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Agonist and antagonist effects of tobacco-related nitrosamines on human α4β2 nicotinic acetylcholine receptors.

Brusco S, Ambrosi P, Meneghini S, Becchetti A - Front Pharmacol (2015)

Bottom Line: However, the functional effects of these drugs on specific nAChR subtypes are largely unknown.The effects of both NNK and NNN were mainly competitive and largely independent of Vm.The different actions of NNN and NNK must be taken into account when interpreting their biological effects in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Biosciences, University of Milano-Bicocca Milano, Italy.

ABSTRACT
Regulation of the "neuronal" nicotinic acetylcholine receptors (nAChRs) is implicated in both tobacco addiction and smoking-dependent tumor promotion. Some of these effects are caused by the tobacco-derived N-nitrosamines, which are carcinogenic compounds that avidly bind to nAChRs. However, the functional effects of these drugs on specific nAChR subtypes are largely unknown. By using patch-clamp methods, we tested 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) on human α4β2 nAChRs. These latter are widely distributed in the mammalian brain and are also frequently expressed outside the nervous system. NNK behaved as a partial agonist, with an apparent EC50 of 16.7 μM. At 100 μM, it activated 16% of the maximal current activated by nicotine. When NNK was co-applied with nicotine, it potentiated the currents elicited by nicotine concentrations ≤ 100 nM. At higher concentrations of nicotine, NNK always inhibited the α4β2 nAChR. In contrast, NNN was a pure inhibitor of this nAChR subtype, with IC50 of approximately 1 nM in the presence of 10 μM nicotine. The effects of both NNK and NNN were mainly competitive and largely independent of Vm. The different actions of NNN and NNK must be taken into account when interpreting their biological effects in vitro and in vivo.

No MeSH data available.


Related in: MedlinePlus

NNN inhibits α4β2 nAChRs. (A) Typical whole-cell current traces elicited by 10 μM nicotine at −80 mV, in the presence and in the absence of 10 μM NNN. Horizontal bars mark time of application of the indicated compound. (B) Steady state inhibition curves were generated by plotting the residual fractional steady state currents as a function of NNN concentration (in Log10 scale). The different data sets were obtained at the indicated concentration of nicotine. Data points are averages of at least nine determinations. Lines through the data points are best fitting curves, obtained with Equation 3. At 100 μM nicotine, IC50 was > 20 μM, whereas at 10 μM nicotine IC50 was 0.21 ± 0.4 nM. (C) Activation curves in the absence (black squares) or in the presence of the indicated concentration of NNN. Data points are peak whole-cell currents normalized to the current elicited by, 100 μM nicotine. Continuous lines through the data points are best fitting curves, obtained with Equation 1. The corresponding parameters were: EC50 = 14.5 ± 1.34 μM (nH = 0.83), for nicotine alone; EC50 = 58.3 ± 4.4 μM (nH = 0.92), in the presence of 1 nM NNN; EC50 = 109.1 ± 0.14 μM (nH = 1), in the presence of 1 μM NNN.
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Figure 4: NNN inhibits α4β2 nAChRs. (A) Typical whole-cell current traces elicited by 10 μM nicotine at −80 mV, in the presence and in the absence of 10 μM NNN. Horizontal bars mark time of application of the indicated compound. (B) Steady state inhibition curves were generated by plotting the residual fractional steady state currents as a function of NNN concentration (in Log10 scale). The different data sets were obtained at the indicated concentration of nicotine. Data points are averages of at least nine determinations. Lines through the data points are best fitting curves, obtained with Equation 3. At 100 μM nicotine, IC50 was > 20 μM, whereas at 10 μM nicotine IC50 was 0.21 ± 0.4 nM. (C) Activation curves in the absence (black squares) or in the presence of the indicated concentration of NNN. Data points are peak whole-cell currents normalized to the current elicited by, 100 μM nicotine. Continuous lines through the data points are best fitting curves, obtained with Equation 1. The corresponding parameters were: EC50 = 14.5 ± 1.34 μM (nH = 0.83), for nicotine alone; EC50 = 58.3 ± 4.4 μM (nH = 0.92), in the presence of 1 nM NNN; EC50 = 109.1 ± 0.14 μM (nH = 1), in the presence of 1 μM NNN.

