Monoaminergic control of the release of calcitonin gene-related peptide- and substance P-like materials from rat spinal cord slices

OOZE-3908/93t6.00 + 0.00 Copyright 0 1993 Pergamon PressLtd

Neuropharmocology Vol. 32, No. 1, pp. 633440, 1993 Pruned in Great Britain. All rights reserved

MONOAMINERGIC CONTROL OF THE RELEASE OF CALCITONIN GENE-RELATED PEPTIDE- AND SUBSTANCE P-LIKE MATERIALS FROM RAT SPINAL CORD SLICES S.B~URGOIN,M.

POHL, A. MAUBORGNE,J. J. BENOLIEL,E. COLLIN, M. HAMON and

F. CBSELIN

INSERM U 288, Neurobiologie Cellulaire et Fonctionnelle, Faculte de M&decine Piti&Salp&ibre, 75634 Paris Cedex 13, France (Accepted

15 March 1993)

Summary-The possible control by monoamines of the spinal release of substance P- and calcitonin gene-related peptide-like materials (SPLM and CGRPLM, respectively) was investigated in vitro, using slices of the dorsal half of the rat lumbar enlargement superfused with an artificial cerebrospinal fluid. Whereas the spontaneous outflow of SPLM and CGRPLM was changed by none of the agonists/ antagonists of monoamine receptors tested, the overtlow of both peptide-like materials due to 30 mM K+ was differentially affected by a,-adrenoreceptor and dopamine D-l receptor ligands. Noradrenaline (10 PM to 0.1 mM) and clonidine (0.1 mM) significantly reduced the K+-evoked overflow of SPLM, and both effects could be prevented by idazoxan (1OpM) and prazosin (lOfih4) as expected from their mediation through the stimulation of a,-adrenoreceptors. In contrast, CGRPLM. overflow remained unaffected by a,-adrenoreceptor ligands. Dopamine D-l receptor stimulation by SKF 82958 (10-100 nM) significantly increased the K+-evoked overflow of both SPLM and CGRPLM, and this effect could be prevented by the selective D-l antagonist SCH 39166 (1 PM). Further studies with selective ligands of other monoamine receptors indicated that neither a,- and b-adrenergic receptors, dopamine D-2, nor serotonin S-HT,, and 5-HT, receptors are apparently involved in some control of the spinal release of CGRPLM and SPLM. These data are discussed in line with the postulated presynaptic control by monoamines of primary afferent fibres conveying nociceptive messages within the dorsal horn of the spinal cord. Key words-dorsal

horn, substance P, CGRP, in vitro release, adrenergic, DA receptors, 5-HT receptors.

It is well documented that activation of spinopetal noradrenergic, serotoninergic and dopaminergic pathways originating in brainstem nuclei and terminating in the grey matter of the spinal cord can modulate spinal nociceptive sensory processing (Yaksh and Stevens, 1988; Zemlan, Corrigan and Pfaff, 1980; Hamon, Collin, Chantrel, Verge and Bourgoin, 1991). In particular, intrathecal administration of agonists of adrenoreceptors or serotonin (5HT) receptors can affect the processing of spinal inputs evoked by noxious stimuli in rats and cats (Yaksh and Stevens, 1988; Hamon et al., 1991). In addition, a decrease in both the nociceptive reflexes and the nociceptive responses of thalamic neurones has been reported in rats following the intrathecal injection of dopamine (DA) receptor agonists (Jensen and Yaksh, 1984; Clatworthy and Barasi, 1987). In man also, the spinal administration of a,-adrenoreceptor agonists can produce analgesia (Yaksh and Stevens, 1988; Maze and Tranquilli, 1991). Within the dorsal horn, al- and /?-adrenoreceptors and 5-HT receptors are located not only on neuronal elements intrinsic to the spinal cord but also on the

terminals of bulbo-spinal and/or primary afferent neurones (Hamon er al., 1991). As expected from the latter location, the destruction by neonatal capsaicin treatment or dorsal rhizotomy of primary afferent fibres results in a significant decrease in the density of noradrenaline (NA) and S-HT receptor binding sites within the superficial layers of the dorsal horn (Daval, Verge, Basbaum, Bourgoin and Hamon, 1987; Howe, Yaksh and Go, 1987; Patterson and Hanley, 1987; Hamon, Gallissot, MCnard, Gozlan, Bourgoin and Verge, 1989). Accordingly, agonists of these receptors may exert, at least partly, their antinociceptive effects by modulating presynaptically the activity of primary afferent fibres which convey the nociceptive messages to the spinal cord. Indeed, several groups already demonstrated that the local administration of selective ligands of NA and S-HT receptors can alter the excitability of primary alTerent fibres (Jeftinija, Semba and Randic, 1981; Calvillo and Ghignone, 1986; Carstens, Gilly, Schreiber and Zimmermann, 1987). Similarly, FleetwoodWalker, Hope and Mitchell (1988) have shown that the stimulation of DA receptors of the D-2 type within the dorsal horn of the rat spinal cord 633

