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. 2016 Jul;173(14):2263-77.
doi: 10.1111/bph.13507. Epub 2016 Jun 6.

Functional Modulation of Glycine Receptors by the Alkaloid Gelsemine

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Functional Modulation of Glycine Receptors by the Alkaloid Gelsemine

Cesar O Lara et al. Br J Pharmacol. .
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Abstract

Background and purpose: Gelsemine is one of the principal alkaloids produced by the Gelsemium genus of plants belonging to the Loganiaceae family. The extracts of these plants have been used for many years, for a variety of medicinal purposes. Coincidentally, recent studies have shown that gelsemine exerts anxiolytic and analgesic effects on behavioural models. Several lines of evidence have suggested that these beneficial actions were dependent on glycine receptors, which are inhibitory neurotransmitter-gated ion channels of the CNS. However, it is currently unknown whether gelsemine can directly modulate the function of glycine receptors.

Experimental approach: We examined the functional effects of gelsemine on glycine receptors expressed in transfected HEK293 cells and in cultured spinal neurons by electrophysiological techniques.

Key results: Gelsemine directly modulated recombinant and native glycine receptors and exerted conformation-specific and subunit-selective effects. Gelsemine modulation was voltage-independent and was associated with differential changes in the apparent affinity for glycine and in the open probability of the ion channel. In addition, the alkaloid preferentially targeted glycine receptors in spinal neurons and showed only minor effects on GABAA and AMPA receptors. Furthermore, gelsemine significantly diminished the frequency of glycinergic and glutamatergic synaptic events without altering the amplitude.

Conclusions and implications: Our results provide a pharmacological basis to explain, at least in part, the glycine receptor-dependent, beneficial and toxic effects of gelsemine in animals and humans. In addition, the pharmacological profile of gelsemine may open new approaches to the development of subunit-selective modulators of glycine receptors.

