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KAPPAI/MU INTERACTIONS IN THE CONTROL OF THE IN VIVO RELEASE OF NEUROPEPTIDES FROM THE RAT SPINAL CORD F. Cesselin, E. Collin, S. Bourgoin, M. Pohl, A. Mauborgne, J. J. Benoliel and M. Hamon, INSERM U 288, Facult6 de Mddecine Piti6-Salp6tri~re, 75634 Paris cedex 13, France.

The possible modulation by K1 opioid receptor stimulation of the effects of p receptor stimulation on the spinal release of met-enkephelin- (ME), substance P- (SP) and calcitonin gene-related peptide- (CGRP) like materials (LM) was investigated in vivo in the rat. The stimulation of K1 receptors by 10 pM U 50488 H exerted no effect per se on the release processes. The stimulation of p opioid receptors by 10 pM DAGO significantly reduced (-40/,,) the spinal outflow of MELM, enhanced (+44%) that of SPLM and did not affect CGRPLM outflow. In the presence of both DAGO and U 50488 H, the spinal release of MELM was not different from control but those of SPLM and CGRPLM were decreased (-34*/= and -40% respectively). These data indicate that, although inactive on their own, K1 receptors can influence the effects of p receptor stimulation on the release of the three peptides studied, supporting the hypothesis of some coupling between p and K1 receptors in the rat spinal cord.

Opioid receptors of the three major types, p, ¢~ and K, are present in the dorsal horn of the spinal cord, where neurones containing endogenous opioids are also found. These receptors are thought to play a key role in the control of nociception. Opioids affect differently, depending on the receptor type involved, the spinal release of neuropeptides originating from primary afferent fibers (which convey the nociceptive messages to the dorsal horn), such as SP and CGRP, or from spinal interneurones, such as ME. Several lines of evidence indicate that the stimulation of one type of opioid receptor can modulate the effects of a selective agonist of another opioid receptor. This prompted us to examine the possible modulation by K1 receptor stimulation of the effects of DAGO (a p agonist) on the spinal release of ME-, SP- and CGRP-LM. The subarachnoid space of gallamine-paralyzed, halothane-anaesthetized rats was perfused (0.1 ml/min) with a thermostated (37°C) artificial CSF for 30-45 min before collecting 1.5 ml (i. e. 15 min) fractions at 0°C. Perfusate fractions (usually 9 for each experiment) were kept at -30°C until the specific radioimmunoassays of ME, SP and CGRP. Drugs were added to the CSF during the collection of fractions 4-6. The mean rates of spontaneous MELM, SPLM and CGRPLM release were 0.60 + 0.09, 0.33 + 0.03 and 4.11 + 0.10 pg/min, respectively. The stimulation of K1 receptors by 10 pM U 50 488 H exerted no effect per se on the release processes. The stimulation of p opioid receptor by 10 pM DAGO significantly reduced the spinal outflow of MELM and enhanced that of SPLM. In contrast, CGRPLM outflow was unaltered by this treatment (Table 1). Addition of 10 pM naloxone to the perfusing CSF did not modify the release of MELM and SPLM, but increased (+ 40/=) that of CGRPLM. Similarly, the blockade of K receptors by 10 pM nor-binaltorphimine (nBNI) did not change the spinal outflow of the tachykinin and the opioid peptide, but enhanced (+ 50%) that of CGRPLM When both 10 pM DAGO and 10 pM U 50 488 H were added to the CSF, the spinal release of MELM was not different from control, but those of SPLM and CGRPLM were decreased (Table 1). Prevention by U 50 488 H of DAGO-induced inhibition of MELM release could be reversed by the intrathecal application of 10 pM nBNI (Table 1). Similarly, the proper stimulatory effects of DAGO on the release of SPLM and of nBNI on that of CGRPLM were recovered in the presence of DAGO + U 50488 H + nBNI in the perfusing CSF (Table 1). When 10 pM naloxone was added to the association of DAGO + U 50 488 H, the release of SPLM remained unaffected while that of CGRPLM was increased (+ 39%). Taken together, these data indicate that endogenous opioids acting (simultaneously) at p and K 1 receptors exert a tonic inhibitory control of the spinal release of CGRPLM (but not of

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MELM and SPLM release). They also show that the changes in peptides' release due to/J receptor stimulation by DAGO, whatever their direction (a reduction in, an enhancement of or no effect on spinal MELM, SPLM and CGRPLM release, respectively) were altered by the concomitant stimulation of ~1 receptors by U 50 488 H. However, on its own, the latter [1 agonist was inactive on the release of the three peptides. In addition to supporting the hypothesis of some functional coupling between p and =<1 receptors, these data suggest that the simultaneous stimulation of spinal p and •1 receptors could be particularly effective in eliciting analgesia. Indeed, p receptor stimulation alone resulted in a decrease in MELM release and an increase in SPLM release, e. g. changes which could both counteract the otherwise (for instance at a postsynaptic site with respect to primary afferent fibers) antinociceptive action of p agonists. In contrast, the concomitant stimulation of p and ~1 receptors not only abolished the reduction in MELM release and converted the enhancement to a diminution of SPLM release, but also induced a decrease in CGRPLM release.

Table 1. Effects of opioid receptor active compounds on the release of peptides from the spinal cord of anaesthetized rats

release (% of control) of MELM SPLM CGRPLM

DAGO 60 + 1" 144 + 10" 101 ± 3 DAGO + U 50 488 H 97 ± 1 66 ± 9* 60 ± 1 * DAGO + U 50 488 H + nBNI 6 4 ± 2 * 145+12" 131 ±11"

Each drug was used at 10 pM. Values are the means + SEM of results obtained in at least 7 animals. * P < 0.05 when compared to the corresponding control value (100%).