S. EJOURWIN et al.

634

(Demenge, Mouchet, G&t-in and Feuerstein, 1981) hyperpolarizes primary afferent fibres. Biochemical evidence of a possible control by monoamines of primary afferent fibres has also &en reported by several groups. In particular, Vasko and his colleagues (Pang and Vasko, 1986; Ono, Mishima, Ono, Fukuda and Vasko, 1991) found that NA and clonidine inhibit the K+- or verat~dine-evoked release of substance P (SP) from slices of the rat spinal cord, where this peptide is located-in partin primary afferent fibres (Hakfelt, Ljungdahl, Terenius, Elde and Nilsson, 1977). In addition, the spinal release of another peptide contained in these fibres, somatostatin (Hiikfelt, Elde, Johansson, Luft and Arimura, 1975), has also been shown to be modulated by S-HT when it is triggered by capsaicin, which selectively excites unmyelinated primary afferent fibres (Kuraishi, Minami and Satoh, 1991). However, within the dorsal horn, SP and somatostatin are present not only in nerve terminals of primary afferent fibres but also in other neuronal elements [e.g. spinal intemeurones, terminals of bulbospinal fibres (Hokfelt et al., 1977; Stine, Yang and Costa, 1982)], and these peptides can be released from these various sources. Therefore the biochemical evidence of possible monoaminergic controls of primary afferent fibres is not firmly established to date. In contrast to SP and somatostatin, calcitonin gene-related peptide (CGRP) is found almost exclusively in terminals of primary agerent fibres within the dorsal horn of the spinal cord (Lee, Takami, Kawal, Girgis, Hillyard, MacIntyre, Emson and Tohyama, 1985; Skofitsch and Jacobowitz, 1985; Pohl, Benoliel, Bourgoin, Lombard, Mauborgne, Taquet, Carayon, Besson, Cesselin and Hamon, 1990). Consequently, the spinal release of CGRP is probably a better index of the activity of primary aflerent fibres than that of SP or somatostatin. This led us to examine the possible modulatory effects of drugs acting at NA, DA and S-HT receptors on the release of CGRP from rat spinal cord slices. Since SP is colocalized with CGRP in some primary afferent fibres (Lee et uf., 1985; Skofitsch and Jacobowitz, 1985), we also investigated the possible effects of these drugs on the release of SP in the same experiments. METHODS

Chemicals

[2-‘ZSI-Iodohistidyl’o]human CGRP ([rz5T]CGRP, 70 TBq/mmol) was from Amersham international (U.K.). [1251]Tyrs-SP ([‘*‘I]SP, 74 TBq/mmol) was from New England Nuclear (Boston, Massachusetts). Other compounds were: clonidine (BoehringerMannheim, Germany), prazosin (P&ret, Groton, Conn~ticut), isoprote~nol (Aldrich, St Quentin Fallavier, France), ipsapirone (Troponwerke, Cologne, Germany), quinpirole (Eli Lilly, Indiana-

