FIGURE LEGENDS
Figure 1. Exocytosis is increased in the presence of varenicline and nicotine in human chromaffin cells. A) Scheme of the triple step protocol in which Cm is recorded in Steps 1 and 3, and the membrane potential in Step 2. Pulses (10 ms) of 300 μM ACh are applied at 0.2 Hz in the second step. Drug perfusion takes place 15 s before and after, and during the 1 min duration of this step. B-E) Representative recordings of the triple step protocol under control conditions, in the presence of varenicline (100 nM), nicotine (250 nM) and both nicotinic agonists together, respectively. F)Cm increments represented as a dot plot graph (mean ± SEM, n=8-13 from six organ donors, 2-tailed Wilcoxon matched-pairs signed rank test ). G) Fold times of Cm increment of each value normalized with respect to its control in each cell (mean ± SEM; n=8-13, 2-tailed Wilcoxon matched-pairs signed rank test ). H) Representative amperometric recordings obtained under control conditions (left panel) and varenicline (100 nM) and nicotine (250 nM) (right panel) perfusion. I) Total charge and number of spikes (J) of the amperometric recordings obtained under control and varenicline plus nicotine conditions (n=5, from one organ donor, no statistical tests could be run ).
Figure 2. Non-α7 nAChRs in rat, bovine, and human adrenal chromaffin cells are mainly of the α3β4* subtype. A-C)Concentration-response curves were generated for a number of antagonists in order to probe for the expression of various nAChR subtypes in rat (A), bovine (B) and human (C) chromaffin cells. The cells were subjected to patch-clamp electrophysiology as described in Methods. Briefly, cells were incubated in extracellular solution containing α-Bgtx (1 μM) for 5 min to irreversibly inhibit the α7 subtype, and then they were placed in the electrophysiology chamber. Cells were stimulated with 200 ms pulses of 300 μM ACh until a steady baseline was achieved, then perfused with extracellular solution containing increasing concentrations of antagonists to determine the pIC50 values. They are provided in Table 1. The responses to ACh were nearly completely inhibited by TxID (1 μM) suggesting that essentially all of the response was mediated by α3β4* nAChRs. The error bars denote the SEM from at least 4 cells for each experimental determination. Data for human adrenal chromaffin cells were adapted from Hone et al., 2015 and Hone et al., 2017. D-F) Concentration-response curves for select nAChR agonists in rat (D), bovine (E) and human (F) chromaffin cells. Adrenal chromaffin cells were stimulated with 500 ms pulses of increasing concentrations of agonists and the data fit to the Hill equation to determine the pEC50 values for activation; potency and efficacy values are provided in Tables 2 and 3. All agonists responses were normalized to the responses evoked by 300 µM ACh obtained in the same cell. The error bars denote the SEM from 3-8 cells. All experiments in rat and bovine adrenal chromaffin cells were performed in cells that had been treated with 1 µM α-Bgtx for 5 min prior to being placed in the electrophysiology chamber. Experiments in human cells were performed in the sustained presence of 100 nM [Leu11,Asp16]ArIB. Dashed lines in the graph of human data indicate data adapted from Hone et al., 2017.
Figure 3. Exocytosis is increased in the presence of varenicline, nicotine and both drugs together in rat chromaffin cells. A-D) Representative recordings of the triple step protocol under control (A), varenicline (100 nM) (B), nicotine (250 nM) (C) and varenicline plus nicotine (D) conditions in rat chromaffin cells.E) Cm increments are represented as a dot plot graph (mean ± SEM, n=16-21 from ten rats, 2-tailed Wilcoxon matched-pairs signed rank test ). F) Fold times of Cm increment of each agonist with respect to its control in each cell (mean ± SEM; n=16-21 from ten rats, 2-tailed Wilcoxon matched-pairs signed rank test ).
Figure 4. The chronic treatment with nicotine does not lead to further increase of exocytosis. Cells were treated during 24 h with nicotine 110 nM and then the different agonists were applied on top of the continuous application of nicotine 110 nM. Representative recordings of the triple step protocol under control (A), varenicline (100 nM) (B), nicotine (250 nM) (C) and varenicline plus nicotine (D) conditions in rat chromaffin cells. E) Cm increments are represented as a dot plot graph graph (mean ± SEM, n=6-8 cells from six rats, 2-tailed Wilcoxon matched-pairs signed rank test ).F) Fold times of Cm increment of each agonist with respect to its control in each cell (mean ± SEM; n=6-8 from six rats, 2-tailed Wilcoxon matched-pairs signed rank test ).
Figure 5. Nicotinic currents and exocytosis elicited by the nicotinic agonists in the absence and presence of ACh in rat chromaffin cells. A ) Representative current traces elicited by 500 ms pulses of ACh (black), varenicline 110 nM (red), 250 nM nicotine (blue), and varenicline and nicotine applied together (grey). B) Dot plot of the currents achieved with the different agonists (mean± SEM, n=10-12 from six rats, 2-tailed paired t-test) .C) Representative Cm recordings corresponding to the currents evoked by the agonists applied in A. D) Dot plot of the Cm increment achieved with the different agonists (mean ± SEM, n=10-12 from six rats, 2-tailed paired test). E) Representative current traces elicited by 500 ms ACh pulses in the presence of the agonists. F) Dot plot of the peak currents achieved by ACh in the presence of varenicline, nicotine or both agonists together (mean ± SEM, n=14-22 from 10 rats, 2-tailed Wilcoxon matched-pairs signed rank test) .