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Abstract

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.

1. Introduction

To foster a fast translation to clinical application, we searched for substances that might have potential effects on glycine sensitive proteins, but that are already used in clinics. We focused on substances that display, by themselves or by one of their metabolites, structural similarities to known GlyT1 substrates (i.e., glycine, sarcosine, or N-ethylglycine). Here, we identified propacetamol, a N,N-diethylglycine ester of acetaminophen that was developed initially to increase its solubility for a possible intravenous application and to reduce hepatotoxicity by prolonged release of acetaminophen [,]. After systemic application of propacetamol, it is cleaved by esterase to acetaminophen and N’N’-Diethyl-glycine (). In this study we assessed the effect of N,N-diethylglycine (DEG) on both glycine transporter (GlyT) and glycine receptor (GlyR) function in a Xenopus laevis oocyte expression system by a two-electrode voltage clamp approach. We demonstrate that DEG, but not acetaminophen or propacetamol, functions as a low affinity alternative substrate for both GlyT1 and GlyT2. Moreover, DEG shows partial agonistic activity on homomeric GlyRα1, but not on GlyRα2 or GlyRα3. Taken together, our data raise the possibility that propacetamol, in addition to its well-characterized function as a COX inhibitor via acetaminophen, might influence glycine-dependent neurotransmission via a multimodal mechanism.

2.4. Electrophysiology

All two-electrode-voltage-clamp (TEVC) recordings were performed using a TurboTec-03X amplifier (npi elctronics, Tamm, Germany), and a custom-made recording chamber with the membrane potential set to −50 mV. Only oocytes showing a stable leak current <300 nA at the beginning of the experiment were used. Oocytes were superfused with ND96-Ca2+. Solutions containing substances at the indicated concentration for 20 s followed by a washout for at least 30 s or until stable baseline currents were observed. All measurements were performed at room temperature (21–23 °C). Current traces were evaluated using the software Cell Works and Cell Works Reader (npi electronics, Tamm, Germany). Excel (Microsoft, Redmond, WA, USA) and Graphpad Prism 7 (Graphpad Software, Inc., San Diego, CA, USA) were used for statistical analysis. Data are presented as mean ± SEM in dose-response curves or boxplots with whiskers from minimum to maximum with median and quartiles.

3.2. GlyT Responses to Propacetamol and Paracetamol and DEG

To test if propacetamol or acetaminophen had any effect on the activity of GlyT1 or GlyT2, GlyT-expressing oocytes were superfused with each of these substances. Neither propacetamol nor acetaminophen alone produced any current response. Alkaline pretreatment, however, which is predicted to cause hydrolysis of the propacetamol (10 mM) to acetaminophen and DEG, resulted in significant inward currents in both GlyT1 and GlyT2-expressing oocytes, reaching 20–40% of the maximum current amplitude. Currents of slightly larger amplitudes (50–70%) were observed after the application of 10 mM DEG alone. Adding 20 µM glycine to 10 mM acetaminophen and 10 mM propacetamol solution each did not significantly alter the current response of any GlyT compared to 20 µM glycine solution alone (A,B).