polis, Indiana), SCH 39166 [( -)trans*6,7,7a,8,9,13bhexahydro-3-chloro-2-hydroxy-N-methyl-SH-benzo[d]-naphto-{2,1-b}azepine] (Schering-Plough, Bloomfield, New Jersey), SKF 82958 [( _+)6-chloro,7,8-dihydroxy-3-allyl-l-phenyl-2,3,4,5-tetrahydro-lH-3benzazepine], idazoxan, 8OH-DPAT [&hydroxy2-(~-~-propylamino)tetralin] and 2-methyl-S-HT (Research Bi~hemi~ls Inc., Natick, Massachu~tts), phenylephrine, NA and S-HT (Sigma, St Louis, Missouri). In vitro perfiion Three-month-old male SpragueDawley rats (Centre d’Elevage R. Janvier, Le Genest, France) were killed by decapitation, and the lumbar enlargement of the spinal cord was immediately dissected in the cold (WC) as described in detail elsewhere (Cesselin, Bourgoin, Artaud and Hamon, 1984). The dissected tissue was then divided in two halves by a horizontal cut passing through the e~ndymal canal. The dorsal halves from 12 rats were collected and suspended in an artificial cerebrospinal fluid (ACSF, in mM:NaCl, 136; NaHCOs, 16.2; KCl, 5.4; NaH2PG,, 1.2; CaClr, 2.2; MgCl,, 1.2; glucose, 5) adjusted to pH 7.3 by bubbling with an O,:CO, mixture (95: 5%). Tissues were then transferred to a filter paper and sliced (thickness: 0.3 mm) using a McIlwain tissue chopper. The pooled slices were resuspended in 6.5 ml of ACSF, dispersed equally into 12 thermostated (37°C) chambers (0.5 ml of the tissue suspension per chamber) and then superfused at a flow rate of 1 ml/4 min with the same medium (see Mauborgne, Lutz, Legrand, Hamon and Cesselin, 1987, for details). Following a 20min washing period, superfusate fractions (1 ml) were collected in polystyrene tubes maintained at 0°C and then kept at -30°C until the measurement of their CGRP-like material (LM) and SPLM contents. Fifteen fractions (corresponding to 60min of perfusion) were collected for each experiment. No loss of CGRPLM or SPLM was detected in samples stored under these conditions for up to 2 months. During collection of fractions 3 and 4 (Kl) and that of fractions 12 and 13 (K2), KC1 con~ntration was increased from 5.4 to 30 mM while NaCl concentration was reduced to 111.4mM in order to maintain the isotonicity of the superfusing medium. The effects of compounds to be tested on the release of CGRPLM and SPLM were investigated under control ([K+ ] = 5.4 mM) and depolarizing ([K+] = 30 mM) conditions. They were added to the superfusing ACSF from the beginning of fraction 8 up to the end of the experiment. Since the ratio of K+-evoked overflow of the peptide-like materials during the second depolarization (K2) to that during the first pulse (Kl) was constant in the absence of drugs (see Results), any change in this ratio in the presence of a given substance could be ascribed to the effect of this particular substance on the Ca2+-dependent

Monoaminergic control of spinal SP and CGRP release release

of CGRPLM

or SPLM (see Cesselin et al.,

1984). Radioimmunoassay of CGRPLM

CGRPLM contents of the perfusate fractions were measured using the radioimmunoassay procedure described in detail elsewhere (Pohl, Lombard, Bourgoin, Carayon, Benoliel, Mauborgne, Besson, Hamon and Cesselin, 1989). All reagents were diluted in 0.05 M Na,HPO, supplemented with 0.01 M EDTA and 0.2% bovine serum albumin (BSA) and adjusted to pH 6.8 with HCl. Two hundred ~1 of each collected fraction were mixed with 400 ~1 of the diluent and 100 ~1 of a CGRP antiserum (1: 80,000 final dilution). This antiserum was produced by a rabbit treated with Try”-a-CGRP-(23-37) coupled to bovine thyroglobulin with glutaraldehyde. After 18 hr at 4°C 100 11 of a [‘251]CGRP solution (corresponding to 150&2000 cpm) were added and the incubation continued for a further 42 hr. The assay was stopped by adsorbing unbound [1251]CGRP onto active dextran T70-coated charcoal. [125I’jCGRP adsorbed to charcoal was spun down at 6000g for 15 min at 4°C and the labelled peptide bound to antibodies was measured in the supematant using a Beckman 5500 gamma counter. Standard curves were obtained by incubating in the same conditions 200 ~1 of rat a -CGRP-( l-37) solutions in the ACSF instead of perfusate samples. Each time a compound was added to the ACSF, a complete standard curve was drawn from radioimmunoassays of 1-2OOpg of rat a-CGRP performed in the presence of this compound at the same concentration as that used for the perfusion experiments. This allowed accurate determination of CGRPLM released in fractions containing an excess of K+ and/or the various drugs used. The detection limit of the assay was 1 pg of CGRP per tube, and half displacement of [1251]CGRP bound to antibodies was obtained with 25 pg of the peptide. Several peptides were tested for their possible cross-reactivity under the radioimmunoassay conditions described above. Besides rat CGRP-(23-37) (cross-reactivity: 225%, as compared to 100% with rat a-CGRP-(l-37)) none of the peptides including amylin, cholecystokinin (CCK-8), porcine and human calcitonins, enkephalins and derived peptides, dynorphins and SP showed a cross-reactivity higher than 0.002% (at concentrations up to 1 pg per tube). In all cases, CGRPLM content was expressed as CGRP equivalents, i.e. in pg of a-CGRP-(1-37) producing the same displacement of bound [‘251]CGRP under standard radioimmunoassay conditions. Radioimmunoassay of SPLM

SPLM in perfusates was radioimmunoassayed as previously described (Mauborgne et al., 1987). Briefly, 500 ~1 of each collected fraction were mixed

