Characterization of dynorphin A-induced antinociception at spinal level

European Journal of Pharmacology, 110 (1985) 21-30

21

Elsevier

C H A R A C T E R I Z A T I O N OF D Y N O R P H I N A-INDUCED A N T I N O C I C E P T I O N AT S P I N A L LEVEL SANTI SPAMPINATO * and SANZIO CANDELETTI Institute of Pharmacology, University of Bologna, Via lrnerio 48, 40126 Bologna, Italy

Received 2 August 1984, revised MS received 27 November 1984, accepted 18 December 1984

S. SPAMPINATO and S. CANDELETTI, Characterization of dynorphin A-induced antinociception at spinal level, European J. Pharmacol. 110 (1985) 21-30. Dynorphin A (DYN A) injected intrathecally in the rat produced a significant elevation of the nociceptive threshold, measured by the tail flick test. The highest dose of DYN A (25 nmol) produced maximal elevation of tail flick latency to radiant heat together with hindlimb paralysis and tail flaccidity lasting several hours, thus confirming several previous reports. A lower dose of DYN A (12.5 nmol) produced only a smaller, not constant, short-lasting change in the nociceptive threshold. The vocalization test (electrical stimulation of the tail) gave a different result: the time course curve showed that the antinociceptive effect had worn off 60 min after DYN A 25 nmol. Thus it can be assumed that the prolonged depression of the tail flick reflex was related to motor dysfunction and did not completely reflect the animal's response to painful stimuli. Tolerance to the antinociceptive and motor effects developed after the chronic intrathecal infusion of DYN A with osmotic minipumps. Intrathecal MR 1452 (30 nmol), a purported r-receptor blocker, fully prevented the effects of DYN A but not morphine-induced antinociception. Naloxone antagonized DYN A only at a 4 fold higher dose. MR 1452 (90 nmol) administered after DYN A reversed the elevation of the vocalization threshold while tail flick latency remained unmodified. Analysis by high performance liquid chromatography of intrathecally injected radiolabelled DYN A revealed that DYN A was largely broken down about 10 min after its administration. Our results seem to indicate that DYN A in the spinal cord causes alterations in nociception and motor function, clearly distinguishable in time and both mediated by an opioid receptor, probably of the r type. However different mechanism(s), possibly non-opioid in nature, may contribute to the prolonged depression of the tail flick. Dynorphin A

Opioid antagonists

Tolerance

1. Introduction Dynorphin A ( D Y N A) and the other structurally related peptides derived from the same comm o n precursor, designated pre-pro-dynorphin (Rossier, 1982), have recently been detected throughout the neuraxis and in peripheral tissues of experimental animals and humans (Goldstein and Ghazarossian, 1980; H611t et al., 1980; Botticelli et al., 1981; Khachaturian et al., 1982; Maysinger et al., 1982; Spampinato and Goldstein, 1983). There is considerable evidence that D Y N A may be an endogenous ligand for the r-receptor (Chavkin et al., 1982). * To whom all correspondence should be addressed. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.

Antinociception

Motor dysfunction

Intrathecal

Intracerebroventricular (i.c.v.) administration of D Y N A and dynorphin-related peptides produces excessive grooming (Walker et al., 1980), hypothermia (Petrie et al., 1982), reduction of gastric secretion (Ferri et al., 1984) and catalepsy (Herman et al., 1980) but only very weak analgesia (Herman et al., 1980). According to Friedman et al. (1981) D Y N A i.c.v, in mice not only failed to produce antinociception but also attenuated morphine and fl-endorphin analgesia. However, D Y N A displays antinociceptive properties after intrathecal administration to mice (Piercey et .al., 1982) and rats (Herman, 1982; Han and Xie, 1982; Przewlocki et al., 1983a; Kaneko et al., 1983). Its mode of action in this regard is not clear

22

and interpretation of these studies remains difficult as there are several reports that intrathecal administration of D Y N A causes disturbances of motor function with hindlimb paralysis in unanesthetized rats (Herman, 1982; Faden and Jacobs, 1983; Przewlocki et al., 1983a). According to Przewlocki et al. (1983a) the antinociceptive activity of intrathecal D Y N A could be secondary to the alteration of motor function. Moreover, these authors report that only a high dose of naloxone blocked the antinociceptive and motor effects of D Y N A while Han and Xie (1982) reported that naloxone was ineffective in antagonizing the effects of intrathecal DYN A. The purpose of the present study was to extend previous work and further investigate the antinociceptive effects of intrathecal D Y N A in r~ts by using two different analgesimetric procedures: the tail flick test and electrical stimulation of the tail. In the latter, the reaction threshold to an electrical stimulus is assessed from rat vocalization and the end-point response does not involve any motor component. Moreover. since Herman et al. (1980) and Leslie and Goldstein (1982) found that [~25I]dynorphin(1-13) underwent rapid enzymatic breakdown in vivo after i.c.v, injection to the rat and in vitro in the presence of rat brain membranes, we studied the distribution and stability of radiolabelled D Y N A after intrathecal administration.

