of the tachy~~n receptors.

We have recently developed highly selective synthetic agonists for 2 of the 3 known SP receptor types [14,15,41]. These are septide ([Glu6,Pro9]SP,_,,), a sub- stance which activates the SP-P receptor at molar concentrations 520 times lower than the SP-N receptor and more than 100,000 times lower than the SP-E receptor, and senktide (succinyl-[Asp6,Me-Phe’]SP,_,,), a substance which activates the SP-N receptor at concentrations at least 70,000 times lower than either of the other 2 receptors (Table I). Both of these substances activate their respective SP receptor subtypes with far greater selectivity than any of the naturally occurring tachykinins (see Table I), and therefore they provide useful tools for analyzing the functional contribution of the SP-P receptor, the SP-N receptor, and SP itself. In this report we describe the behavioral effects of intrathecal injection of septide and senktide in adult rats.

Methods

Subjects and drug injection The experiments were carried out using adult male rats (250-350 g) of the

Wistar-derived Sabra strain. The animals were housed in groups of 2-4 in solid-bot- tomed cages covered with pine shavings, provided with food and water ad libitum, and maintained at 24” C with a 6.00 on : 18.00 off day : night cycle.

For intrathecal drug injection, each animal in turn was lightly anesthetized with ether and the fur on the back shaved. With the spinal column arched, a 25-gauge hypodermic needle was inserted into the subarac~oid space at the level of the lower sacral spinal cord-cauda equina by puncturing on the midline at an angle of = 45 o between the L4 and L5 (It 1) vertebrae. A 3Q-pl bolus of drug or dilution vehicle (0.1 mg/ml bovine serum albumin (BSA, Sigma) in sterile Hepes-buffered Krebs solution (pH 7.6)) was then injected intrathecally, and the needle was withdrawn [9,10,28]. In skilled hands the injection procedure takes about 3 min, from the beginning of ether administration to withdrawal of the hypodermic needle. Animals regained consciousness within 2 min of injection, and behavioral testing began after 5 min, by which time they were fully awake. Drugs used were: (1) the water soluble derivative of septide (acetyl-[Arg6,Pro9JSP~_r1), prepared by us using a modification of methods described elsewhere [14]. This derivative has agonist

266

properties virtually identical to those of the original septide [R. Laufer, unpublished data]; and (2) senktide, also prepared by us [see 151. In both cases the pre-dilution concentration was 0.1 mg/ml. Rats were tested using 30-~1 volumes of septide and senktide undiluted (3 pg), or following stepwise dilution by factors of 10 until behavioral scores did not differ from those of vehicle controls (see Results). Three

rats were tested at each dose level. Individual rats were given only a single intrathecal injection at a single dose. That is, no animal was injected more than

once. To assure that injected drugs reach the lower lumbar spinal cord, a preliminary

study was carried out, using 20 rats, in which drugs were injected in various volumes, mixed with 1% methylene blue. In post-mortem dissections performed about 30 min after injection, the dye was always localized within the subarachnoid space, with the extent of spread dependent on the volume injected. A 30-~1 bolus spread rostrally as far as the lower thoracic spinal cord, about 40 mm from the injection site. Spread caudally was substantially less. The hind limb and tail spinal representations were therefore definitely flooded with drug, and while we cannot rule out some spread as far as the forelimb representation, the dose there would necessarily be very low (see Results).

Behavioral observations Behavioral observations consisted of a battery of 8 standardized tests, which

required about 5 min to administer. The test battery was developed on the basis of qualitative observations made during pilot trials with septide and senktide. Each experiment began with 2 pre-injection tests, administered 10 and 5 min before ether anesthesia and intrathecal drug injection. There were then 8 post-injection tests, administered 5, 10, 15, 20, 25, 30, 60 min and 24 h from the time of drug injection. Statistical evaluations are based on 2-tailed Student’s t tests where a continuous variable was used (tests 3, 4, 5, 6, 7 and S), or Mann-Whitney U tests where ordinal data were involved (tests 1 and 2). Comparisons were made between drug- and vehicle-treated groups at the same post-injection time, except where noted. All means are expressed k standard error of the mean. The tests, always given in

standard sequence, were the following. (I) Hind limb posture. This was rated on a 5-point scale with the rat at rest on a

flat surface, where 4 = hyperextension, 3 = normal weight supporting stance, 2 = hypoextension with weight support, 1 = no weight support (rat resting on its belly) but with plantar surface of the foot maintained against the surface, and 0 = no weight support. The hind limbs were usually splayed, and the foot was not moved

when laid dorsal surface down. The right and left limbs were scored separately, but since there were no consistent differences results were averaged.