635

with 50~1 of 0.025 M sodium phosphate buffer, pH 6.4, containing histones (1 g/l), BSA (1 g/l) and sodium azide (0.2 g/l), and 50 ~1 of a SP antiserum (l/100,000 final dilution). After 15-18 hr at 4°C. 100~1 of a [*251]SP solution (corresponding to 2500-3000 cpm) were added and the incubation continued for a further 15-l 8 hr. The assay was stopped by adsorbing free [1251]SPonto dextran T70-coated charcoal. The detection limit of the assay was 0.5 pg of SP per sample, and half displacement of [‘2sI]SP bound to antibodies was obtained with 20 pg of the peptide. Each time a compound was added to the ACSF, a complete standard curve was drawn from radioimmunoassays of OS-100 pg of SP performed in the presence of this compound at the same concentration as that used for the superfusion experiments. The SP antiserum was directed toward the carboxyterminal moiety of the SP molecule. Indeed, C-terminal fragments up to SP-5-11 showed 100% cross-reactivity [the lowest C-terminal fragment recognized was SP-7-11 (25% cross-reactivity)] and SP-1-l l-COOH and N-terminal fragments did not show any crossreactivity (i.e. less than 0.01% as compared to 100% for SP-l-11). Neurokinins A and B were practically devoid of any immunoreactivity (0.4 and 0.03%, respectively) (see Mauborgne et al., 1987, for details). In all cases, SPLM content was expressed as SP equivalents, i.e. in pg of SP-l-11 producing the same displacement of bound [1251]SPunder standard radiommunoassay conditions. Data analysis

Statistical analyses were performed using the Student’s t-test. When the P value was higher than 0.05, a difference was considered as being nonsignificant. RESULTS Characteristics of the in vitro release of CGRPLM and SPLM from rat spinal cord slices

Under control conditions ([K+] = 5.4 mM), about 200 pg of CGRPLM were found in the first fraction (204.5 f 9.8 pg/ml, mean f SEM, n = 23) collected after a 20min washing of the spinal cord tissues. Thereafter, the rate of CGRPLM outflow decreased slowly and regularly down to lo&l20 pg/ml in the fifteenth fraction (106.5 + 7.0 pg/ml, mean + SEM, n = 23) collected at the end of the superfusion experiments (1 hr). K+-induced depolarization produced a marked enhancement of CGRPLM release since the mean levels of the peptide in fractions (nb 3, 4 and 5) corresponding to the Kl pulse reached 459 + 40% (mean + SEM, n = 14) of those found in the same fractions under control conditions ([K+] = 5.4 mM). CGRPLM outflow then returned to the baseline (control) level, and a second exposure to 30 mM K+

636

S.

et al.

EWRG~IN

44min after the first one also induced a highly significant enhancement of CGRPLM release. However, the absolute overflow produced by the second depolarization (K2) was regularly less than that due to the first one (Kl), and the ratio K2/Kl (see Methods) was remarkably constant (0.35 _+0.02, mean f SEM, n = 31) from one perfusion chamber to another. Under resting conditions, SPLM outflow from spinal cord slices remained essentially stable during the whole superfusion experiments. Therefore, a mean rate of SPLM outflow could be calculated: 11.8 + 1.4 pg/fraction (mean + SEM, n = 12), i.e. about 2.9pg/min. The first K+-induced depolarization (Kl) produced an N 5-fold enhancement of SPLM release followed by a rapid return to the spontaneous level. A significant but less pronounced increase in SPLM release was also induced by the second exposure (K2) to 30 mM K+ leading to a ratio K2/Kl of 0.60 f 0.03 (mean _+SEM, n = 22). Effects of NA-, DA- and S-HT-receptor active drugs on K +-evoked CGRPLM and SPLM release

Under control conditions, none of the drugs tested affected the spontaneous outflow of CGRPLM or SPLM from spinal cord slices (not shown). In contrast, the K+-evoked overllow of these peptide-like materials exhibited significant changes in the presence of some ligands of monoamine receptors. Efects of adrenoreceptor ligancis: NA, phenylephrine, clonidine, boproterenol, idazoxan and pra zosin. The K+-evoked release of CGRPLM from