2. Materials and methods

2. l. Animals Male Sprague-Dawley rats (Nossan, Correzzana, Italy) weighing 300-350 g were used. They were housed individually with a 12 h light-dark cycle and allowed free access to food and water.

2.2. Surgery The rats were implanted with chronic intrathecal catheters utilizing a modification of the method described by Yaksh and Rudy (1976). Briefly, catheters (PE 10) were sterilized by immersion in a solution of benzalkonium chloride

and flushed with a sterile osmotically balanced solution adopted as drug vehicle (NaC1 7.46 g: KCI 0.19 g; MgCl 2 0.19 g; CaCI2 0.14 g in 1000 ml of distilled water). The rats were anesthetized with sodium pentobarbital and the catheters were inserted through a slit in the atlanto-occipital membrane and pushed 8.5 cm down to the rostral edge of the lumbar enlargement. All animals were allowed to recover for 1 week before the experiment and any rats that showed neurological or motor deficits were discarded. Each animal was used only once. At the end of the experiment the rats were randomly selected to verify the correct position of the catheter by injection of Evans blue.

2.3. Evaluation of antinociceptive activity The response to nociceptive stimuli was determined in conscious, restrained animals first by the vocalization test (Paalzow, 1978) and immediately after the tail flick assay ( D ' A m o u r and Smith, 1941), before and at appropriate times after administration of drugs or vehicle (control). The mean of two trials was recorded. For determination of the vocalization threshold, two stainless steel 30 gauge electrodes were inserted into the middle section of the tail with the positive pole in the proximal position. Electrical stimulation was delivered to the tail from a high frequency square wave constant current generator, using a frequency of 125 pulses/s, with pulse width 1.6 ms and 1 s train. The intensity of the current (measured in mA) necessary for induction of vocalization was taken as a measure of nociception. Each animal's individual threshold for vocalization was determined in three preliminary tests 20 min before drug administration. The maximal intensity of the current delivered was 2 mA. In the tail flick test the radiant heat source was adjusted to produce a baseline tail flick of 2 - 4 s; the time required for the rat to remove its tail from the path of focused rays from a 300 W quarz bulb projection lamp was recorded by an automated device. In animals tested before drug injection, tail flick latencies were typically 3.4 to 4.0 s with S.E.M. of 0.3 to 0.5 s. A cut-off time of 10 s was used in order to avoid tissue damage.

23

2.4. Tolerance experiments Tolerance to DYN A was induced in rats bearing a chronic intrathecal catheter by continuous infusion of DYN A with an osmotic minipump (Alza Corp., Palo Alto, CA; mod. 2001), a system capable of delivering a small volume at a constant rate (0.90-0.95 /~l/h) over 7 days. Osmotic minipumps were prefilled with a solution of DYN A (1 nmol//~l) or vehicle alone, then coupled to the intrathecal catheter, previously filled with the same solution, by means of PE 60 tubing and inserted subcutaneously between the scapulae in the etheranesthetized rats. On the 7th day of infusion, each minipump was removed under ether anesthesia and the intrathecal catheter was washed with 10/~1 of vehicle. DYN A 25 nmol was injected 1 h after removal of the minipumps and the degree of tolerance was assessed in the two analgesimetric tests.

2.5. In vivo breakdown of [zeSI]DYN A For assessment of in vivo stability, monoiodinated DYN A was obtained as described by Leslie and Goldstein (1982). Four rats received 25 nmol of DYN A intrathecally and about 10 000 000 cpm of mono-iodinated DYN A dissolved in the vehicle and were killed after 8 min. Within 2-3 min the brain (subdivided into hindbrain, midbrain and forebrain) and spinal cord were dissected out and homogenized (Ultra-Turrax, Janke and Kunkel) in 10 vol. of 1 M acetic acid at 90°C. The homogenates were incubated at this temperature for 15 min, cooled on ice then centrifuged at 11 000 × g for 20 rain at 4°C. As a comparison, to see whether there was degradation of the peptide during the extraction procedure, 4 untreated rats were decapitated, their brains and spinal cords were removed, dissected and immediately placed in hot acetic acid solution to which 10 000 000 cpm of mono-iodinated DYN A and 25 nmol of DYN A were added. The tissues were then processed as described above. Supernatant solutions were passed through octadecylsilylsilica cartridges (Sep-Pak C~8, Waters Associated), prepared as previously reported (Spampinato and Goldstein, 1983). The cartridges were washed with 20 ml of 5 mM trifluoroacetic

(TFA). [125I]DYN A and its radiolabelled metabolites were then eluted with 3 ml of a mixture (1:1, v/v) of acetonitrile and 5 mM TFA, lyophilized, taken up in methanol/1 N HC1 (1 : 1, v/v) and analyzed by reverse-phase high-performance liquid chromatography (HPLC) using a Waters Associates system with a /~Bondapak C~8 column. Initial conditions: 25% acetonitrile in 5 mM TFA. Simultaneously with sample injection, a linear gradient was started, to 45% acetonitrile in 5 mM TFA in 30 min at 1.5 ml/min, fraction volume, 0.75 ml.

