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Fig. 1. Chemical structures of diltiazem and the terpenoids used in this study.
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Fig. 2. Electrophysiological characterization of the actions of citral (Cit), eucalyptol (Euc), and linalool (Lin) at 5-HT3 receptors. (A) Stable currents could be evoked by 10 µM 5-HT applied at 1-minute intervals. (B) At 10 µM 5-HT, stable levels of inhibition by citral, eucalyptol, or linalool were only seen following preapplication but remained stable thereafter [also see (F)]. (C) Following inhibition, responses were slow to recover. For all three compounds, full recovery was achieved after 3 minutes and was independent of the frequency of 5-HT activation during the recovery period. (D) Receptors were inhibited in the closed state as shown by the comparable levels of inhibition seen when the compounds were applied in the absence of 5-HT or during 5-HT application. Inhibition persisted after washout, and the recovery time was unaltered by the timing or frequency of subsequent 5-HT applications. (E) The slow washout of the compounds is highlighted by the absence of a rebound current following the removal of citral, eucalyptol, or linalool. By contrast, rebound was clearly seen following the removal of diltiazem, a channel blocker with faster recovery from inhibition. (F) At a concentration of 300 µM, the oils required 30-second preapplication to stably inhibit the 10 µM 5-HT response. At lower concentrations, the effect of preapplication was less apparent, since the 5-HT response took longer to reach a stable peak current. (G) The level of inhibition at +40 mV or â60 mV was the same for all three compounds.
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Fig. 3. Inhibition of the 5-HT3 receptor by citral, eucalyptol, or linalool. 5-HTâinduced currents were measured at â60 mV in the absence or presence of various concentrations of citral (A), eucalyptol (B), or linalool (C) following a 30-second preapplication. The data were analyzed as described in the text, and the output of this nonlinear mixed-effects modeling is shown in Table 1. The main features of the structural model are illustrated here. The dotted lines show the effect of the compounds on the EC50. The insets show normalized data at the same inhibitor concentrations; for citral and linalool, the apparent shifts in the EC50 seen in this normalized data are artifacts caused by the normalization. Additional data at 10, 30, 300, 600, and 1000 µM citral and 200 and 660 µM eucalyptol were also collected, but the curves are omitted for clarity.
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Fig. 4. Nonlinear mixed-effects modeling. (A) The relationship between observed and predicted peak current values for the best fit model described in Table 1 (n = 532). The values cluster around the line of unity with no apparent systematic deviation, showing that data are well fitted by the model. (B) Modeled Max0 and pEC50,0 parameter values for individual oocytes (n = 55 oocytes). The clear and strong relationship between these parameter estimates (correlation = 0.869 ± 0.054) suggests that oocytes with a high maximum peak current are more sensitive to 5-HT. This relationship is obscured by data normalization.
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Fig. 5. Competition of a fluorescent 5-HT3 receptor competitive antagonist. (A) Competition of 10 nM G-FL with the agonist 5-HT (pIC50 = 6.10 ± 0.03, nH = 2.1, IC50 = 0.79 µM, n = 4) and the competitive antagonist granisetron (pIC50 = 8.42 ± 0.03, nH = 1.1, IC50 = 3.8 nM, n = 6). (B) The test compounds citral (n = 3), eucalyptol (n = 3), and linalool (n = 3) show no competition with G-FL.
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Fig. 6. Inhibition of the 5-HT3 receptor using a dual-application approach. 5-HT3 receptors were activated with a supramaximal (100 µM) concentration of 5-HT. Concentrations of BB, DTZ, and the terpenoids were preselected to inhibit the response by approximately 62% when used alone. Results are shown in the white bars. These same concentrations were then applied in dual combinations, giving results shown in the black bars. The gray bars are the allotopic (allo) and syntopic (syn) predictions based on the levels of inhibition caused by the compounds alone. Data are the mean ± S.E.M. Two-way analysis of variance with Dunnettâs post-hoc test was used (IBM SPSS Statistics 20) to compare actual and predicted dual application responses. (A) Citral (n = 7). For BB, P = 0.019 (analysis of variance), P = 0.95 (H0: BB+citral = allo), P = 0.031 (H0: BB+citral = syn); and for DTZ, P = 0.0005 (analysis of variance), P = 0.42 (H0: DTZ+citral = allo), P = 0.0004 (H0: DTZ+citral = syn). (B) Eucalyptol (n = 6). For BB, P = 0.015 (analysis of variance), P = 0.38 (H0: BB+eucalyptol = allo), P = 0.009 (H0: BB+eucalyptol = syn); and for DTZ, P = 0.00008 (analysis of variance), P = 0.013 (H0: DTZ+eucalyptol = allo), P = 0.00004 (H0: DTZ+eucalyptol = syn). (C) Linalool (n = 5). For BB, P = 0.0006 (analysis of variance), P = 0.023 (H0: BB+linalool = allo), P = 0.0004 (H0: BB+linalool = syn); and for DTZ, P = 0.003 (analysis of variance), P = 0.056 (H0: DTZ+linalool = allo), P = 0.002 (H0: DTZ+linalool = syn). In all experiments, stable levels of inhibition were achieved by applying the compounds for 1 minute before 5-HT was added.
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Fig. 7. Predicted binding locations for citral, eucalyptol, and linalool in a homology model of the human 5-HT3 receptor. Using a loosely defined binding site (see Materials and Methods), 10 docked poses were generated for each ligand. Examples from the docked pose clusters (sphere representation) are shown at each of the predicted binding sites. The transmembrane domains of the five subunits that form the functional 5-HT3 receptor are shown as different colored ribbons viewed from the extracellular side, with both the intracellular and extracellular domains removed for clarity. (A) A single binding site was predicted for linalool with the docked pose cluster differing by only 2.29 Ã
root-mean-square deviation. The white arrowhead indicates the origin from which (D) is viewed. (B) Two sites were predicted for eucalyptol, but were similarly located at the boundaries of adjacent subunits. (C) For citral, two potential binding sites were identified, a major (6/10) site at the interface of adjacent subunits and a minor site located at the lipid-exposed interface at the intracellular ends of M1 and M4. (D) In four of the 10 docked poses for linalool, PyMol 1.3 predicted hydrogen bonds (blue dotted line) between the ligandâs terminal hydroxyl and the backbone of the channel-lining 6' Thr residue.
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Fig. 8. The effects of OELa. (A) Concentration inhibition of 2 µM 5-HTâinduced currents by OELa in Xenopus oocytes expressing the 5-HT3 receptor (n = 5). (B) Rat tracheal contraction in response to 5-HT in the absence (â¾, n = 6) and following a 10-minute preapplication with 10 µM granisetron (â½, n = 3). (C) Concentration-inhibition of the 10 µM 5-HTâevoked tracheal contractile response by OELa (n = 4). (D) Inhibition by OELa of 10 µM 5-HTâevoked guinea pig ileum contractions (n = 6). (E). Example recordings from 5-HTâevoked (black bars) contractions of guinea pig ileum and their inhibition by 30, 60, and 100 µg mlâ1 OELa. At concentrations â¥60 µg mlâ1 OELa, the 5-HTâevoked contractions became increasingly slow to recover. Following a control 5-HT response (0.6 µM, black bar), the addition of 100 µg mlâ1 OELa continued to inhibit the 5-HTâevoked contractions 50 minutes later, although an acetylcholine (1 µM, gray bar) response was unaltered. Parameters defining the curves are shown in the text.
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