spinal cord slices was not significantly altered when NA (0.1 /IM to 0.1 mM) was added to the perfusing fluid (Fig. 1). Similarly, no change in K+-evoked CGRPLM overflow was observed in the presence of clonidine (1 PM to 0.1 mM), isoproterenol (0.1-10 PM) or phenylephrine (3 PM) (Table 1). In contrast, NA at 10 PM to 0.1 mM (Fig. 1) and clonidine at 0.1 mM (Table 1) significantly reduced (-20 to 25%) the K+-evoked SPLM overflow from spinal cord slices, whereas phenylephrine (3 p M) and isoproterenol (0.1-10 PM) were inactive (Table 1). The inhibitory effects of both NA (10pM) and clonidine (0.1 mM) on SPLM release were abolished by either idazoxan or prazosin at 10pM (Table 1). When added alone to the perfusing fluid, the latter drugs did not significantly affect the K+-evoked overflow of SPLM and CGRPLM (Table 1). Eflects of DA receptor ligands: quinpirole, SKF 82958 and SCH 39166. As shown in Fig. 2, the

selective DA D-l receptor agonist SKF 82958 (O’Boyle, Gaitanopoulos, Brenner and Waddington, 1989) significantly enhanced (+ 30-50%) the release of both CGRPLM and SPLM when added at 10-100 nM into the superperfusing fluid. In contrast, the D-2 receptor agonist quinpirole (10 nM to 1 p M) (Walters, Bergstrom and Carlson, 1986) altered neither the spinal release of CGRPLM nor that of SPLM (not shown).

0.8

0.5

KS/K1 0.4

C

-7

-5

-6

4

@JWAI~

Fig. 1. Effects of increasing concentrations of NA on the K+-evoked release of CGRPLM and SPLM from rat spinal cord slices. Slices of the dorsal half of the lumbar enlargement of the spinal cord were depolarized twice (K I, K2) by 30 mM K+ in the course of superfusion with ACSF, and NA (0.1 PM to 0.1 mM) was added to the superfusing medium from the beginning of the collection of fraction nb 8 up to the end of the experiments. SPLM and CGRPLM were measured in superfusate fractions and the ratio K2/K1 was calculated as described in Methods. Each point is the mean + SEM of at least 6 independent determinations. Grey areas indicate the range of K2/KI values for each peptidelike material in the absence of NA (C on abscissa). *P < 0.01, +*P -c 0.001 when compared to K2/KI value for SPLM overflow in the absence of NA.

Although inactive alone, the selective D-l receptor antagonist, SCH 39166 (1 PM) (Chipkin, Iorio, Coffin, MC Quade, Berger and Barnett, 1988), completely prevented the stimulatory effect of 100 nM

Table

I. Effects of various

release of CGRPLM

adrenoreceptor and SPLM

ligands on the K+-evoked

from

rat spinal

cord

slices

I

KZ/K Addition

CGRPLM

None

0.33 + 0.02

0.58 f 0.03

0.31 f 0.04

0.44 + 0.03’

NA

0.1 mM

Clonidine

I /r M IOpM

ldazoxan +NA

0.32 f 0.02

0.55 f 0.03

0.37 f 0.03

0.56 + 0.03

0.1 mM

0.32 f 0.03

0.47 * 0.02*

IO CM

0.39 +_ 0.08

0.56 +_ 0.03

nd

0.60 k 0.02i

nd

0.62 f 0.03:

0.1 mM

+clonidine Prazosin +NA

SPLM

0. I mM

IO 1M

0.29 + 0.02

0.55 f 0.02

0.1 mM

nd

0.59 + 0.03t

nd

0.55 * 0.02:

Phenylephrine

3 PM

0.32 + 0.01

0.60 f 0.03

Isoproterenol

0. I fl M

0.33 f 0.03

0.50 + 0.04

+clonidine

0. I mM

1PM IOuM Compounds

were added

ning of fraction end

of

the

as described < 0.01

when

0.61 k 0.10

0.34 z!z0.02

0.5 I f 0.07

to the superfusing

8 (twenty-ninth

experiments.

minute

The

in Methods.

least 6 independent *P

0.36 ? 0.05

Each

ratio value

ACSF

from

of superfusion) K2/KI

was

the begin-

up to the calculated

is the mean +_ SEM

of at

determinations.

compared

to

the

respective

control

value

(no

addition). tP

< 0.01 when compared

to K2/K

I in the presence of 0. I mM

NA

alone. $P < 0.01

when

clonidine nd: Not

compared

alone.

determined.

lo

KZ/KI

in the presence

of 0.1 mM

Monoaminergic control of spinal SP and CGRP release

63-l

Table 3. Effects of 5-HT and other ligands of 5-HT receptors on the K+-evoked release of CGRPLM and SPLM from rat spinal cord slices K2/Kl

Ml

0.6

7

0.3

C

/

4

I -10 log

-0 FKF

-6

-7

=@WM

Fig. 2. Effects of increasing concentrations of the selective D-l receptor agonist, SKF 82958, on the K+-evoked release of CGRPLM and SPLM from rat spinal cord slices. The same protocol as that described in the legend to Fig. 1 was used except that 0.1 nM to 0.1 PM SKF 82958 instead of 0.1 PM to 0.1 mM NA was added to the superfusing ACSF. Each point is the mean _+SEM of at least 6 independent determinations of the K2/Kl ratio. Grey areas indicate the range of K2/Kl values for CGRPLM and SPLM overtlow in the absence of SKF 82958 (C on abscissa). lP ~0.01, **P < 0.001 when compared to the respective control values (C on abscissa).

