RESULTS
In the present study, we investigated the effect of varenicline, a drug
successfully used in the treatment of tobacco addiction, on the
exocytosis elicited by ACh in human chromaffin cells obtained from organ
donors. At the beginning of the treatment with varenicline, patients
often continue smoking for at least 2 weeks. Thus, here we investigated
the effect of 100 nM varenicline alone and in combination with nicotine
(250 nM or 110 nM nicotine were used to investigate its acute and
chronic consumption, respectively) on the exocytotic process that leads
to catecholamine secretion. The concentration of varenicline (100 nM)
was the maximum plasma concentration achieved in patients consuming a
therapeutic dose of 1 mg twice a day (Faessel, Gibbs et al. ,
2006; Ravva, Gastonguay et al. , 2009; Kikkawa, Maruyama et
al. , 2011). Nicotine at 250 nM was the arterial plasma concentration
achieved the first 10 min after smoking 1-3 cigarettes, one puff/min for
10 min (Gourlay & Benowitz, 1997). The chronic treatment of nicotine
was performed using 110 nM concentration of the drug because this was
the average steady-state plasma concentration achieved by healthy adult
smokers (Faessel, Gibbs et al. , 2006).
In this study, a modification of the triple-step protocol reported by
Pérez‐Alvarez & Albillos (2007) was employed to measure the
agonist-induced exocytosis by means of Cm changes. This
method combines the voltage-clamp and the current-clamp configurations
of the patch-clamp technique. First, the potential of the cell was fixed
at -60 mV in human cells and at -55 mV in rat cells. The initial
Cm was recorded in the voltage-clamp configuration (Step
1) for 60 seconds. Then by switching to the current-clamp configuration,
11 pulses (10 ms) of 300 μM ACh at 0.2 Hz were applied to closer mimic
the physiological stimulation in which the ACh released by the
splanchnic nerve basally stimulates chromaffin cells (Hone, McIntoshet al. , 2017) (Step 2). Drugs were perfused 15 s before, during
and 15 s after the current-clamp part of the protocol. Finally, by
returning to the voltage-clamp mode, the increase produced in
Cm could be recorded, reflecting the overall exocytosis
evoked by the agonist (Step 3) (Fig. 1A).