(2) Hind limb jlexion. With the rat held in the left hand, each hind limb was grasped in turn with the right, and pulled into extension. Counterflexion was rated on a 5-point scale where 4 = hyperflexion, 3 = normal flexion, 2 = weak flexion, 1 = flexor twitch but without detectable flexor force, and 0 = no flexion.

(3) Hot plate test. The rat was placed on a 53.3 + 0.3”C hot plate by rapid withdrawal of an insulating card, and latency to licking a hind paw was measured

267

with a stopwatch. We made a note of the first limb licked, but there was no consistent left-right preference. The maximal exposure time allowed was 25 sec.

(4) Tail immersion. The distal half of the tail was immersed in 55.0 &- 0.1” C water

and latency to flick removal or vocalization was measured. Exposure time was limited to 25 sec.

(5) Noxiouci mechanica stimulation. The left then right foot was placed in a prone

posture under the conical teflon probe (rounded 2 mm tip) of a Randall-Selitto Analgesy-meter (Ugo Basile) and the force applied was increased linearly (48 g/set, maximum 750 g) until the rat withdrew its limb or vocalized.

(6) Tail pinch. A metal compression clip was applied to the tail about 5 cm from the base, and time to tail flick or vocalization was measured. Reaction times < 1 set

were scored as 1 set, and the test was ended if there was no response by 10 sec. The clip, which applied compressive force between 2 smooth jaws, was adjusted so as to produce moderate pain when applied to the 5th digit of the experimenter (force =2

kg). (7) Open-field activity. The rat was placed at the center of an elevated 60 X 45 cm

platform marked off in a 7.5-cm grid. We counted the number of grid-line crossings of a point near the base of the tail during a 30-set observation period and noted

whether the rat used the hind limbs to locomote, or dragged them passively. If the rat reached the edge of the platform it was returned to the center.

(8) Stereotyped behaviors. The rat was replaced in its cage and observed passively for 60 set, while the number of seconds engaged in stereotyped behaviors such as

licking and scratching the hind limbs was measured.

Electrophysiology In the light of the behavioral observation of marked flaccid paralysis produced

by set&tide, we carried out some preliminary electrophysiological observations to determine whether this substance acts as a local anesthetic. In these experiments we applied senktide (0.1 mg/ml) to the desheathed sciatic and sural nerves and to lower lumbar dorsal and ventral roots in nembutal anesthetized rats (50 mg/kg i.p.) and

looked for changes in hind limb muscle EMGs and nerve compound action potentials evoked by electrical stimulation of the sciatic nerve and the L4 and L5 spinal roots.

Results

By 5 min after intrathecal drug injection the rats appeared fully awake and active, and with the exception of open-field activity which declined, scores did not change following vehicle injection on any of the behavioral measures tested (Fig. 1). Since a decline in open-field exploration also occurred in rats that had not been injected, we presume that this simply reflects familiarization with the test arena. The

small apparent increase in hot plate latency (Fig. 1, upper right) did not reach statistical significance (P > 0.2). We conclude that vehicle effects, and any lingering effects of the ether, were insubstantial. Septide and senktide, in contrast, produced

Ca

ud

al

Scr

atch

/it&

F

,eld

Crossmgs

Hin

d ,,m

b

(SK

/

60

set)

(p

er

30

Se-

C)

Fle

xIO

n

c i

Tal

l P

tnch

(s

et)

T I .

269

Figs. 4 and 5. Dose-response relations for senktide (left) and septide (right) measured 10 min after intrathecal drug injection. Symbols indicate mean j, standard error (N = 3).

obvious and contrasting behavioral changes. The results are summarized in Figs. 2-5.