SKF 82958 on the K+-evoked overflow of CGRPLM and SPLM from spinal cord slices (Table 2). Eflects of 5-HT receptor ligandr: SHT, g-OHDPAT, ipsapirone and 2-methyl-5-HT. Neither S-HT

(0.1-10 PM) nor agonists acting selectively at 5-HT,, (10 nM to 1 PM &OH-DPAT, 10 nM to 1 PM ipsapirone) or S-HT, (l-100 PM 2-methyl-5-HT) receptors (see Hoyer, 1991) significantly affected the K+-evoked release of SPLM or CGRPLM from rat spinal cord slices (Table 3). Table 2. Prevention by SCH 39166 of the stimulatory effect of SKF 82958 on the K+-evoked release of CGRPLM and SPLM from rat soinal cord slices

None

SCH 39166 1 pM

0.35 * 0.02 (31) 0.54 f 0.05+ (12) 0.36 + 0.03

SKF 82958 0.1 pM + SCH 39166 I gM

0.41 * 0.03t (16)

SKF 82958 0.1 /IM

(22)

0.60 f 0.03 (22) 0.84 f 0.04* (12) 0.54 f 0.03

(22) 0.60 + 0.05t (11)

SKF 82958 (0. I p M) and/or SCH 39166 (1 p M) were added to the superfusing ACSF from the beginning of fraction 8 (twentyninth minute of superfusion) up to the end of the experiments. The ratio K2/Kl was calculated as described in Methods. Each value is the mean _+SEM of the number of individual determinations indicated in parentheses. *P e 0.001 when compared to the respective control value (no addition). tP -z 0.05 when compared to K2/Kl in the presence of SKF 82958 alone.

Addition

CGRPLM

None 5-HT 0.1 PM 1pM 1OpM 8-OH-DPAT 10 nM 0.1 pM 1CM lpsapirone 10 nM 0.1 pM IpM 2-methyl-5-HT I JIM lOjiM 0.1 mM

0.33 * 0.31 f 0.32 + 0.38 + 0.36 + 0.36 f 0.33 * 0.36 k 0.34 f 0.38 f 0.39 + 0.36 f 0.38 *

0.02 0.06 0.04 0.03 0.05 0.04 0.05 0.08 0.06 0.04 0.05 0.04 0.03

SPLM 0.58 f 0.03 0.52 & 0.08 0.57 f 0.08 0.54 * 0.07 0.51 f 0.07 0.55 f 0.07 0.57 * 0.09 0.60 f 0.03 0.64 f 0.05 0.56 + 0.07 0.59 * 0.07 0.56 k 0.05 0.51 +_0.08

Each compound was added to the superfusing ACSF from the beginning of fraction 8 up to the end of the experiments. Each value of the ratio K2/KI is the mean + SEM of at least 6 independent determinations. None of these compounds significantly affected the K+-evoked overtlow of CGRPLM or SPLM (P > 0.05).

DISCUSSlON

In agreement with previous data (Kuraishi, Hirota, Sato, Kaneko, Satoh and Takagi, 1985; Pang and Vasko, 1986; Go and Yaksh, 1987; Ono et al., 1991), we found here that NA could decrease the spinal release of SPLM. This action appears to be mediated by a,-adrenoreceptors since it could be mimicked by clonidine, but not by agonists acting at a,- and j?-adrenoreceptors such as phenylephrine and isoproterenol, respectively (see Yaksh, 1985). Although the concentrations (2 10 PM) of NA and clonidine that were needed to significantly reduce SPLM release are in the same range as those used in other studies (Kuraishi et al., 1985; Pang and Vasko, 1986; Go and Yaksh, 1987; Ono et al., 1991), they appear to be relatively high when compared to those for the occupancy of a,-adrenoreceptor binding sites in brain membranes (see Ruffolo, Nichols, Stadel and Hieble, 1991). However, it has to be pointed out that affinity measured in binding studies with brain homogenates in the absence of guanine nucleotides, ions etc., is optimal and may not reflect the actual situation in intact tissues such as spinal cord slices superfused with ACSF. Furthermore, drugs in the superfusion medium have to diffuse into the slices to reach the receptors, and their actual concentration within tissues is probably less than that in the medium. Indeed, as expected from the selective interaction of clonidine with a,-adrenoreceptors under such in vitro conditions, the a, antagonist idazoxan (Ruffolo et al., 1991) completely prevented the inhibitory effect of the former drug on SPLM release. Furthermore, idazoxan also prevented the effect of 0.1 mM NA, indicating that a,-adrenoreceptor stimulation entirely accounted for the inhibitory action of the catecholamine on the peptide release. Although prazosin was initially considered as a selective a, antagonist (Ruffolo et al., 1991), it is also presently known as a high affinity antagonist of the a,,-adrenoreceptor