2.6. Drugs Drugs were dissolved in the vehicle and injected intrathecally in a volume of 20 /tl. The catheter was cleared by subsequent injection of 8 /~1 of vehicle. Drugs used in these experiments were DYN A (Peninsula Laboratories, San Carlos, CA), naloxone HC1 (a gift from Endo Laboratories, Garden City, NY) and MR 1452 [(-)-N-(3-furylmethyl)-a-normetazocine methanesulfonate] a purported K-opioid receptor antagonist (a gift from Boehringer, Ingelheim), morphine HCI (Carlo Erba, Milan). DYN A purity was at least 99% and only freshly made-up solutions were used.

2.7. Statistical analysis Statistical analysis of the data was performed by using t-tests. Reciprocal transformed latencies of tail flick test and log transformed current intensity values of vocalization test were submitted to two separate one-way analyses of variance (ANOVAs) followed by control vs. drug single comparisons and by regression analysis of dose-response relationships.

3. Results

3.1. Effects of intrathecal D Y N A on nociceptive threshold Intrathecal injection of DYN A elevated the nociceptive threshold to a different extent in the tail flick and the vocalization tests.

24

(A) Fig. 1 (top panel) reports the time-effect curves for the tail flick effect after injection of 6.25, 12.5 and 25 nmol of DYN A. The highest dose of DYN A (25 nmol) produced maximal elevation of the tail flick latency in 5 min; the increase lasted the entire observation period (2 h). The 12.5 nmol dose produced an effect in some animals only. Tail flick latency after intrathecal DYN A, 6.25 nmol, was the same as after administration of the vehicle. This observation was confirmed by the dose-response curve (fig. 2, top panel).

10

5

o -,~l

TAIL FLICK o :a:

10 :r w-

v

•

1

d

lie

:>

o

5.

L

/ /

I

R',~994

.,o

39

2'5

log DOSE DYN A, nmol

J_ ...I

r'~ ~ - v -

,

m

1

i

VOCALI ZATION TEST

2D

v

1.o N

TIME AFTER INTRATHECAL INJECTION (min)

Fig. 1. Time course of the effect of DYN A injected intrathecally at three different doses on tail flick latency (top panel) and vocalization threshold (bottom panel). (O) Vehicle, ((i)) DYN A 6.25 nmol, ( I ) DYN A 12.5 nmol, (D) DYN A 25 nmol. Standard errors for pre-injection values are not indicated for the sake of clarity. Each curve represents the mean and S.E.M. from 8-9 animals. * P < 0.01, ** P < 0.05 vs. vehicletreated group.

Fig. 2. Log dose-response curves for the effects of DYN A on tail flick latency (top panel) and vocalization threshold (bottom panel) determined 5 min after intrathecal injection. ( O ) Vehicle-treated rats, (O) DYN A-treated rats. Each point represents the mean and S.E.M. from 8-9 animals. For statistical evaluation see text.

Anova showed a highly significant overall difference between vehicle and drug-treated groups (F = 11.01; DF--- 1/30; P < 0.0025). The log dose-response relationship followed a highly significant linear trend (F = 34.55; DF = 1/30; P < 0.0001) without significant deviations from linearity (F < 1). Single comparisons showed that the response to the dose of 6.25 nmol of DYN A did not significantly differ from the controls (F < 1). All rats treated with 25 nmol of DYN A exhibited flaccid hindlimb paralysis within 5-10 min, with flaccidity of the tail which gradually disappeared within 4 h of injection. At the dose of 12.5 nmol, DYN A induced flaccidity of the tail, either alone or associated with hindlimb paralysis only in some animals (and lasting within 1 h). The 6.25 nmol dose had no appreciable effects on motor behavior.

