Inhibition of opioid release in the rat spinal cord by serotonin 5-HT(1A) receptors

Abstract

Neurotransmitter receptors that inhibit the release of opioid peptides in the spinal cord may play an important role in modulating pain. Serotonin is an important neurotransmitter in bulbospinal descending pathways, and 5-HT(1) receptors have been shown to inhibit synaptic transmission. Our goal was to determine whether 5-HT(1A) receptors inhibit opioid release in the spinal cord. Opioid release was evoked from rat spinal cord slices by electrically stimulating one dorsal horn, and measured in situ through the internalization of micro-opioid receptors in dorsal horn neurons. Stimulation with 1000 square pulses at 500 Hz produced internalization in 60% of the mu-opioid receptor neurons in the stimulated dorsal horn, but not in the contralateral one. The selective 5-HT(1A) receptor agonist 8-hydroxy-2-dipropylaminotetralin (8-OH-DPAT) inhibited the evoked mu-opioid receptor internalization by about 50%, with an approximate IC(50) of 50 nM. The effect of 8-OH-DPAT was attributed to inhibition of opioid release and not of the receptor internalization process, because 8-OH-DPAT did not inhibit the internalization induced by incubating the slices with a micro-opioid receptor agonist (endomorphin-2, 100 nM). The selective 5-HT(1A) receptor antagonist WAY100135 (10 microM) blocked the inhibition produced by 1 microM 8-OH-DPAT. These results show that 5-HT(1A) receptors inhibit opioid release in the spinal dorsal horn, probably from a subpopulation of enkephalin-containing presynaptic terminals. Therefore, 5-HT(1A) receptors likely decrease the analgesia produced by endogenously released opioids.

Figure 1. MOR internalization evoked by electrical stimulation was inhibited by 8-OH-DPAT

MOR internalization was evoked by dorsal horn stimulation with 1000 electrical pulses at 500 Hz. 8-OH-DPAT inhibited the internalization with Log IC₅₀ = −7.30 ± 0.55 (IC₅₀ = 50 nM). Data are the mean ± SEM of 3−12 replicates. The inhibition was partial, decreasing MOR internalization from 57 ± 4 % (“top”) to 31 ± 6 % (“bottom”). The data were fitted significantly better by a dose-response function than by a horizontal line (p=0.003, F-test). Two-way ANOVA revealed significant effects of stimulation (p<0.0001) and drug (p=0.002). Bonferroni's post-test: ** p < 0.01, *** p<0.001 compared with control.

Figure 2. A 5-HT_1A receptor antagonist reversed the inhibition produced by 8-OH-DPAT

MOR internalization was evoked by dorsal horn stimulation with 1000 electrical pulses at 500 Hz. 8-OHDPAT (1 μM) decreased the evoked MOR internalization. This effect was reversed by the 5-HT_1A antagonist WAY-100135 (10 μM). Data are the mean ± SEM of 3−12 replicates. Two-way ANOVA revealed a significant effect of stimulation (p<0.0001) and drugs (p<0.001). Bonferroni's post-test: *** p<0.001 compared with control, † p<0.05 compared to 8-OH-DPAT.