|
|
|
Fig. 1. Chemical structures of monoterpenoid compounds used in the present study.
|
|
Fig. 2. Agonist effects of monoterpenoid compounds on levamisole sensitive O. dentatum nAChR. A. Sample traces for the agonist experiment. B. Bar chart (expressed as mean ± SEM%) showing agonistic effects of the monoterpenoid compounds: menthol (6.5 ± 2.4%, n = 6), geraniol (0.9 ± 0.2%, n = 6), cinnamaldehyde (0.6 ± 0.2%, n = 6), citronellol (0.4 ± 0.2%, n = 6), menthyl acetate (0.3 ± 0.1%, n = 6), eugenol (0.3 ± 0.2%, n = 6), phenethyl propionate (0.3 ± 0.1%, n = 6), limonene oxide (0.3 ± 0.2%, n = 6), carvacrol (0.2 ± 0.2%, n = 6), menthone (0.2 ± 0.1%, n = 6), pulegone (0.1 ± 0.1%, n = 6) and carvone (0.0 ± 0.0%, n = 6).
|
|
Fig. 3. Effect of monoterpenoid compounds as antagonists on levamisole sensitive O. dentatum receptor acetylcholine mediated responses. A. Bar graph showing the rank order potency of monoterpenoid compounds as antagonists. Results were expressed as % mean inhibition ± SEM of currents elicited by 100 μM ACh: limonene oxide (36.0 ± 3.2%, n = 6) > citronellol (18.0 ± 1.6%, n = 6) > carvone (14.0 ± 0.6%, n = 6) > carvacrol (11.0 ± 1.6%, n = 6) = pulegone (11.0 ± 2.6%, n = 6) = eugenol (11.0 ± 1.0%, n = 6). Inset: image showing predicted acetylcholine response in the absence of limonene oxide (dotted line) and inhibition of acetylcholine mediated response in the presence of limonene oxide (highlighted in green). B. Acetylcholine current responses, recorded from the Xenopus oocyte expressing the levamisole sensitive channel, alone and in the presence of 100 μM carvacrol (n = 6) & 100 μM limonene oxide (n = 5). C. Concentration-response plots for acetylcholine alone (n = 6, black) and acetylcholine in the presence of 100 μM carvacrol (n = 6, maroon) & 100 μM limonene oxide (n = 5, green). pEC50 (mean ± SEM), EC50 (mean, μM), Hill slope (nH) (mean ± SEM) and Imax (mean ± SEM%) values were respectively: 5.3 ± 0.0, 5.3 μM, 2.0 ± 0.2 and 104.6 ± 1.6 for acetylcholine alone; 5.4 ± 0.2, 4.0 μM, 2.2 ± 1.7 and 62.8 ± 7.0 in the presence of limonene oxide; 5.7 ± 0.1, 2.0 μM, 1.7 ± 1.1 and 74.1 ± 3.2 in the presence of carvacrol. Bottom was constrained to zero for curve fitting. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
|
|
Fig. 4. Effects of menthol as a PAM on levamisole sensitive O. dentatum nAChR acetylcholine responses. A. Sample traces for two-electrode voltage-clamp recording showing inward currents in response to ascending concentrations of acetylcholine alone, acetylcholine in the presence of 0.1 μM and 10 μM menthol. B. Concentration-response relationships for acetylcholine alone (n = 7, black), acetylcholine in the presence of 0.1 μM (n = 6, blue) and 10 μM menthol (n = 7, purple). pEC50 (mean ± SEM), EC50 (mean, μM), Hill slope (nH) (mean ± SEM) and Imax (mean ± SEM%) values were respectively: 5.3 ± 0.0, 5.0 μM, 1.4 ± 0.1 and 107.5 ± 2.6 for acetylcholine alone; 6.4 ± 0.1, 0.4 μM, 1.0 ± 0.1 and 109.0 ± 3.6 in the presence of 0.1 μM menthol; 6.5 ± 0.1, 0.3 μM, 1.0 ± 0.1 and 110.5 ± 3.7 in the presence of 10 μM menthol. Bottom was constrained to zero for curve fitting. C. Bar chart showing comparison of pEC50 (expressed as mean ± SEM) for acetylcholine in the presence and absence of menthol. ****P < 0.0001; significantly different as indicated; extra sum of squares F-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
|
|