Septide The most obvious effect of septide (3 pg, 3.5 nmol) was compulsive stereotyped

scratching, biting and licking of the hind limb (but never the forelimb), which lasted for about 30 mm after drug injection (Fig. 2, lower right). The animals were not irritable when handled and did not vocalize or give any other indication of being in severe pain. During the period of intense scratching and licking of the hind limb, posture and movements were normal, as were responses to noxious thermal and mechanical stimulation of the hind paws and tail (P > 0.2). The slight increase in hot plate latency lo-20 min post-injection (Fig. 3, right) was nearly identical to that produced by vehicle injection (Fig. 1, right). Dilution of septide to 0.3 pg (in the standard 30-yl bolus, 0.35 nmol) eliminated its effect (Figs. 4, 5, right).

Senktide Rats injected intrathecally with senktide behaved very differently from those

injected with septide. The most obvious effect of senktide 3.0 pg (3.6 nmol) and 0.3 pg (0.36 nmol) was a profound, but transient, flaccid paralysis of both hind limbs (Fig. 6). Beginning immediately on awakening and lasting for about 20 min (Fig. 2, left), the rats did not support their weight on their hind limbs, made no flexion movements when the hind limb was manually extended, and dragged the backs of their bodies along when locomoting with the forelimbs. These behaviors returned

270

Fig. 6. Photograph of a rat about 10 min after intrathecal injection of 3 pg senktide. Note splaying of the

flaccid hind limbs.

between 15 and 30 min post-injection, often with surprising suddenness. The forelimbs were unaffected. At these doses there were no obvious stereotyped movements of the sort seen with septide, and there was no struggling, vocalization

or other signs during handling that might have suggested that the animals were in pain.

Responses to noxious thermal and mechanical stimuli on the hind paw and tail

were reduced for the first 15-30 min after injection (Fig. 3, left). For mechanical stimuli the tail was more profoundly affected than the foot. Responses gradually returned to normal, although scores for (foot) hot plate and tail pinch did not return to normal until 30-60 min post-injection. The forelimb was not affected. It must be kept in mind that during the tests at 5 and 10 min post-injection, the animals had no motor control of the hind limb or tail, and therefore responses to noxious stimuli during this period could only be expressed by vocalization and by struggling with the trunk and forelimbs. By 15-20 min, however, there had already been substantial motor recovery. Curiously, dilution of senktide from 3 pg to 0.3 pg produced a slight exaggeration in 3 of the 4 measures of nociception (Fig. 5, left). Individually, however, the increases did not achieve statistical significance (P > 0.2). There were no major effects on motor tone or noxious threshold on the forelimbs.

Senktide did not produce significant flaccidity at 0.03 pg (36 pmol), but this dose did produce a large elevation in hot plate latency (P < 0.01) reduced sensitivity to noxious mechanical stimulation of the foot (0.05 < P < O.l), and total insensitivity to tail pinch in 1 of the 3 rats tested. Indeed, the response to tail pinch was low (in 2 of 3 rats) even at a senktide dose of 0.003 pg (3 ng. 3.6 pmol, Fig. 5. left).

271

A curious exception to the monotonic decline in symptoms with decreasing doses was the appearance of stereotyped ‘wet-dog shakes’ which occurred in all 3 of the rats tested with intrathecal injection of 0.003 pg senktide. This behavior was not seen at higher or lower dose levels and was not seen with septide. ‘Wet-dog shakes’ occurred in brief convulsive bouts repeated at irregular intervals l-11 times/mm. They began as the animal emerged from the anesthetic, intensified during the period

lo-25 min post-injection (mean 4.5/min) and then gradually declined over the next hour.

Electrophysiologv Sex&tide (0.1 mg/ml) applied on a soaked cotton pledget to the desheathed

sciatic or sural nerves, or bath applied to the L4 and L5 dorsal and ventral roots, did not affect evoked EMGs or compound action potentials (5 experiments). Substitution of 2% lignocaine blocked both.