subtype (Bylund, Ray-Prenger and Murphy, 1988). Therefore, the blockade by prazosin of the inhibitory action of NA and clonidine on the K+-evoked release of SPLM from rat spinal cord slices suggests that a,,-adrenoreceptors mediate this effect, in agreement with previous observations {Kuraishi et al., 1985; On0 et al., 1991). In contrast to that observed on SPLM release, the K+-overflow of CGRPLM was affected neither by NA nor by clonidine. Similarly, France-Cereceda, Rydh and Dalsgaard (1992) recently observed that 1 PM NA does not significantly modify the nicotineor capsaicin-evoked release of CGRPLM from guinea pig spinal ganglia in culture. Thus, although CGRP and SP are both contained in primary afferent fibres and colocalized in some of them (Lee ef al., 1985; Skofitsch and Jacobowitz, 1985; FrancoCereceda, Henke, Lundberg, Petermann, Hiikfelt and Fischer, 1987), there is a discrepancy between the effects of a~-adrenor~ptor stimulation on K +-induced release of CGRPLM and SPLM. Since virtually all SPergic primary afferent fibres contain CGRP (Lee et al., 1985; Skofitsch and Jacobowitz, 1985; Franco-Cereceda er al., 1987), this suggests that the a,-adrenergic control of SPLM release concerns SPcontaining neuronal elements distinct from primary afferent terminals, i.e. SP-containing intemeurones and/or bulbo-spinal projections (Hokfelt et al., 1977; Pohl et al., 1990). The modulation by qadrenergic agonists of only part of the SP-containing terminals within the dorsal horn could explain why the reduction in SPLM release due to NA and clonidine plateaued at m -25%. Thus, maximal inhibition of SPLM release would be achieved with 0.1 mM of each agonist, and the remaining SPLM release would originate from SP-containing fibres and terminals unaffected by adrenergic receptor ligands. The lack of effect of NA and clonidine on spinal CGRPLM release suggests that the antinociceptive action of intrathecally administered tlZadrenoreceptor agonists (see Yaksh, 1985) does not involve a presynaptic control of CGRP-containing primary afferent tibres. In fact, anatomical data did not reveal noradrenergic terminals in contact with primary afferent fibres in the spinal cord (Satoh, Kashiba, Kimura and Maeda, 1982). Furthermore, the presynaptic location on primary afferent fibres of about 20% of spinal qadrenoreceptors that was reported by Howe ef al. (1987) was not confirmed by Wikberg and Hajos (1987) who concluded that all o+,-adrenoreceptors are located on capsaicininsensitive neurones within the dorsal horn of the spinal cord. Therefore, in agreement with electrophysiological data (Willcockson, Chung, Hori, Lee and Willis, 1984; Fleetwood-Walker, Mitchell, Hope, Molony and Iggo, 1985; Villanueva, Chitour and Le Bars, 1988) the antin~i~ptive action of a*adrenergic agonists is very probably due to the stimulation of postsynaptic receptors with respect to primary afferent fibres.