Mentions: Differently from NNK, NNN did not elicit any whole-cell current even at concentrations (100 μM) much higher than those encountered in physiological conditions. Figure 2D shows a typical experiment at−80 mV, comparing the effects of 10 μM nicotine and 10 μM NNN, in a cell expressing a large nicotinic current. Once again, nicotine was applied before and after NNN, to exclude channel rundown. Given that NNN produced no nAChR activation, we studied its possible inhibitory effect in the presence of nicotine. We tested NNN concentrations between 1 pM and 100 μM on currents activated by concentrations of nicotine ranging between 10 nM and 100 μM. A representative current trace is shown in Figure 4A, in which 10 μM NNN was applied in the presence of 10 μM nicotine, at −80 mV. NNN was generally applied until the effect had reached the steady state. Next, the drug was removed. After the current had recovered from inhibition, nicotine was also washed out. This experimental procedure (Buisson et al., 1996; Palma et al., 1996) was preferred to the alternative procedure of pre-conditioning with NNN and then applying simultaneously the agonist and the antagonist (as we did with DHβE). The former method allows to directly compare the effect of NNN with the one produced by nicotine. In this way, the possible artifacts occurring in repetitive consecutive trials (such as channel rundown, poor solution exchange, precise estimation of the current peak, etc.) are avoided or immediately recognized. Moreover, the blockade kinetics can be directly appreciated. For each concentration, the steady state current in the presence of NNN was divided by the current remaining after NNN was removed. These fractional currents were plotted in Figure 4B, as a function of the NNN concentration, for the indicated nicotine concentrations (0.5, 10, and 100 μM). Notice that higher nicotine concentrations decreased the inhibitory effect of NNN, which suggests a competitive blocking mechanism. For instance, the IC50 value for NNN was >10 μM in the presence of 100 μM nicotine, whereas it was approximately 1 nM in the presence of 10 μM nicotine (full statistics are reported in the figure legend). Conversely, the concentration-response relations for nicotine in the presence of the indicated NNN concentrations are plotted in Figure 4C. Data points were fitted with Equation 1 (continuous lines through the data points). The right-shift of the curves produced by NNN is consistent with the notion that this drug tends to produce competitive block of α4β2 nAChRs. From a pathological point of view, it is worth noticing that NNN can exert significant current block at concentrations normally encountered in smokers' plasma (<100 pM; Schuller, 2007).


Agonist and antagonist effects of tobacco-related nitrosamines on human α4β2 nicotinic acetylcholine receptors.

Brusco S, Ambrosi P, Meneghini S, Becchetti A - Front Pharmacol (2015)