25

(B) Intrathecal injection of DYN A caused a dose-related rise in the vocalization threshold (fig. 1, bottom panel). The maximum antinociceptive effect was reached with a dose of 25 nmol 5 min after intrathecal administration; 30 min later the effect was diminished but was still significantly different from the control values. The rise in vocalization threshold had worn off 60 rain after I)YN A 25 nmol but the depressant effect on tail flick latency and the hindlimb paralysis were still present. The magnitude of the antinociceptive effect was dose-related (fig. 2, bottom panel). As with the tail flick test, ANOVA indicated a highly significant overall difference between vehicle and drug-treated groups (F = 54.25; DF = 1/32; P <0.0001). The log dose-response relationship exhibited a highly significant linear trend (F = 29.30; DF = 1/32; P < 0.001; deviations from linearity: F = 2.14; N.S.). In this case the response to the lowest dose of DYN A was significantly different from the controls (F = 13.91; DF = 1/32; P < 0.001).

TAIL FLICK

to

3.31 Antagonism of D Y N A by naloxone and MR 1452 Naloxone abolished both antinociceptive and motor effects only when injected intrathecally at a dose of 120 nmol 5 min before DYN A 25 nmol; lower doses (15, 30 and 60 nmol, data not shown) did not modify the effects of DYN A. In contrast, MR 1452 15 nmol attenuated and 30 nmol completely inhibited the effects on the nociceptive threshold and on motor function produced by intrathecal DYN A, 25 nmol (fig. 4).

-:

(u

5

OVOCALIZATION TEST

~Jl)-

E

o

i--

"tO.

3.2. Tolerance to intrathecal D YN A Tolerance, manifested as a reduction of the antinociceptive efficacy of DYN A on tail flick latency and vocalization threshold, developed after the rats had been chronically infused with DYN A into the intrathecal space (approx. 1 nmol/h) for 7 days. DYN A 25 nmol did not raise the nociceptive threshold in either test in these rats (fig. 3). Moreover, chronic intrathecal DYN A abolished not only the antinociceptive effects but also the motor paralysis produced by DYN A.

--~-J,---~--~-

D

O-

,-ll,

-~o5

•

t'~ :~ L5 ~

6o

TIME AFTER INTRATHECAL INJECTION (min)

Fig. 3. Tolerance to the antinociceptive effects of D Y N A chronically infused into the intrathecal space by osmotic minip u m p s for 7 days. Figure shows the time course of the effect of D Y N A (25 nmol) injected intrathecally 1 h after removal of the minipumps, on tail flick latency (top panel) and vocalization threshold (bottom panel). (m) DYN A in rats chronically infused with D Y N A; (O) D Y N A in control rats chronically infused with drug vehicle. The analgesic thresholds measured in two tests at the end of the infusion were the same as the values before chronic infusion in the controls and D Y N A-treated rats. Each curve represents the mean and S.E.M. from 8-9 animals. * P < 0.01, ** P < 0.05.

MR 1452 (30 nmol) and naloxone (120 nmol) prevented the elevation of the nociceptive threshold produced by 12.5 nmol DYN A (data not shown). MR 1452 injected 5 min after 25 nmol DYN A reversed the DYN A-induced .elevation of the vocalization threshold. On the contrary, the tail flick response remained unmodified (fig. 5) even with the high dose of 90 nmol MR 1452.

26 TAIL FLICi

TAIL FLIC~ 10

10. •e

5"

•

u~

u~

• oe ee

c, z

e*

5-

O. o.

,-~1,

~ i , ,

,

,

,

,

VOCALIZATION TEST

•

20. VOCALIZATION TEST

2.0.

oc Fmc

= o

>

•

0-

0

~

45

60

1~)0

t

~j,,

.2oo5

.20~[~ ~ 15

6'o

MR TIME AFTER INTRATHECAL INJECTION (min)

6o

TIME AFTER INTRATHECAL INJECTION (min)

Fig. 4. The time course of the effect of DYN A injected intrathecally on tail flick latency (top panel) and vocalization threshold (bottom panel) in rats treated 5 rain before with naloxone or MR 1452 intrathecally. ([3) DYN A 25 nmol, (O) naloxone 120 nmol+DYN A 25 nmol, (O) MR 1452 15 nmol +DYN A 25 nmol, (O) MR 30 nmol+DYN A 25 nmol. Standard errors are not indicated for any values for the sake of clarity. Each curve represents the mean+S.E.M, from 8-10 animals. * P < 0.01, ** P < 0.05 vs. pre-drug values.

Fig.

5. T h e t i m e c o u r s e o f t h e e f f e c t o f D Y N

A injected

intrathecally on tail flick latency (top panel) and vocalization threshold (bottom panel) in rats treated 5 min after with MR 1452 intrathecally. Standards errors are not indicated for any values for the sake of clarity. (Q) DYN A 25 nmol, (11) DYN A 25 nmol + MR 1452 90 nmol. Each curve represents the mean + S.E.M. from 8-10 animals. ** P < 0.05 vs. DYN A-treated rats.

