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) .