Abstract
1. The effects of selective 5-opioid agonists and antagonists have been studied using both in vivo and in vitro models of ẟ-opioid receptor function to further investigate the physiology of this receptor. In addition, studies were aimed to explore the possibility that 5-receptors represent a heterogeneous population of receptor sites. 2. Using the warm water tail immersion test three highly selective 5-opioid receptor agonists DSLET, DPDPE and DELTI induced a dose-related antinociception in 25 day old rats following i. p. administration. This antinociception had peak tail immersion latencies of 7. 5, 6. 5 and 6. 0 sec for DSLET, DPDPE and DELT I respectively. Calculation of ED75's for the ẟ-agonists gave values of 0. 65,0. 66 and 0. 032mg/kg for DSLET, DPDPE and DELT I respectively. The antinociception induced by the administration of the ED75 of the ẟ-agonists was fully antagonised by prior injection of the highly selective 5-opioid antagonist naltrindole (NTI) at a dose of 1 mg/kg. Administration of 0. 1mg/kg NTI fully reversed the effect of DSLET, partially reversed the effect of DPDPE but had no effect on the response to DELT I. Further 0. 01mg/kg NTI had no effect on the response to DELT I but partially antagonised the responses to DSLET and DPDPE. The differential antagonism by NTI of the effects of the three selective ẟ-agonists suggests 5-receptor heterogeneity. Further, the lower sensitivity of the response to DELT I suggests that this agent may exert its antinociceptive effects at a different ẟ-receptor subtype from DPDPE or DSLET. 1mg/kg NTI had no effect on the antinociception induced by administration of the u-agonist alfentanil demonstrating that the doses of NTI used are selective for ẟ-opioid receptors. 3. Administration of the highly selective ẟ-opioid antagonist naltriben (NTB) (50ug/kg) fully reversed the antinociception induced by injection of the ED75 of each of the ẟ-agonists. However administration of 5ug/kg had no effect on the responses to DPDPE or DELT I but fully antagonised the antinociception induced by DSLET. Similarly, 0. 5 ug/kg NTB partially reversed the effect of DSLET with no effect on the responses to DPDPE and DELT I. The differential antagonism by NTB of the antinociceptive effects of three selective ẟ-agonists provides further support for ẟ-receptor heterogeneity confirming the results obtained with NTI. Further the profile of antagonism of the ẟ-agonists by NTB is dissimilar to the profile of antagonism by NTI suggesting that these two antagonists have differential affinities for the putative ẟ-receptor subtypes. Administration of 50pg/kg NTB had no effect on the antinociception induced by a submaximal dose of alfentanil indicating that the doses of NTB used were selective for the ẟ-opioid receptors. The relative sensitivity to antagonism by NTI and NTB has been used to classify ẟ-mediated responses into ẟ1 (NTB insensitive) and ẟ2 (NTB sensitive) subtypes. In accordance with this nomenclature responses to DELT I and DSLET in the 25 day old rat can be designated as ẟ1 and ẟ2 respectively. 4. The antinociception induced by the ED75 of the three ẟ-agonists was also antagonised by ICI 174,864, at a dose (2mg/kg) which had no effect on the antinociception induced by a submaximal dose of alfentanil. A lower dose of ICI 174,864 (1mg/kg) also significantly antagonised the effects of the three ẟ-agonists with a differential degree of attenuation, and being significantly less effective at antagonising the effects of DELT I. Thus, in addition to NTI and NTB the peptide ẟ-antagonist ICI 174,864 also shows a differential antagonism of the antinociceptive effects of the three ẟ-agonists. In addition, administration of the non-selective opioid antagonist naloxone (lmg/kg) reversed the effects of all three ẟ-agonists to a similar degree indicating that the antinociception induced by these three agonists is opioid mediated and that naloxone does not distinguish between the putative ẟ-subtypes. 5. To study whether weaning stimulates the development of a subtype of the ẟ-opioid receptor the antinociceptive activity of putative ẟ1 (DELT I) and ẟ2 (DSLET) agonists in weaned and non-weaned rats was investigated. The antinociceptive effects of DSLET and DELT I were investigated in 25 day old weaned and non-weaned rats. Administration of DSLET induced a dose-related antinociception in weaned rats but was without effect in non-weaned animals. In contrast DELT I induced a dose-related antinociception in both weaned and non-weaned animals. The effect of DSLET in weaned rats was antagonised by 5ug/kg NTB whereas the antinociception induced by DELT I in both weaned and non-weaned groups was unaffected by administration of this dose of NTB. The results suggest that the ẟ1 and ẟ2 receptors develop differentially and the expression of ẟ1 responses is independent of weaning whereas expression of ẟ2 responses is weaning dependent. 6. The potency of selective 8-agonists and their reversal by the selective ẟ-antagonists NTI and NTB were studied in the mouse vas deferens of two strains of mice (SCH and TO) and in the hamster vas deferens. DSLET, DPDPE and DELT I all produced an inhibition of the field stimulated contractions of the mouse vas deferens. The inhibition was dose-related and gave IC50 values of 1. 98 and 1. 12nM for DSLET in the SCH and TO mouse respectively, 3. 32 and 4. 20nM for DPDPE in the SCH and TO mouse respectively and 0. 75 and 0. 84nM for DELT I in the SCH and TO mouse respectively. Addition of NTI to the bath induced a parallel rightward shift in the agonist dose-response curves, indicating that NTI acts as a competitive antagonist in this tissue. Following Schild analysis significant differences in the pA2 values for Nil were seen and the slopes of the Schild plots for NTI versus DELT I (SCH) and DSLET (TO) were significantly different from unity raising the possibility that the agonists were exerting their effects via multiple ẟ-sites. The pA2 values obtained following administration of NTB in the SCH mouse vas deferens were similar, but the slope of the Schild plot for NTB versus DSLET was significantly different from unity. 7. All three ẟ-agonists inhibited the field stimulated contractions of the hamster vas deferens in a dose-related manner. Calculation of IC50 values yielded figures of 658, 346 and 86nM respectively for DSLET, DPDPE and DELT I. Addition of NTI or NTB induced a parallel rightward shift in the agonist dose-response curves, indicating that these compounds act as competitive antagonists in this tissue. Ke values for NTI versus the ẟ-agonists were not significantly different, however Ke values for NTB versus DPDPE and DELT I were significantly different with NTB being more effective at antagonising the response to DELT I, again raising the possibility that the effects of these compounds are mediated via a heterogeneous receptor population. 8. In order to investigate if functional ẟ-receptors were present in the VMH the action of ẟ-selective agonists and antagonist were studied, by recording extracellularly the effects on neuronal firing from single neurones in the rat VMH in vitro. Perfusion of rat VMH in vitro with DSLET, DPDPE and DELT I led to a dose-related inhibition of neuronal firing. ED75 values of 65, 177 and 35nM were calculated for DSLET, DPDPE and DELT I respectively. The inhibition induced by DSLET and DPDPE was antagonised by perfusing the slice with 10nM NTI. This dose of NTI had no effect on the inhibition induced by the u-agonist DAGO nor the non-opioid depressant GABA. The reversibility of the NTT antagonism was also investigated, and a full recovery of the DSLET response was achieved 120min after reperfusion with NTI-free aCSF. These results show the presence of functional ẟ-receptors in rat VMH.