3.4. Breakdown of [J25I]D YN A after injection into the spinal cord

A s e x p e c t e d , m o r p h i n e (15 /~g) e x h i b i t e d antin o c i c e p t i v e a c t i v i t y in the tail flick a n d v o c a l i z a t i o n tests w h e n i n j e c t e d i n t r a t h e c a l l y . P r e t r e a t m e n t w i t h n a l o x o n e (30 n m o l ) c o m p l e t e l y b l o c k e d the effects o f m o r p h i n e w h i l e M R 1452 (30 n m o l ) was n o t e f f e c t i v e (fig. 6).

L a b e l l e d D Y N A i n j e c t e d i n t o the rat spinal c o r d was a l m o s t c o m p l e t e l y b r o k e n d o w n w i t h i n a few m i n u t e s . A s s h o w n in fig. 7 (top panel), the H P L C p r o f i l e o f l y o p h i l i z e d e x t r a c t s f r o m spinal c o r d o f c o n t r o l rats (see M a t e r i a l s a n d m e t h o d s ) e l u t e d f r o m the c o l u m n in the s a m e p o s i t i o n as i n t a c t m o n o - i o d i n a t e d D Y N A ( r e c o v e r y o f the r a d i o a c t i v i t y a d d e d to the tissues p r i o r to h o m o -

27

TAIL FLICK

40-

10-

u~ v

/

20 _.1

±

?

>

d', ~,

0

c_)

O.

VOCALIZATION TEST

20

i

,- Q5

25

4's

35

ACETONITRILE

(%)

N

TIME AFTER INTRATHECAL INJECTION (min)

Fig. 6. The time course of the effect of morphine injected intrathecally on tail flick latency (top panel) and vocalization threshold (bottom panel) in rats treated 5 rain before with naloxone or MR 1452. (O) Vehicle, (O) morphine 15 /~g, (O) naloxone 30 nmol+morphine 15 #g, (11) MR 1452 30 nmol+ morphine 15 /~g. Standard errors are not indicated for any values for the sake of clarity. Each curve represents the mean + S.E.M. from 5-6 animals. ** P < 0.05 vs. vehicle-treated group.

genization was 84 + 2.1%, n = 4), indicating that there was no degradation of mono-iodinated DYN A during extraction. The HPLC profile of the radioactivity extracted from the spinal cord of rats killed 8 min after treatment showed a major peak (80 + 3% of the total radioactivity recovered, n = 4) behaving identically to tyrosine, as confirmed by thin layer chromatography of the material collected, and a second peak (15 + 2%, of the total radioactivity recovered, n = 4) eluting in the same position as intact mono-iodinated DYN A (fig. 7 bottom

Fig. 7. Representative elution profile of radioactivity extracted from the spinal cord from reverse-phase HPLC of monoiodinated DYN A added to control samples before extraction (top panel) and of mono-iodinated DYN A added intrathecally (bottom panel). Samples were processed as described in Methods. Radioactivity collected from the first peak (bottom panel) was analyzed by thin layer chromatography as described by Leslie and Goldstein (1982) and found to correspond to [t25I]tyrosine. Arrows indicate the elution position of standard mono-iodinated DYN A.

panel). Recovery of radioactivity from the spinal cord after intrathecal injection was 16.5 + 6.5% (n = 4). No radioactivity was detected in any of the brain areas examined (data not shown).

4. Discussion

These results indicate that DYN A injected intrathecally in the rat produced a significant, dose-related elevation in the nociceptive threshold, as measured by the vocalization test. The antinociceptive effect was relatively brief. The tail flick test gave different results. The highest dose of DYN A produced a maximal elevation of tail flick latency lasting several hours

28 while a lower dose (12.5 nmol) produced only a smaller, not constant, short-lasting change in the nociceptive threshold and the dose of 6.25 nmol was ineffective, Przewlocki et al. (1983a) also reported wide variability in the analgesic response after low intrathecal doses of DYN A. In analyzing the mechanisms through which intrathecal DYN A alters the nociceptive threshold, we have to consider that 25 nmol of the peptide caused motor impairment with hindlimb paralysis and tail flaccidity (as already shown by Herman, 1982; Faden and Jacobs, 1983; Przewlocki et al., 1983a) that disappeared gradually within about 4 h. Thus we could assume that the prolonged depression of the tail flick reflex was related to the motor dysfunction and did not completely reflect the animal's response to painful stimuli. In fect, the vocalization threshold returned to pre-drug values within 60 rain of intrathecal injection of DYN A, indicating that rats were again reactive to noxious stimuli. lntrathecal studies have clearly demonstrated the relevance of spinal p,-, 6- and ~-opioid receptors in modulation of the reactions to various types of nociceptive stimuli. Several groups (Przewlocki et al. 1983b; Schmauss et al., 1983; Schmauss and Yaksh, 1984) have suggested that K-agonists, including DYN A, may be involved in the control of visceral pain, while kt- and 8-receptors mediate the nociception produced by cutaneous thermal stimuli. Hayes et al. (1983) reported that DYN A raised the nociceptive pressure thresholds in the rat after intracerebroventricular administration but had no such effect when a noxious heat stimulus was applied. These data could help to explain the low efficacy of small doses of DYN A in the tail flick test. In addition, we showed that DYN A administered into the spinal cord interfered with the transmission of nociceptive input, assessed by vocalization induced by an electrical stimulus which requires a well organized response by the animal (Hoffmeister and Kroneberg, 1966). This analgesimetric procedure is suitable for detecting antinociceptive activity of opioids given intrathecally (in agreement with previous observations of Wiesenfeld-Hallin and Persson, 1984). Opioids given intrathecally not only block spinally coordinated