Figures

Figure 1
Figure 1
Modulation of recombinant glycine receptors (GlyR) by gelsemine. (A). Whole‐cell current traces evoked by 15–20 μM glycine before and after the application of gelsemine (10 μM or 50 μM) to HEK293 cells expressing α1GlyRs or α1βGlyRs. The graph summarizes the effect of different gelsemine concentrations (0.01–300 μM) on the glycine‐activated currents. (B.) Current traces evoked by 40–60 μM glycine before and after the application of gelsemine (50 μM) to HEK293 cells expressing α2GlyRs or α2βGlyRs. The plot summarizes the effects of different gelsemine concentrations (0.1–300 μM) on the glycine‐activated currents. (C.) Current traces evoked by 50–70 μM glycine before and after the application of gelsemine (50 μM) to HEK293 cells expressing α3GlyRs or α3β GlyRs. The plot summarizes the effects of different gelsemine concentrations (0.1–300 μM) on the glycine‐activated currents. (D). Summary of the gelsemine effects (25 μM) on homomeric and heteromeric GlyRs. *, P < 0.05; significant difference between α1GlyRs and the other receptors studied; ANOVA followed by Bonferroni post hoc test, F(5,55) = 12.37. The values of the gelsemine‐induced inhibition of homomeric and heteromeric GlyRs were not significantly different. Data are means ± SEM. of cells expressing α1(n = 12), α1β(n = 13), α2(n = 8), α2β(n = 10), α3(n = 12) and α3β (n = 8).
Figure 2
Figure 2
Effects of gelsemine on the agonist sensitivity and on the desensitization rates of different glycine receptor (GlyR) subunits. A–C. Concentration–response curves to glycine in the absence and the presence of gelsemine from cells expressing α1 (panel A, n = 12), α2 (panel B, n = 10) or α3 (panel C, n = 13) GlyRs. (D). Examples of current traces from GlyRs using a saturating concentration of glycine (1000–3000 μM) in the absence or presence of gelsemine (10 μM for α1GlyRs and 50 μM for α2/α3GlyRs). (E). The graphs summarize the effects of gelsemine on the percentage of desensitized current and on the decay time constant of the glycine‐evoked currents. The alkaloid did not significantly influence the desensitization rates. Data are means ± SEM. of cells expressing α1(n = 12), α2(n = 10) and α3(n = 13).
Figure 3
Figure 3
The positive and negative effects of gelsemine on the glycine‐evoked currents in different glycine receptor (GlyR) subunits are voltage‐independent. A–C. Voltage–current relationships (I–V plots) for α1 (panel A), α2 (panel B) or α3 (panel C) GlyRs in the absence and presence of gelsemine. The recordings were performed using membrane potentials ranging from −90 to +60 mV with steps of 30 mV. The test concentration of the alkaloid was 10 μM for α1GlyRs and 50 μM for α2/α3GlyRs. (D–F). The plots summarize the mean percentage of potentiation (α1GlyR, panel D) or the mean percentage of inhibition (α2/α3GlyRs, panels E, F) elicited by gelsemine on glycine‐evoked currents recorded at different membrane potentials. Both the positive and negative effects of the alkaloid were not significantly influenced by the membrane potential (ANOVA followed by Bonferroni post hoc test). Data are means ± SEM. of seven cells for each glycine receptor subunit.
Figure 4
Figure 4
Gelsemine effects on the single‐channel activity of glycine receptor (GlyR) α1, α2 and α3 subunits. (A). Single‐channel recordings in the outside‐out configuration from cells expressing α1GlyRs before and during the application of 10 μM of gelsemine. (B, C). The graphs show that gelsemine significantly enhanced the open probability of α1GlyRs, but did not modify the main conductance. * P < 0.05, paired Student t‐test. (D). The bar graph summarizes the percentage change elicited by 10 μM of gelsemine on the single‐channel activity of α1GlyRs. Differences were significant. * P < 0.05; paired Student t‐test. (E). Recordings from cells expressing α2GlyRs before and during the application of 50 μM of gelsemine. (F, G). The plots summarize the effects of gelsemine on the open probability and the main conductance of α2GlyRs. * P < 0.05, paired Student t‐test. (H). The bar graph summarizes the percentage change elicited by 50 μM of gelsemine on the single‐channel activity of α2GlyRs. * P < 0.05; paired Student t‐test. (I). Single‐channel recordings in the cell‐attached configuration from cells expressing α3GlyRs before and in the presence of 50 μM of gelsemine. (J, K). Gelsemine significantly decreased the open probability of α3GlyRs, but did not modify the main conductance. * P < 0.05; paired Student t‐test. (L). The bar graph summarizes the percentage change elicited by 50 μM of gelsemine on the single‐channel activity of α3GlyRs. * P < 0.05; paired Student t‐test. Data were obtained from eight patches in each condition. γ, main conductance; f, frequency; NPo, open probability.
Figure 5
Figure 5
Effects of gelsemine on spinal glycine, GABAA and AMPA receptors. (A). Glycine concentration–response curve obtained from cultured spinal cord neurons (n = 7). (B). Glycine‐activated currents from a spinal neuron in the absence and presence of increasing concentrations of gelsemine. (C). The graph describes the concentration‐dependent inhibition of the neuronal glycine‐evoked current by gelsemine (0.1–300 μM, n = 8). (D). Current traces from spinal glycine receptors (GlyRs) activated by 1000 μM of glycine in the absence or the presence of 50 μM of gelsemine. (E). The graphs summarize the effects of gelsemine on the fraction of desensitized current and on the decay time constant of the glycine‐evoked currents. Differences were not significant. (F). Concentration–response curves to AMPA (n = 8) or GABA (n = 6) from spinal cord neurons. (G). Examples of AMPA or GABA‐activated currents from spinal neurons in the absence and presence of 50 μM of gelsemine. (H). Sensitivity of spinal GABAA and AMPA receptors to gelsemine. Because the two data sets did not fit properly to the Hill equation, the IC50 values were not calculated (n = 8 for both receptors). (I). Summary of the gelsemine effects (50 μM) on spinal glycine, GABAA and AMPA receptors. The effects of gelsemine on GlyRs were significant. * P < 0.05; significant effect of gelsemine;. The gelsemine‐induced effects on glycine, GABAA and AMPA receptors were also significantly different. + P < 0.05; ANOVA followed by Bonferroni post hoc test; F (5,48) = 12.00. Data are means ± SEM. of glycine activated‐currents (vehicle (n = 5) and gelsemine (n = 8)), GABA‐activated currents (vehicle (n = 8) and gelsemine (n = 11)) and AMPA‐activated currents (vehicle (n = 8) and gelsemine (n = 13)) obtained from spinal neurons.
Figure 6
Figure 6
Gelsemine modulation of glycinergic neurotransmission. (A). Examples of current traces showing the glycinergic synaptic activity before and during the application of 50 μM gelsemine from a single spinal neuron. (B). The scatter graph depicts the effect of 50 μM of gelsemine on the frequency of synaptic events in 10 spinal neurons. The alkaloid completely abolished the presence of glycinergic mIPSC (i.e. frequency value equals zero) in 6/10 of the neurons examined. (C). The graph summarizes the effects of 10 and 50 μM of the alkaloid on the average frequency of glycinergic mIPSCs. The average frequency of the glycinergic synaptic events was significantly diminished only by the application of 50 μM of gelsemine. * P < 0.05, significant difference; ANOVA followed by Bonferroni post hoc test; F (2,29) = 5.27. Control (n = 17), 10 μM (n = 8) and 50 μM (n = 10) of gelsemine. (D). Cumulative probability of the inter‐event intervals of glycinergic mIPSCs in the absence or the presence of 50 μM of gelsemine. The distribution was significantly altered by the alkaloid. * P < 0.05; Kolmogorov–Smirnoff test. (E). The bar plot summarizes the effects of 10 and 50 μM on the amplitude of glycinergic mIPSCs. Differences were not significant. (F). Cumulative probability of the amplitudes of glycinergic mIPSCs in the absence or the presence of 50 μM of gelsemine. Differences were not significant. (G). Representative average current traces before and during the application of 50 μM of gelsemine. (H). The bar graphs summarize the effects of gelsemine on the mIPSC kinetics. Differences were not significant; ANOVA followed by Bonferroni post hoc test.
Figure 7
Figure 7
Modulation of glutamatergic neurotransmission by gelsemine. (A). Current traces showing the glutamatergic synaptic activity before and during the application of 50 μM gelsemine. (B). The bar plot summarizes the effects of gelsemine (10–50 μM) on the amplitude of glutamatergic mEPSCs. Differences were not significant. (C). Cumulative probability of the amplitudes of glutamatergic mEPSCs in the absence or the presence of 50 μM of gelsemine. Differences were not significant. (D). The scatter graph depicts the effect of 50 μM of gelsemine on the frequency of glutamatergic events on eight spinal neurons. (E). The graph summarizes the effects of 10 and 50 μM of the alkaloid on the average frequency of glutamatergic mEPSCs. The frequency of the synaptic events was significantly diminished by the application of 50 μM of gelsemine. * P < 0.05; ANOVA followed by Bonferroni post hoc test; (F (2,22) = 4.89. Control (n = 8), 10 μM (n = 5) and 50 μM (n = 8) of gelsemine. (F). Cumulative probability of the inter‐event intervals of glutamatergic synaptic events in the absence or the presence of 50 μM of gelsemine. The distribution was significantly modified by the alkaloid. * P < 0.05; Kolmogorov–Smirnoff test.

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