Fig. 5. Effects of menthol on levamisole sensitive nAChR levamisole responses. A. Representative current traces for two-electrode voltage-clamp recording showing inward currents in response to ascending application of levamisole alone, levamisole in the presence of 0.1 μM and 10 μM menthol. B. Concentration-response relationships for levamisole alone (n = 7, steel gray) and in the presence of 0.1 μM (n = 6, light green) and 10 μM menthol (n = 7, dark green). pEC50 (mean ± SEM), EC50 (mean, μM), Hill slope (nH) (mean ± SEM) and Imax (mean ± SEM%) values were respectively: 6.3 ± 0.1, 0.5 μM, 1.0 ± 0.2 and 96.3 ± 5.2 for levamisole alone; 6.5 ± 0.1, 0.3 μM, 1.1 ± 0.2 and 94.6 ± 5.0 in the presence of 0.1 μM menthol; 6.8 ± 0.2, 0.2 μM, 1.0 ± 0.2 and 89.2 ± 6.0 in the presence of 10 μM menthol. Bottom was constrained to zero for curve fitting. C. Bar chart summarizing the results showing comparison of pEC50 (expressed as mean ± SEM) for levamisole in the presence and absence of menthol. *P < 0.05; significantly different as indicated; extra sum of squares F-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
|
|
Fig. 6. Effects of menthol and carvacrol on nicotine sensitive A. suum ACR-16 nAChR acetylcholine responses. A. Representative current traces for two-electrode voltage-clamp recording showing inward currents in response to increasing acetylcholine concentrations in the presence of menthol (10 μM) and carvacrol (10 μM and 100 μM) B. Concentration-response plot for acetylcholine alone (n = 7, black) and acetylcholine in the presence of menthol (n = 6, red). pEC50 (mean ± SEM) and EC50 (mean, μM) values were respectively: 5.0 ± 0.1 and 11.1 μM for acetylcholine alone, 5.0 ± 0.1 and 9.5 μM in the presence of 10 μM menthol. Hill slope (nH) (mean ± SEM) values were: 1.6 ± 0.3 for acetylcholine alone; 2.5 ± 1.2 in the presence of 10 μM menthol. Imax (mean ± SEM%) values were: 109.1 ± 6.6 for acetylcholine alone; 101.6 ± 7.5 in the presence of 10 μM menthol. Bottom was constrained to zero for curve fitting. C. Concentration-response plots for acetylcholine alone (n = 7, black) and in the presence of carvacrol (n = 6, blue). pEC50 (mean ± SEM), EC50 (mean, μM), Hill slope (nH) (mean ± SEM) and Imax (mean ± SEM%) values were respectively: 5.1 ± 0.1, 8.1 μM, 2.5 ± 0.8 and 94.6 ± 5.9 in the presence of 10 μM carvacrol; 5.1 ± 0.1, 8.2 μM, 2.0 ± 0.7 and 55.8 ± 4.4 in the presence of 100 μM carvacrol. Bottom was constrained to zero for curve fitting. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
|
|
Fig. 7. A. Sample trace for the A. suum muscle flap contraction experiments. Inset: concentration-response plots for acetylcholine alone (n = 6, black) and in the presence of 0.1 μM menthol (n = 6, red). pEC50 (mean ± SEM) and EC50 (mean, μM) values were respectively: 5.2 ± 0.2 and 7.0 μM for acetylcholine alone; 5.2 ± 0.2 and 6.5 μM in the presence of 0.1 μM menthol. Rmax (mean ± SEM, g) values were: 1.7 ± 0.2 g for acetylcholine alone and 2.0 ± 0.2 g in the presence of 0.1 μM menthol. Hill slope (nH) (mean ± SEM) values were: 0.8 ± 0.2 for acetylcholine alone and 0.8 ± 0.1 in the presence of 0.1 μM menthol. Bottom was constrained to zero for curve fitting. B. Bar chart summarizing the results showing significant potentiation in amplitude of acetylcholine mediated contractions in the presence of 0.1 μM menthol. *P < 0.05, ***P < 0.001; significantly different as indicated; paired t-tests. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
|