reflexes but can also alter the response elicited by the nociceptive input which reaches supraspinal structures starting from the spinal cord itself (see review by Yaksh, 1981). Chronic intrathecal infusion of DYN A induced tolerance to the antinociceptive effect of DYN A, confirming that this peptide behaves like other opioid peptides or opiates chronically infused into the subarachnoid space (Yaksh et al., 1977; Tseng, 1982; Wiesenfeld and Gustafsson, 1982). Tolerance also developed to motor effects (though we did not evaluate hindlimb function according to an ordinal grading scale). The opioid antagonist MR 1452, a purported K-receptor blocker, fully prevented DYN A's effects but failed to prevent the antinociception elicited by 15 #g of morphine given intrathecally. The morphine-induced effect was, on the contrary, inhibited by naloxone (30 nmol). In agreement with these results, Przewlocki et al. (1983b) found that MR 2266, a K-antagonist similar to MR 1452, was much more effective than naloxone in antagonizing the inhibition of writhing induced by DYN A in mice. However, naloxone antagonized DYN A only at a 4 fold higher dose (120 nmol). These data confirm previous reports of low affinity of naloxone for the receptor mediating DYN A's effects in vitro on guinea-pig ileum longitudinal muscle (Goldstein et al., 1979), and in vivo on catalepsy and antinociception (Herman et al., 1980; Przewlocki et al., 1983a; Han and Xie, 1984). The fact that a K-antagonist fully antagonized DYN A is consistent with the results of other studies suggesting that DYN A has a higher affinity for •-opioid receptors than for kt- or 8-receptors. Mouse vas deferens made tolerant to sufentanyl (a relatively pure p~-agonist) and [DAla2,D-LeuS]enkephalin (a 8-agonist) did not display significant cross-tolerance to DYN A (W~ster et al., 1980). Furthermore, as we reported previously DYN A still elevates tail flick latency and produces motor impairment after its intrathecal administration in rats made tolerant to morphine (a relatively pure /~-agonist) (Spampinato et al., 1984). Recently, in agreement with this hypothesis, Han et al. (1984) reported that the antinociception produced by dynorphin B, an opioid peptide structurally related to DYN A, was significantly

29

decreased in rats tolerant to ethylketazocine (a relatively pure •-agonist). The hypothesis that the effects of intrathecal DYN A derive from an action in the spinal cord was also supported by the results of injecting radiolabelled DYN A. Ten min after administration of mono-iodinated DYN A, when the maximal antinociceptive effect had developed, we could not detect any radioactivity in the various brain areas. Similar results have been obtained with several radiolabelled analgesic compounds that do not reach supraspinal structures in significant amount in the first few hours after their intrathecal injection (Yaksh, 1981). About 80% of the total radioactivity extracted from the spinal cord was due to [125I]tyrosine. Thus, a few minutes after intrathecal injection, mono-iodinated DYN A is largely broken down. Similar results were obtained after i.c.v. [125I]dynorphin-(1-13) in the rat by Herman et al. (1980) and [3H][Met]enkephalin in mice (Craves et al., 1978). The rapid degradation of DYN A could to some extent explain its shorter antinociceptive activity in comparison to that of other opioid peptides such as intrathecal fl-endorphin (Yaksh and Henry, 1978). The longer lasting effect on the tail flick and on motor function could represent the result of persistent activity on the complex spinal neural circuits, no longer dependent on the integrity of the DYN A molecule. This hypothesis is further supported by our data showing that MR 1452 administered after DYN A at a dose 3 fold higher than that preventing DYN A-induced effects in either test reversed the elevation of the vocalization threshold only. This agrees with the observations of Han and Xie (1984) who found that a narcotic antagonist (naloxone) was more effective in antagonizing the action of intrathecal DYN A on tail flick latency in the rabbit when given prior to the peptide. Further research will be necessary to find the neurochemical substrates with which DYN A interacts and to determine whether its action on nociception and motor function is simply pharmacological, or reproduces a physiological effect of an endogenous peptide. In actual fact the amino acid sequence(s) of

