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.