Glycine Transporter 1 Inhibitors Minimize the Analgesic Tolerance to Morphine

Abstract

Opioid analgesic tolerance (OAT), among other central side effects, limits opioids' indispensable clinical use for managing chronic pain. Therefore, there is an existing unmet medical need to prevent OAT. Extrasynaptic N-methyl D-aspartate receptors (NMDARs) containing GluN2B subunit blockers delay OAT, indicating the involvement of glutamate in OAT. Glycine acts as a co-agonist on NMDARs, and glycine transporters (GlyTs), particularly GlyT-1 inhibitors, could affect the NMDAR pathways related to OAT. Chronic subcutaneous treatments with morphine and NFPS, a GlyT-1 inhibitor, reduced morphine antinociceptive tolerance (MAT) in the rat tail-flick assay, a thermal pain model. In spinal tissues of rats treated with a morphine-NFPS combination, NFPS alone, or vehicle-comparable changes in µ-opioid receptor activation, protein and mRNA expressions were seen. Yet, no changes were observed in GluN2B mRNA levels. An increase was observed in glycine and glutamate contents of cerebrospinal fluids from animals treated with a morphine-NFPS combination and morphine, respectively. Finally, GlyT-1 inhibitors are likely to delay MAT by mechanisms relying on NMDARs functioning rather than an increase in opioid efficacy. This study, to the best of our knowledge, shows for the first time the impact of GlyT-1 inhibitors on MAT. Nevertheless, future studies are required to decipher the exact mechanisms.

Keywords: NFPS; NMDARs; glycine transporter-1; opioid analgesic tolerance.

Figure 1

The antinociceptive effect of sc. morphine, NFPS, and Org-25543 and their combination acutely (A,C) and after 10-day treatment (B,D). Means ± S.E.M. of tail-flick latency are presented at the tested time points. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to the corresponding vehicle group (morphine vs. saline; NFPS or Org-25543 vs. 10% DMSO; combination vs. saline and 10% DMSO, showing the stricter) in a Mixed-effect model (for panel D) and two-way ANOVA (panel, A–C), followed by Tukey’s multiple comparison test, n = 5–12 per group.

Figure 2

Motor coordination effects of test compounds, morphine, NFPS, and their combination acutely (A) and after 10-day treatment (B) 30 min after subcutaneous treatment. Columns represent latency times of animals on the rotarod test, shown as mean ± S.E.M., *** p < 0.001, **** p < 0.0001 compared to saline; Kruskal–Wallis test with uncorrected Dunn’s post hoc test, n = 5–17 per group. Day 1: H = 45.06; Day 10: H = 40.59.

Figure 3

CSF glycine (A) and glutamate (B) content of animals after 10-day subcutaneous treatments with morphine, NFPS, or their combination. Data are presented as means ± S.E.M. in µM concentration. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. vehicles, one-way ANOVA followed by Fisher’s LSD test, n = 6–22 per group. ((F (4, 28) = 5.116 for panel A; F(4, 53) = 2.610 for panel B).

Figure 4

The effect of chronic treatment with NFPS (0.6 mg/kg, sc.) alone or in combination with morphine (10 mg/kg sc.) on MOR agonist-induced G-protein activity in morphine-induced [³⁵S]GTPγS binding in homogenized L4-6 sections of spinal cord from corresponding treated animals (A). Concentration–response curves of morphine-induced [³⁵S]GTPγS binding measured in samples from the indicated treatment groups. Figure 4A represents the specific binding of [³⁵S]GTPγS in the presence of increasing concentrations (0.1 nM–10 μM) of morphine. Points represent means ± S.E.M. for at least three experiments performed in triplicate. “Basal” on the x-axis indicates the basal activity of the monitored G-protein, which is measured in the absence of morphine and also represents the total specific binding of [³⁵S]GTPγS. The level of basal activity was defined as 100%, indicated by dotted line (B). Maximum efficacy of morphine over basal activity calculated from concentration–response curves. Columns represent means ± S.E.M. ***/###: p < 0.001, one-way ANOVA with Dunnett’s multiple comparisons test (compared to saline-treated group (F (4, 50) = 12.16); compared to morphine 10 mg/kg alone-treated group (F (6, 77) = 10.66)).

Figure 5

Spinal cord MOR levels relative to GAPDH after 10-day treatment. Representative bands are taken from the same membrane. Data are presented as means ± S.E.M. p > 0.05 vs. saline, one-way ANOVA followed by Dunnett’s post hoc test, n = 9–10 per group.

Figure 6

The effect of 10-day morphine, NFPS and combination treatment on spinal cord Oprm1 and GluN2B expression relative to GAPDH. Data is presented as means ± S.E.M. For statistical analysis One-way ANOVA followed by Dunnett’s post hoc test was used, n = 5 per group.