immunoreactive dynorphin from the spinal cord is still unknown, though preliminary data reported by Botticelli et al. (1981) indicate the presence of several molecular forms. More accurate in vivo studies will be possible when all these peptides have been sequenced. In summary, we demonstrated that DYN A at the spinal cord level caused alterations in nociception and motor function, clearly distinguishable in time, both mediated by an opioid receptor probably of the x-type since they were antagonized by pretreatment with a K-antagonist. However different mechanism(s), possibly nonopioid in nature, may contribute to the prolonged depression of the tail flick as DYN A appears to be rapidly broken down.

Acknowledgements These studies were supported by grants from MPI and the National Research Council (No. 83. 2640.56), Italy. We are indebted to Dr. H. Merz, Boehringer, lngelheim Am-Rhein) for the gift of MR 1452. The authors thank Prof. N. Montanaro for his kind help in performing statistical analysis.

References Botticelli, L., B.M. Cox and A. Goldstein, 1981, Immunoreactive dynorphin in mammalian spinal cord and dorsal root ganglia, Proc. Natl. Acad. Sci. U.S.A. 78, 7783. Chavkin, C., I.F. James and A. Goldstein, 1982, Dynorphin is a specific endogenous ligand of the kappa opioid receptor, Science 215, 413. Craves, F.B., P.Y. Law, C.A. Hunt and H.H. Loh, 1978, The metabolic disposition of radiolabelled enkephalins in vitro and in situ, J. Pharmacol. Exp. Ther. 206, 492. D'Amour, F.E. and D.L. Smith, 1941, A method for determining loss of pain sensation, J. Pharmacol. Exp. Ther. 72, 74. Faden, A.J. and T.P. Jacobs, 1983, Dynorphin induces partially reversible paraplegia in the rat, European J. Pharmacol. 91, 321. Ferri, S., S. Candeletti, E. Cavicchini, P. Romualdi, C. Spadaro, E. Speroni and S. Spampinato, 1984, Effects of opioid peptides on gastric secretion and ulceration, in: Central and Peripheral Endorphins: Basic and Clinical Aspects, eds. E.E. Muller and A.R. Genazzani (Raven Press, New York) p. 217. Friedman, H.J., M.F. Jen, J.K. Chang, N.M. Lee and H.J. Loh, 1981, Dynorphin: a possible modulatory peptide on morphine or B-endorphin analgesia in mouse, European J. Pharmacol. 69, 357.

30 Goldstein, A. and V.E. Ghazarossian, 1980, Immunoreactive dynorphin in pituitary and brain, Proc. Natl. Acad. Sci. U.S.A. 77, 6207. Goldstein, A., S. Tachibana, L.I, Lowney, M. Hunkapiller and L. Hood, 1979, Dynorphin-(l-13), an extraordinarily potent opioid peptide, Proc. Natl. Acad. Sci. U.S.A. 76, 6666. Han, J.S. and C.W. Xie, 1982, Dynorphin: potent analgesic effect in spinal cord of the rat, Life Sci. 31, 1781. Han, J.S. and G.X. Xie, 1984, Dynorphin: important mediator for electroacupuncture analgesia in the spinal cord of the rabbit, Pain 18, 367. Han, J.S., G.X. Xie and A. Goldstein, 1984, Analgesia induced by intrathecal injection of dynorphin B in the rat, Life Sci. 34, 1573. Hayes, A.G., M. Skingle and M.B. Tyers, 1983, Antinociceptive profile of dynorphin in the rat, Life Sci. 33, Suppl. 1,657. Herman, B.H., 1982, Intrathecal dynorphin induces antinociception and paralysis, Soc. Neurosci. Abst. 8, 91. Herman, B.H., F. Leslie and A. Goldstein, 1980, Behavioral effects and in vivo degradation of intraventricularly administered dynorphin-(1-13) and D-Ala2-Dynorphin-(1-11) in rats, Life Sci. 27, 883. Hoffmeister, F. and G. Kroneberg, 1966, Experimental studies in animals on the differentiation of analgesic activity, in: Methods in Drug Evaluation, eds. P. Mantegazza and F. Piccinini (Elsevier/North-Holland, Amsterdam) p. 270. H611t, V., 1. Haarmaan, K. Bovermann, M. Jerlicz and A. Herz, 1980, Dynorphin-related immunoreactive peptides in rat brain and pituitary, Neurosci, Lett. 18, 149. Kaneko, T., T. Nakazawa, M. Ikeda, K. Yamatsu, T. lwama, T. Wama, M. Satoh and H. Takagi, 1983. Sites of analgesic action of dynorphin, Life Sci. 33, Suppl. 1,661. Khachaturian, H., S.J. Watson, M.E. Lewis, D. Coy, A. Goldstein and H. Akil, 1982, Dynorphin immunocytochemistry in the rat central nervous system, Peptides 3, 941. Leslie, F.M. and A. Goldstein, 1982, Degradation of dynorphin-(l-13) by membrane-bound rat brain enzymes, Neuropeptides 2, 185. Maysinger, D., V. H611t, B.R. Seizinger, P. Mehraein, A. Pasi and A. Herz, 1982, Parallel distribution of immunoreactive a-neo-endorphin and dynorphin in rat and human tissue, Neuropeptides 2, 211. Paalzow, G., 1978, Development of tolerance to the analgesic effect of clonidine in rats: cross-tolerance to morphine, Naunyn-Schmiedeb. Arch. Pharmacol. 304, 1. Petrie, E.C., S.T. Tiffany, T.B. Baker and J.L. Dahl, 1982, Dynorphin (1-13): analgesia, hypothermia, cross-tolerance with morphine and /3-endorphin, Peptides 3, 41. Piercey, M.F., K. Varner and L.A. Schroeder, 1982, Analgesic activity of intraspinally administered dynorphin and ethylketocyclazocine, European J. Pharmacol. 80, 283.

