DISCUSSION
Varenicline is a drug widely used in smoking-cessation therapy. It inhibits the reinforcing effects of nicotine and reduces craving and withdrawal symptoms by modulating dopaminergic function through partial agonism of α4β2* nAChRs (Coe, Brooks et al. , 2005; Gonzales, Rennard et al. , 2006; Jorenby, Hays et al. , 2006; Rollema, Chambers et al. , 2007). However, varenicline therapy is associated with increased frequency of some cardiovascular side effects such as heart attack. Because the drug acts also on α3β4* and α7 nAChRs, the main receptors expressed in human chromaffin cells (Pérez-Alvarez, Hernández-Vivanco et al. , 2012a; Hone, McIntosh et al. , 2015), it is possible that varenicline potentiates the release of catecholamines from the adrenal gland, contributing to those undesirable effects. Two previous studies support this idea. First, therapeutic concentrations of varenicline (100 nM) increase the firing of action potentials in chromaffin cells in the presence of nicotine (Hone, McIntosh et al. , 2017). Second, a case of varenicline-triggered pheochromocytoma crisis in a smoking subject when the patient took the second tablet of varenicline the next day (Hukkanen, Ukkola et al. , 2010). Varenicline is 10-fold more potent and a full agonist relative to ACh, and nicotine is 2-fold more potent and behaved as a partial agonist with respect to ACh (Rollema, Chambers et al., 2007; Hone et al. , 2017). This might explain why varenicline could trigger pheochromocytoma crisis in the case report, whereas nicotine derived from smoking did not (Hukkanen, Ukkola et al. , 2010). Moreover, the patient of the case report had no other known crisis triggers, and she had a documented visit to the emergency department for palpitations six years before the admission, suggesting that she may have suffered pheochromocytoma for years without crisis. Furthermore, two 0.5 mg doses of varenicline results in plasma concentrations of about 75 nM (Kikkawa, Maruyama et al. , 2011), which increases the electrical activity when nicotine is present (Hone, McIntosh et al. , 2017) and the exocytosis and catecholamine release in human chromaffin cells (present study), something that happens in a smoker that begin smoking cessation therapy.
In this study, we confirmed our hypothesis by measuring Cm as an index of exocytosis elicited by very short pulses of ACh applied at 0.2 Hz in the presence of varenicline, nicotine or both drugs together. We determined that varenicline, at concentrations that could be achieved in smoking cessation therapy, increased exocytosis in human chromaffin cells obtained from organ donors when it was perfused together with nicotine at concentrations that would be achieved in the plasma after smoking, or/and during chronic consumption of tobacco. Furthermore, this enhanced exocytosis corresponds to an increase in the release of catecholamines as determined by carbon-fiber amperometry. Although these last data were obtained from only a single donor, it shows a tendency to increase catecholamine release by varenicline and nicotine. This is shown by the rise in the total charge and the number of amperometric spikes elicited by the application of both drugs (each spike reflects the content of a single secretory vesicle being released as shown by Wightman and colleagues, 1991).
Next we wanted to investigate whether chronic administration of nicotine (in order to mimic the situation of a smoker) could enhance the exocytosis elicited by varenicline. Also, we wanted to know whether varenicline and nicotine could be increasing the nicotinic currents, explaining the increase in exocytosis. However, the COVID-19 pandemic severely limited the number of organ transplants preventing the continuation of this study in human cells. Therefore, we aimed at investigating these issues in non-human species such as rat or bovine. We first characterized the non-α7 nAChRs in these species using a panel of agonist and antagonist ligands. We performed experiments with different nAChR antagonists that confirmed the presence of an α3β4* nAChR subtype in the three species. Also, the DHβE and the PeIA analog responses suggest very few α4- and α6- containing receptors in the three species. Thus, as the differences found were not large among these species, we performed dose-response curves with different agonists that would help us to determine which species was the most similar to the human in relation to the nicotinic response. We found that the α3β4* nAChR subtypes of the three species exhibited also similar sensitivity to agonists. Therefore, we chose the rat species to continue with the experiments due to its greater availability.
We first performed the same protocol in rat chromaffin cells to that conducted in human chromaffin cells, to determine the effects of the drugs on exocytosis. We found that varenicline in the presence of nicotine, as well as varenicline and nicotine separately, increased exocytosis. This might be due to the fact that young rats were used in the experiments (one month and a half), while the experiments in human were performed in 50-70 years old subjects. Chronic treatment with nicotine at a concentration achieved in plasma by smokers (110 nM) (Faessel, Gibbs et al. , 2006) resulted in an increase in the exocytosis elicited with varenicline or nicotine applied independently or together.
On the other hand, varenicline elicited smaller currents than ACh at the concentrations used in the present study (100 nM varenicline, 300 µM ACh), which did not correspond with an increase in exocytosis. Neither the effect of varenicline or nicotine perfusion while pulses of ACh were applied can explain the increase in exocytosis obtained, because these agonists inhibited the ACh-evoked currents. Further research will enable understanding of the mechanisms by which these agonists increased the exocytotic response and whether those processes depend on age.
In summary, the present study reports that the combination of varenicline and nicotine increases the exocytosis of chromaffin vesicles and the release of catecholamines in human chromaffin cells. Exocytosis was also increased in rat chromaffin cells. This finding might help to explain the increased number of heart attacks reported by the FDA (2011) in subjects taking varenicline.