Discussion

The receptor-selective substance P analogues septide and senktide produced dramatically different behavioral effects when injected intrathecally. Septide, the selective SP-P receptor agonist, evoked intense, compulsive scratching, biting and licking of the hind limb without measurable effects on responses to noxious stimulation of the foot or tail, and with no sign of motor flaccidity. In contrast,

senktide, the selective SP-N receptor agonist, produced a combination of profound motor flaccidity and reduced response to noxious stimuli.

In this study we made direct intrathecal injections in rats using a method similar to that of Hylden and Wilcox [9] in mice. This procedure, which mimics clinical lumbar puncture, avoids the spread of drugs to brain-stem structures and facilitates intersegmental comparisons (especially hind limb versus forelimb) in the same animal. It has several advantages over the more widely used spinal catheterization technique [42], such as eliminating the need for a prior surgical preimplant, together with the attendant risks of brain-stem or cord injury and infection, prolonged CSF leak, and faulty catheter localization in the rostrocaudal or dorsoventral planes. Furthermore, since it is simple and quick, it eliminates the temptation of using the same animal for more than one ‘drug type or dose. On the other hand, since we felt obliged to use light ether anesthesia during the actual injection, the first few minutes of drug effects were obscured. This drawback is particularly severe for natural substances which are rapidly metabolized, but presents less of a problem for the synthetic analogues used here [14-161. It is not unlikely, however, that we missed the early, transient phase of vocalization and hyperalgesia that may occur in immediate response to intrathecal injection of SP and related tachykinins [e.g., 3,431.

Septide produced a period of hind limb biting, scratching and licking that

probably indicates that the animal was feeling distress, perhaps intense itch. However, there was no change in response to noxious thermal or mechanical stimuli during this period.

272

Senktide produced 2 distinctly different symptoms, flaccidity and hypoalgesia. Could the reduced response to noxious stimuli have been due to the inability of the animal to express its distress rather than to true hypoalgesia? At least 3 lines of evidence suggest not. First, the hypoalgesia involved a failure to vocalize and struggle in addition to failure to withdraw the (paralyzed) limb. For comparison, the animals injected with vehicle or septide always vocalized upon tail pinch. Second, it

outlasted the motor flaccidity by a substantial period of time. And third, it persisted at a drug dose 2 orders of magnitude below that required to produce flaccidity. It seems likely, therefore, that the SP-N receptor plays an important and distinct role in both noxious sensation and motor control.

SP and neurokinin A occur in primary afferent terminals in the dorsal horn, and all 3 known mammalian tachykinins occur in interneurons within the dorsal and

intermediate gray matter. In addition, a descending SP-containing fiber system has been described in the ventral horn [e.g., 8,251. We do not yet have sufficient data to

comment on the mechanism whereby senktide produces motor flaccidity, other than to state that it is not a local anesthetic and therefore presumably acts at synaptic junctions and/or conceivably on spinal vasculature. We note, however, that the flaccidity was global, including block of voluntary (supraspinal) motor control. This suggests that senktide generates inhibition in spinal interneuronal pools. SP bath applied in vitro has been shown to depolarize and excite spinal motoneurons [e.g., 12,261, a result that seems inconsistent with flaccid paralysis. This, of course, may be the outcome of simultaneous activation of several different SP receptor types and would explain the fact that flaccid paralysis is not generally seen following intrathe- cal injection of SP itself. Interestingly, repetitive stimulation of some classes of afferent C fibers, which presumably release a mix of tachykinins and other peptides in the spinal cord, produces a prolonged heterosynaptic facilitation of the polysyn- aptic ventral root reflex [39,40].

On the sensory side, there is specific evidence that the inhibition that yields hypoalgesia involves inhibitory interneurons. Specifically, the delayed (‘ rebound overshoot’) hypoalgesia produced by intrathecal SP, an effect that closely resembles the hypoalgesia that we saw following senktide injection, is blocked by systemic naloxone [4,10,43]. This suggests that SP in this instance may be exciting an inhibitory enkephalinergic interneuron. In vitro, the tachykinins as a group tend to be powerfully excitatory on (unidentified) dorsal horn neurons [33].

