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Neuroscience Letters, 134 (1992) 238-242 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 03.50

NSL08314

x-Opioid receptor stimulation abolishes #- but not 6-mediated inhibitory control of spinal Met-enkephalin release E. Collin, S. Bourgoin, L. Ferhat, M. H a m o n a n d F. Cesselin INSERM U 288, FacultO de Mbdecine PitiO-Salp~tri&e, Paris' (France) (Received 29 August 1991; Revised version received 4 October 1991; Accepted 4 October 1991)

Key words: Rat spinal cord; In vivo release; Met-enkephalin-like material; Opioid receptor; x/p Interaction The possible opioid control through ~,/t and x receptors of the spinal release of Met-enkephalin-like material (MELM) was investigated in halothane-anaesthetized rats. The intrathecal perfusion of the 6 agonist DTLET (10 pM) or the/1 agonist DAGO (10 #M) resulted in a marked inhibition of MELM release, which could be prevented by the selective antagonists naltrindole and naloxone, respectively. Although the ~c agonist U 50488 H (10/~M) was inactive per se, it completely suppressed the inhibitory effect of DAGO, without affecting that of DTLET. As the selective x antagonist norbinaltorphimine blocked the action of U 50488 H, it can be concluded that x receptors modulate the ,u- (but not the 6-) mediated feed back control of spinal enkephalinergic neurones.

Opioid receptors of the three major types, 8,/~ and x, are present in the spinal cord [15, 20], particularly in the superficial layers of the dorsal horn, where neurones containing proenkephalin-A and -B derivatives are also found [3, 11]. In addition to playing a key role in the control of nociception (see ref. 10), these receptors were recently shown to participate in a local feedback regulation of enkephalin release. Thus, in vitro as well as in vivo in the rat, a marked decrease in the spinal release of Met-enkephalin-like material (MELM) occurred upon the stimulation of local/z or 6 opioid receptors [4, 7]. Several lines of evidence have indicated that the stimulation of one type of opioid receptor can modulate the effects of a selective agonist of another opioid receptor, and a model of an opioid receptor complex composed of distinct, yet interacting, p, 6 and t¢ binding sites has been described [12]. For instance, X agonists can alter the pharmacological (e.g. antinociceptive) effects of morphine and p selective opioid agonists [9, 16--19, 21], and electrophysiological studies revealed that the inhibition of spinal neurons by /z agonists was less pronounced upon ~c opioid receptor stimulation [8]. In addition, K opioid receptor stimulation also prevented the inhibitory effect of DAGO, a selective/~ agonist [25], on the release of cholecystokinin from slices of the rat spinal cord [2]. Correspondence: E. Collin, INSERM U 288, Facult4 de Mrdecine Piti4-Sal!~trirre, 91 Boulevard de I'H6pital, 75634 Paris Cedex 13, France. Fax: (33) 1 40 77 97 90.

As the selective x agonist, U 50488 H [22], was by its own totally inactive on the outflow of M E L M from the rat spinal cord in vivo [4], we thought that this drug should be particularly useful to examine the possible modulatory effects of h: opioid receptor stimulation on the #- or g-mediated inhibitory control of the peptide release. Thus, opioid ligands acting selectively on/~, 6 or x receptors were administered intrathecally in halothane-anaesthetized rats, and the in vivo release of M E L M from the whole spinal cord was monitored through the perfusion of the subarachnoid space with an artificial cerebrospinal fluid (ACSF). Male Sprague-Dawley rats (Centre d'Elevage R. Janvier, Le Genest, France) weighing 320-380 g were kept under controlled environmental conditions (22 +_ I°C, 12 h alternate light-dark cycles, food and water ad libiturn) for at least 7 days before being used for the experiments. The surgical procedures were performed under deep anaesthesia (2% halothane in a nitrous oxide-oxygen mixture [2:1]). Following tracheal cannulation and insertion of a catheter in a jugular vein, the head was immobilized in a ventroflexed position, the body lying on a sloping surface. Then, the animals were artificially ventilated, the rate and the volume being adjusted to maintain a normal acid-base equilibrium. The pC02 was continuously monitored (30 +__ 5 mmHg) and the body temperature was kept constant (37.0 + 0.5°C) by means of an homeothermic blanket system. Preparation of the animals for the intrathecal perfusion was essentially as

239 described by Yaksh and Tyce [23]. A transverse incision was made over the external occipital bump and on the midline overlying the cisterna magna. Muscles were drawn aside from the skull and atlas, and the occipitoatlantoidal membrane was retracted from the cisterna dura. A small incision of the dura and the arachnoid was made over the obex. A nylon inflow catheter (0.50 mm i.d., 0.63 mm o.d.) was then carefully inserted and conveyed 85 mm into the subarachnoid space. Then an outflow catheter was inserted parallel to the former, with its extremity overlying the lower medulla. Following the surgical procedure, rats were paralyzed by slow i.v. infusion of gallamine triethiodide and the level of halothane was lowered to 0.5% for the remainder of the experiment. The intrathecal space was then perfused with an ACSF (see ref. 5) maintained at 37°C at the output of the inflow catheter. The flow rate was 0.1 ml/min. Thirty min after the beginning of the perfusion, 9 perfusate fractions (1.5 ml each corresponding to 15 min) were collected at 4°C, and immediately frozen at -20°C in three aliquots of 500/al until the radioimmunoassay of their MELM content (see ref. 5). Each time a compound was added to the ACSF, a complete standard curve was drawn from radioimmunoassays of 0.1-500 pg of authentic ME performed in the presence of this compound at the same concentration as that used for the perfusion experiments. This allowed the accurate determination of MELM contents of fractions containing opioid receptor active drugs. MELM contents of the perfusate fractions were expressed as ME equivalents, i.e. in pg ME producing the same displacement of bound [125I]ME (New England Nuclear) under standard radioimmunoassay conditions. In order to determine the significance of the effect produced by a given treatment, the mean value of MELM content in the first three fractions, always collected before the addition of any compound to the ACSF, was calculated in each experiment and used as the control value (100%). MELM content of each successive fraction was then expressed as a percentage of this control value (see ref. 4). In the absence of drugs, perfusion of the subarachnoid space with ACSF at a flow rate of 0.1 ml/min yielded a constant outflow of MELM for at least 135 min (9 fractions). Thus, the mean MELM content of collected fractions was equal to 9.0 + 1.4 pg/fraction (mean +_ S.E.M., n = 9 rats), that corresponded to a rate of 0.60 + 0.09 pg/min for the spontaneous MELM outflow from the whole spinal cord in halothane-anaesthetized rats. When 10/aM DAGO, a selective/1 opioid receptor agonist [25], was added to the perfusing ACSF (fractions 4-6), an immediate decrease in MELM outflow was observed (Fig. 1A). This inhibitory effect of DAGO persisted throughout the 45 rain of its application, and

MELM levels in fractions 4-6 (not significantly different from each other) amounted to 60 + 1% (P

Kappa-opioid receptor stimulation abolishes mu- but not delta-mediated inhibitory control of spinal Met-enkephalin release.

The possible opioid control through delta, mu and kappa receptors of the spinal release of Met-enkephalin-like material (MELM) was investigated in hal...
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