Exocytosis elicited by ACh was increased by varenicline and nicotine in human chromaffin cells
Exocytosis was determined as the difference in the Cmvalues obtained between the Steps 3 and 1 in human chromaffin cells obtained from adrenal glands of six donors (three males and three females). Representative recordings obtained under control conditions (Fig. 1B), or after the different treatments with 100 nM varenicline or 250 nM nicotine applied separately (Fig. 1C and D, respectively), or both together (Fig. 1E), are displayed. Absolute average values and the corresponding normalized values with respect to matched controls are plotted in Fig. 1F and G, respectively. The exocytosis achieved in control cells was 121.2 ± 31.6 fF (n=13) and ranged from 17.5 fF to 437 fF. Varenicline alone evoked 119.3 ± 44.9 fF (n=10) and nicotine elicited 124.2 ± 40.4 fF (n=8) which represented a 1 ± 0.2 and 1.1 ± 0.4-fold increment with respect to the matched control values, respectively (p>0.05). However, 100 nM varenicline in the presence of 250 nM nicotine triggered a Cm increase of 371.7 ± 118 fF (n=13; *p). These values were normalized, and represented a 3.3 ± 0.9-fold increase with respect to the control condition (*p).
The membrane potential of the cells did not change after perfusing the different drugs. Initially under control conditions it exhibited a value of -57.8 mV ± 1.4 mV (n=13). Then it amounted to -58.3 ± 5.1 mV (n=10), -58.5 ± 1 mV (n=8), and -53.3 ± 2.3 mV (n=13) with varenicline and nicotine alone, and in the presence of both drugs, respectively.
Additionally, a series of experiments to determine the release of catecholamines triggered by varenicline (100 nM) and nicotine (250 nM) was performed. Amperometric carbon fiber electrodes were used in human chromaffin cells obtained from one donor (26 year old, female). The protocol consisted of 11 pulses of 300 μM ACh applied at 0.2 Hz. Drugs were perfused 15 s before, during and 15 s after the ACh pulses were applied. Representative original recordings of the amperometric spikes recorded under control and nicotinic stimulation conditions are shown in Fig. 1H. The total charge of the catecholamines released by varenicline in the presence of nicotine (Fig. 1I), and the number of spikes triggered (Fig. 1J), showed a tendency to increase with respect to the control condition.