Przewlocki, R., G.T. Shearman and A. Herz, 1983a, Mixed opioid/non opioid effects of dynorphin and dynorphin related peptides after their intrathecal injection in rats, Neuropeptides 3, 233. Przewlocki, R., L. Stala, M. Greczek, G.T. Shearman, B. Przewlocka and A. Herz, 1983b, Analgesic effects of /~-. 6and g-opiate agonists and, in particular, dynorphin at the spinal level, Life Sci. 33, Suppl. 1,649. Rossier, J., 1982, Opioid peptides have found their roots, Nature (London) 298, 221. Schmauss, C. and T.L. Yaksh, 1984, In vivo studies on spinal opiate receptor systems mediating antinociception, lI. Pharmacological profiles suggesting a differential association of mu, delta and kappa receptors with visceral chemical and cutaneous thermal stimuli in the rat, J. Pharmacol. Exp. Ther. 228, 1. Schmauss, C., T i . Yaksh, Y. Shimohigashi, G. Harty, T. Jensen and D. Rodbard, 1983, Differential association of spinal p., ~ and K opioid receptors with cutaneous thermal and visceral chemical nociceptive stimuli in the rat, Life Sci. 33, Suppl. 1,653. Spampinato, S., S. Candeletti, P. Romualdi, E. Speroni, E. Cavicchini, C. Spadaro, G. Murari, G. Giobbi and S. Ferri, 1984, Opioid receptors mediating the antinociceptive effect of dynorphin at spinal level, Ann. Ist. Super. SanitY. 20, 309. Spampinato, S. and A. Goldstein, 1983, Immunoreactive dynorphin in rat tissues and plasma, Neuropeptides 3, 193. Tseng, L.F. 1982, Tolerance and cross-tolerance to morphine after chronic spinal D-Ala2-D-LeuS-enkephalin infusion, Life Sci. 31, 987. Walker, J.M., R.J. Katz and H, Akil, 1980, Behavioral effects of dynorphin-(l-13) in the mouse and rat: initial observations, Peptides 1,341. Wiesenfeld, Z, and L.L. Gustaffson, 1982, Continuous intrathecal administration of morphine via an osmotic minipump in the rat, Brain Res. 247, 195. Wiesenfeld-Hallin, Z. and A. Persson, 1984, Subarachnoid injection of salmon calcitonin does not induce analgesia in rats, European J. Pharmacol. 104, 375. Wi~ster, M., R. Schulz and A. Herz, 1980, Opiate activity and receptor selectivity or dynorphin 1-13 and related peptides, Neurosci. Lett. 20, 79. Yaksh, T.L., 1981, Spinal opiate analgesia: characteristics and principles of action, Pain 11,293. Yaksh, T.L. and J.L. Henry, 1978, Antinociceptive effects of intrathecally administered human fl-endorphin in the rat and cat, Can. J. Physiol. Pharmacol. 56, 754, Yaksh, T.L., R.L. Kohl and T.A. Rudy, 1977, Induction of tolerance and withdrawal in rats receiving morphine in the spinal subarachnoid space, European J. Pharmacol. 42, 275. Yaksh, T.L. and T.A. Rudy, 1976, Chronic catheterization of the spinal subarachnoid space, Physiol. Behav. 17, 1031.