As expected from a DA-mediated control of nociceptive messages within the dorsal horn of the spinal cord, eleetrophysiological investigations have shown that nociceptive responses are reduced following intrathecal administration of quinpirole, a selective DA D-2 receptor agonist (Clatworthy and Barasi, 1987). This drug, however, was unable to si~ficantly modify the spinat release of either CGRPLM or SPLM, therefore favouring a postsynaptic rather than a presynaptic action with respect to primary afferent fibres. To date, the presence of DA D-2 receptors on primary afferent fibres has not yet been investigated in radioligand binding studies but is only suggested from electrophysiological data (Fleetwood-Walker et al., 1988). In contrast to the lack of effect of DA D-2 receptor stimulation on the spinal release of CGRPLM and SPLM, the selective stimulation of D-l receptors by SKF 82958 @‘Boyle et al., 1989) enhanced the release of both peptides. The potent D-l antagonist SCH 39166 (Chipkin et al., 1988) blocked the stimulatory effect of SKF 82958, as expected from its mediation through the activation of D-l receptors. Such a facilitation of the release of neurotransmitters from primary afferent fibres might account, at least partly, for the hyperalgesic effects of D-l receptor agonists (Rooney and Sewell, 1989) as well as in the capacity of these drugs to reduce the antinociceptive action of morphine and baclofen (Zarrindast and Moghaddampour, 1989, 199 1). Concerning the possible modulation by 5-HT receptors of the spinal release of SPLM and CGRPLM release, we focused our studies on the 5-HT,, and 5-HT, subtypes (Hoyer, 1991) because it was previously demonstrated that primary afferent fibres entering the dorsal horn of the rat spinal cord are endowed with both of them (Daval et al., 1987; Hamon et al., 1989; Laporte, Kidd, Verge, Gozlan and Hamon, 1992). However, neither 5-HT nor selective agonists of these two 5-HT receptor subtypes affected the spinal release of SPLM and CGRPLM. Similarly, Pang and Vasko (1986) in the rat and Go and Yaksh (1987) in the cat reported that 5-HT exerts no influence on the spinal release of SP. At variance with these findings, Yonehara, Shibutani, Imai, Ooi, Sawada and Inoki (1991) observed that 5-HT reduces the release of SPLM in the trigeminal nucleus of the rabbit. The latter effect can be antagonized by methysergide (Yonehara et al., 1991) which blocks indifferently 5-HT, and S-HT,, but not 5-HT, receptor subtypes (Hoyer, 1991). As the 5-HT,, receptor subtype does not seem to be involved in a possible control by S-HT of SP release (see Table 3), one can propose that 5-HT,a, 5-HT,, and/or 5-HT, receptors mediate this action of the indoleamine, in line with the antinociceptive effects of their selective agonists injected via the intrathecal route in rats (see Hamon et al., 1991). However further studies have to be performed in order to investigate whether selective agonists of 5-XT,,, !i-HT,c and/or 5-HT, receptors

Monoaminergic control of spinal SP and CGRP release really affect the release of SP from primary afferent fibres within the dorsal horn. In contrast to Saria, Javorsky, Humpel and Gamse (I990) who concluded that S-H1; receptor stimulation exerts a facilitatory influence on the release of CGRPLM from rat spinal cord slices, we did not find any increase in the K+-evoked release of this peptide from tissues superfused with the potent 5HT3 agonist 2-methylJ-HT (Hoyer, 1991). As 5-HT, receptors may desensitize rapidly (Hoyer, 1991), it can be speculated that the discrepancy between our results and those of Saria et al. (1990) originates in possible differences between the desensitization rate by 5-HT, receptor agonists in the two studies. Indeed, the superfusion method of Saria et al. (1990) markedly differed from ours as, in particular, they applied one K+ pulse only instead of two in the present study. In any case, the data reported herein do not support that 5-HT exerts its antinociceptive action at the spinal level (see Hamon et al., 1991) through a presynaptic control of primary afferent fibres containing SP and/or CGRP. Instead, el~trophysiological studies have concluded that the 5-HT action on spinal neurones located postsynaptically with respect to primary afferent fibres very probably accounts for its inhibitory influence on the transfer of nociceptive messages (Giesler, Gerhat, Yezierski, Wilcox and Willis, 1981; Alhaider, Lei and Wilcox, 1991). In conclusion, our data show that the K+-evoked release of CGRPLM from rat spinal cord slices remained unaffected by cc,-adrenoreceptor, DA D-2, 5-HT,, and 5-HT, receptor agonists. Therefore, the antinociceptive effects of these drugs injected via the intrathecal route (see Yaksh and Stevens, 1988; Hamon et al., 1991) do not probably involve a presynaptic inhibitory control of CGRP-containing primary afferent fibres. The decrease in spinal SPLM release elicited by NA and clonidine very likely results from an action of these compounds on other targets than SP-containing primary afferent fibres, and is therefore probably unrelated to their antinoci~ptive effects. Finally, the increased release of both CGRPLM and SPLM (probably from primary afferent terminals) due to DA D-l receptor stimulation may account-at least partly-for the pronociceptive effects of agonists acting selectively at this receptor type (Rooney and Sewell, 1989; Zarrindast and Moghaddampour, 1989, 1991). Acknowledgemenrs-This research has been supported by grants from INSERM and Universite Paris VI (DRED). We are grateful to pharmaceutical companies (Eli Lilly, Schering-Plot& Troponwerke) for their generous gifts of drugs.

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