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).