Previous attempts have been made to analyze the diverse functional effects of intrathecal SP in terms of different ligand receptors by taking advantage of the relative selectivity of the various natural tachykinins for the SP-P, SP-N and SP-E receptors [3,5,6,21,35]. For example, thermal hyperalgesia as measured by the tail-flick test appears to result from activation of the SP-P receptor, on the grounds that it is produced at slightly lower doses by SP and physalaemin than by eledoisin, eledoisin-related peptide or neurokinin A [3,21]. The dose-response relationship for distress vocalization was not noted in these studies. Scratch/bite/lick behavior probably involves either the SP-P or the SP-E receptor (or both) on the grounds that neurokinin B (SP-N selective, Table I) produces this behavior only at much higher doses than SP and other tachykinins [35]. Unfortunately, the molar selectivity of the

273

natural tachykinins is relatively poor (Table I) and the results obtained using this

strategy are therefore somewhat uncertain. A priori, there is no reason to expect that any given behavioral symptom will be

associated with a single receptor subtype. Indeed, this notion originally struck us as naive. Nonetheless, with the use of 2 synthetic SP agonists that display an extremely high degree of selectivity for the SP-P and the SP-N receptors respectively (Table I), we have achieved what we believe to be a clear-cut receptor-specific dissection of much of the SP-induced behavioral repertoire. The compulsive scratch/bite/lick syndrome produced by SP and its non-selective agonists appears to be related to activation of the SP-P receptor as it is evoked by septide but not senktide. Both substances are extremely poor SP-E receptor agonists. Selective activation of the SP-N receptor by senktide, on the other hand, appears to be responsible for delayed thermal hypoalgesia, or at least that form of thermal hypoalgesia measured in the hot plate and tail immersion tests, and for hypoalgesia to noxious mechanical stimuli. Activation of the SP-N receptor also appears to be responsible for motor flaccidity. The rapid and complete reversal of hind limb paralysis suggests that we are dealing with a specific, receptor-mediated effect and not non-specific tissue necrosis. Histological analysis, however, has not yet been carried out. Assuming that senktide-induced hypoalgesia and motor flaccidity are indeed specific effects of

SP-N receptor activation, it would obviously be of great practical interest to find a way to functionally dissociate them, and particularly to obtain the powerful hypoal- gesic effect without paralysis. Finally, ‘wet-dog shakes,’ which appeared only after senktide injection, are probably also related to the SP-N receptor. Since they were not observed consistently, however, we hesitate to draw firm conclusions. Elliott and Iversen [5] suggested that ‘wet-dog shakes’ may be related to the SP-E receptor.

Based on the conviction that the primary behavioral effect of SP is the produc- tion of hyperalgesia and pain, a large number of SP analogues have been synthe-

sized and tested as potential analgesics, and indeed, several have been identified which produce hypoalgesia and/or block SP-induced hyperalgesia [e.g., 1,5,18,28,29,32]. Unfortunately, many of these agents also tend to produce motor blockade. In light of the present results, we suspect that some or all of these presumed ‘selective SP antagonists’ have, among other things, SP-N agonist activity. Similar mixed receptor actions may account for the fact that many of the behavioral effects of intrathecally administered tachykinins, including paralysis, are shared by many naturally occurring non-fachykinin peptides including bombesin, calcitonin

gene-related peptide, dynorphin, neuropeptide Y, neurotensin, oxytocin, somatosta- tin, thyrotropin releasing factor, and vasopressin [6,19,22,23a,34,37,39].

The functional dissection that we have described here was made possible by the development of synthetic substances with extremely high selectivity for known tachykinin receptors that occur in the mammalian PNS and CNS, and we believe that the effects of the substances indeed result from selective activation of these

receptors. Unfortunately, in the absence of satisfactory receptor-specific antagonists, we cannot rigorously exclude the possibility that septide and senktide interact with other, as yet unidentified, receptor types. A more thorough understanding of the effects of naturally occurring and synthetic peptides on the various SP receptor

274

subtypes, and the development of subtype-selective SP receptor antagonists, is urgently needed.

Acknowledgements

This work was supported by grants from the Charles E. Smith Family Founda- tion for Psychobiology, the Israel Ministry of Health, and the United States-Israel Binational Science Foundation.

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