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.