Rat and bovine chromaffin cells were tested as alternative
species to further investigate the effect of varenicline and nicotine:
antagonists and agonists effects on rat, bovine and human chromaffin
cells
Due to the scarce availability of human cells, we also investigated the
effect of varenicline, nicotine and other nAChR agonists on rat and
bovine chromaffin cells in order to find a non-human species in which
these compounds elicit the most similar results to human. This would
allow further investigation of this and related issues without the
limits of cell availability.
First, we characterized the non-α7 nAChR in these species by testing
different antagonists in cells pretreated with α-bungarotoxin (α-BgTx)
to inhibit α7 nAChR. These compounds were α-conotoxins (α-CTxs) TxID,
[Ala5,Hyp6]BuIA and
[Val7,His9,Ala10,Arg11,Ala14]
PeIA, that target human (Hone et al. , 2015; Hone et al. ,
2017) and rat (Luo et al. , 2013) α3β4, β4*-, and α6*-containing
nAChRs, respectively, and DHβE, that targets human and rat α4β2 and α4β4
nAChRs (Chavez-Noriega, Crona et al. , 1997).
The activities of the antagonists were determined by applying 200 ms
pulses of ACh once every 2 min until a steady baseline response was
achieved. The control solution was then switched to one containing the
antagonist of interest and perfused until a steady state level of
inhibition was observed. The curves obtained for rat and bovine are
shown in Fig. 2A and B. The α-Ctxs
[Ala5,Hyp6]BuIA,
[Val7,His9,Ala10,Arg11,Ala14]
PeIA and TxID were investigated previously in human chromaffin cells in
our lab (Pérez-Alvarez, Hernández-Vivanco et al. , 2012b; Hone,
McIntosh et al ., 2015; Hone, McIntosh et al ., 2017). Data
were plotted here for comparison with the other species (Fig. 2C). The
IC50 values obtained for the various antagonists are
shown in Table 1. In the presence of 1 μM TxID, the ACh-evoked responses
were inhibited by 98.0 ± 0.5% (n=4) in rat (A) and 98.7 ± 0.6% (n=6)
in bovine (B). They were inhibited by 98.8 ± 0.3% in human (Hone,
McIntosh et al ., 2017). Very little inhibition was observed with
DHβE at concentrations expected to inhibit β2-containing nAChRs. These
results indicated that the predominant non-α7 nAChR subtype expressed by
all three species is α3β4*.
In a different set of experiments, increasing concentrations of various
nicotinic agonists (ACh, varenicline, nicotine, DMPP and cytisine) were
tested in chromaffin cells of the three species. The dose-response
curves of the different agonists and the representative recordings of
the nicotinic currents elicited in the three species by them are plotted
in Fig. 2D-F. The data of ACh, varenicline and nicotine were previously
reported in human cells (Hone, McIntosh et al. , 2017), and showed
here for ease of comparison with the other species curves. Agonist
potencies and efficacies are summarized in Tables 2 and 3, respectively.
Potency and efficacy values of ACh, varenicline and nicotine in the
three species do not differ substantially to justify to choose rat or
bovine. Therefore, the greater availability of rats as a model organism
was the final criteria to select this species to perform the rest of the
experiments of this study.