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Biomolecules
2021 Mar 25;114:. doi: 10.3390/biom11040493.
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Modulation of Glycinergic Neurotransmission may Contribute to the Analgesic Effects of Propacetamol.
Barsch L
,
Werdehausen R
,
Leffler A
,
Eulenburg V
.
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Treating neuropathic pain remains challenging, and therefore new pharmacological strategies are urgently required. Here, the enhancement of glycinergic neurotransmission by either facilitating glycine receptors (GlyR) or inhibiting glycine transporter (GlyT) function to increase extracellular glycine concentration appears promising. Propacetamol is a N,N-diethylester of acetaminophen, a non-opioid analgesic used to treat mild pain conditions. In vivo, it is hydrolysed into N,N-diethylglycine (DEG) and acetaminophen. DEG has structural similarities to known alternative GlyT1 substrates. In this study, we analyzed possible effects of propacetamol, or its metabolite N,N-diethylglycine (DEG), on GlyRs or GlyTs function by using a two-electrode voltage clamp approach in Xenopus laevis oocytes. Our data demonstrate that, although propacetamol or acetaminophen had no effect on the function of the analysed glycine-responsive proteins, the propacetamol metabolite DEG acted as a low-affine substrate for both GlyT1 (EC50 > 7.6 mM) and GlyT2 (EC50 > 5.2 mM). It also acted as a mild positive allosteric modulator of GlyRα1 function at intermediate concentrations. Taken together, our data show that DEG influences both glycine transporter and receptor function, and therefore could facilitate glycinergic neurotransmission in a multimodal manner.
Figure 1. Chemical structure of (A) propacetamol, (B) acetaminophen, (C) N,N-diethylglycine (DEG), (D) sarcosine, (E) glycine. Propacetamol is hydrolysed to acetaminophen and DEG, which shares structural similarities to the neurotransmitter glycine.
Figure 2. Functional analysis of glycine transporters (GlyTs) with glycine-induced dose-dependent current responses. (A) Representative current traces of original electrophysiological recordings from Xenopus laevis oocytes expressing GlyT1 or GlyT2, compared to non-injected oocytes after superfusion with glycine solution in increasing concentrations. Substance application is indicated by open box above the trace. (B) Dose–response curves of GlyTs showing the glycine-induced currents in relation to the maximum observed current induced by 10 mM glycine (GlyT1 n = 10–22, GlyT2 n = 13–23). (C) Maximum glycine-induced current amplitudes (nA) determined on oocytes expressing transporter individually taken together from all measurements (GlyT1 n = 148; GlyT2 n = 130; data presented as boxplots).
Figure 3. N,N-diethylglycine (DEG), but not propacetamol or acetaminophen, functions as an alternative substrate on GlyT1 and GlyT2. (A) Representative extract of original electrophysiological traces in Xenopus laevis oocytes expressing GlyT1 and GlyT2 individually compared to a non-injected oocyte after superfusion with glycine solution (20 µM, 1 mM), DEG (10 mM), acetaminophen (10 mM), propacetamol (10 mM), hydrolysed propacetamol (10 mM) and co-application with glycine (20 µM). (B) Relative substance-induced currents of GlyT1 and GlyT2 in relation to the maximum observed current induced by 1 mM glycine (GlyT1 n = 29–51; GlyT2 n = 13–44; data presented as boxplots, p < 0.001 (***), one-way ANOVA with Bonferroni post-hoc correction).
Figure 4. DEG functions as a full agonist at GlyT1 and GlyT2. (A) Representative original traces of electrophysiological measurements in Xenopus laevis oocytes expressing GlyT1 and GlyT2 individually after superfusion with DEG solution in increasing substance concentration (100 µM, 333 µM, 1 mM, 3.3 mM, 10 mM), sarcosine solution (1 mM) and glycine solution (1 mM) and after superfusion with different glycine solutions (20 µM, 200 µM, 2 mM) additionally containing distinct amounts of DEG (0 mM, 3.3 mM, 10 mM) to create co-application conditions. (B) Dose–response curves of GlyTs showing DEG and sarcosine-induced currents in relation to the maximum observed current induced by 1 mM glycine in comparison to the construct’s glycine dose–response curve (Figure 2B) (GlyT1: DEG n = 8–26, sarcosine n = 19; GlyT2: DEG n = 17, sarcosine n = 16). (C) Relative substance-induced currents of GlyT1 and GlyT2 in relation to the maximum observed current induced by 2 mM glycine + 0 mM DEG solution (GlyT1 n = 13–23; GlyT2 n = 15–31; data shown as boxplots, p < 0.001 (***), one-way ANOVA with Bonferroni post-hoc correction).
Figure 5. Functional analysis of the used glycine receptor (GlyR) constructs with glycine-induced dose-dependent current responses. (A) Representative extract of original electrophysiological traces in Xenopus laevis oocytes expressing GlyR subunits (α1-3) individually with glycine solution in increasing substance concentration (1 µM, 10 µM, 33 µM, 100 µM, 333 µM, 1 mM, 3.3 mM, 10 mM). Substance application is indicated by black bars. Note that due to limited perfusion channel number and differing substance concentrations in each experiment, not every measurement contained all concentrations. (B) Dose-response curves of GlyRs showing the glycine-induced currents in relation to the maximum observed current induced by 10 mM glycine (GlyRα1 n = 12, GlyRα2 n = 3–15, GlyRα3 n = 11). (C) Maximum glycine-induced current amplitudes [nA] determined on oocytes expressing transporter or receptor proteins individually taken together from all measurements (GlyRα1 n = 47, GlyRα2 n = 45, GlyRα3 n = 40; data shown as boxplots).
Figure 6. Current response of GlyRα1-3 after DEG application with and without glycine. (A) Original traces of electrophysiological measurements in Xenopus laevis oocytes expressing GlyRα1-3 individually after superfusion with DEG solution in increasing substance concentration (100 µM, 333 µM, 1 mM, 3.3 mM, 10 mM), sarcosine solution (1 mM) and glycine solution (1 mM) and after superfusion with different glycine solutions (20 µM, 200 µM, 2 mM) additionally containing amounts of DEG (0 mM, 3.3 mM, 10 mM). (B) Relative substance-induced currents of GlyRα1-3-expressing oocytes in relation to the maximum observed current induced by 2 mM glycine (GlyRα1 n = 24–37; GlyRα2 n = 14–30; GlyRα3 n = 12–29; data shown as boxplots, p < 0.001 (***), one-way ANOVA with Bonferroni post-hoc correction).
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