NNN inhibits α4β2 nAChRs. (A) Typical whole-cell current traces elicited by 10 μM nicotine at −80 mV, in the presence and in the absence of 10 μM NNN. Horizontal bars mark time of application of the indicated compound. (B) Steady state inhibition curves were generated by plotting the residual fractional steady state currents as a function of NNN concentration (in Log10 scale). The different data sets were obtained at the indicated concentration of nicotine. Data points are averages of at least nine determinations. Lines through the data points are best fitting curves, obtained with Equation 3. At 100 μM nicotine, IC50 was > 20 μM, whereas at 10 μM nicotine IC50 was 0.21 ± 0.4 nM. (C) Activation curves in the absence (black squares) or in the presence of the indicated concentration of NNN. Data points are peak whole-cell currents normalized to the current elicited by, 100 μM nicotine. Continuous lines through the data points are best fitting curves, obtained with Equation 1. The corresponding parameters were: EC50 = 14.5 ± 1.34 μM (nH = 0.83), for nicotine alone; EC50 = 58.3 ± 4.4 μM (nH = 0.92), in the presence of 1 nM NNN; EC50 = 109.1 ± 0.14 μM (nH = 1), in the presence of 1 μM NNN.
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Figure 4: NNN inhibits α4β2 nAChRs. (A) Typical whole-cell current traces elicited by 10 μM nicotine at −80 mV, in the presence and in the absence of 10 μM NNN. Horizontal bars mark time of application of the indicated compound. (B) Steady state inhibition curves were generated by plotting the residual fractional steady state currents as a function of NNN concentration (in Log10 scale). The different data sets were obtained at the indicated concentration of nicotine. Data points are averages of at least nine determinations. Lines through the data points are best fitting curves, obtained with Equation 3. At 100 μM nicotine, IC50 was > 20 μM, whereas at 10 μM nicotine IC50 was 0.21 ± 0.4 nM. (C) Activation curves in the absence (black squares) or in the presence of the indicated concentration of NNN. Data points are peak whole-cell currents normalized to the current elicited by, 100 μM nicotine. Continuous lines through the data points are best fitting curves, obtained with Equation 1. The corresponding parameters were: EC50 = 14.5 ± 1.34 μM (nH = 0.83), for nicotine alone; EC50 = 58.3 ± 4.4 μM (nH = 0.92), in the presence of 1 nM NNN; EC50 = 109.1 ± 0.14 μM (nH = 1), in the presence of 1 μM NNN.
Mentions: Differently from NNK, NNN did not elicit any whole-cell current even at concentrations (100 μM) much higher than those encountered in physiological conditions. Figure 2D shows a typical experiment at−80 mV, comparing the effects of 10 μM nicotine and 10 μM NNN, in a cell expressing a large nicotinic current. Once again, nicotine was applied before and after NNN, to exclude channel rundown. Given that NNN produced no nAChR activation, we studied its possible inhibitory effect in the presence of nicotine. We tested NNN concentrations between 1 pM and 100 μM on currents activated by concentrations of nicotine ranging between 10 nM and 100 μM. A representative current trace is shown in Figure 4A, in which 10 μM NNN was applied in the presence of 10 μM nicotine, at −80 mV. NNN was generally applied until the effect had reached the steady state. Next, the drug was removed. After the current had recovered from inhibition, nicotine was also washed out. This experimental procedure (Buisson et al., 1996; Palma et al., 1996) was preferred to the alternative procedure of pre-conditioning with NNN and then applying simultaneously the agonist and the antagonist (as we did with DHβE). The former method allows to directly compare the effect of NNN with the one produced by nicotine. In this way, the possible artifacts occurring in repetitive consecutive trials (such as channel rundown, poor solution exchange, precise estimation of the current peak, etc.) are avoided or immediately recognized. Moreover, the blockade kinetics can be directly appreciated. For each concentration, the steady state current in the presence of NNN was divided by the current remaining after NNN was removed. These fractional currents were plotted in Figure 4B, as a function of the NNN concentration, for the indicated nicotine concentrations (0.5, 10, and 100 μM). Notice that higher nicotine concentrations decreased the inhibitory effect of NNN, which suggests a competitive blocking mechanism. For instance, the IC50 value for NNN was >10 μM in the presence of 100 μM nicotine, whereas it was approximately 1 nM in the presence of 10 μM nicotine (full statistics are reported in the figure legend). Conversely, the concentration-response relations for nicotine in the presence of the indicated NNN concentrations are plotted in Figure 4C. Data points were fitted with Equation 1 (continuous lines through the data points). The right-shift of the curves produced by NNN is consistent with the notion that this drug tends to produce competitive block of α4β2 nAChRs. From a pathological point of view, it is worth noticing that NNN can exert significant current block at concentrations normally encountered in smokers' plasma (<100 pM; Schuller, 2007).

Bottom Line: However, the functional effects of these drugs on specific nAChR subtypes are largely unknown.The effects of both NNK and NNN were mainly competitive and largely independent of Vm.The different actions of NNN and NNK must be taken into account when interpreting their biological effects in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Biosciences, University of Milano-Bicocca Milano, Italy.

ABSTRACT
Regulation of the "neuronal" nicotinic acetylcholine receptors (nAChRs) is implicated in both tobacco addiction and smoking-dependent tumor promotion. Some of these effects are caused by the tobacco-derived N-nitrosamines, which are carcinogenic compounds that avidly bind to nAChRs. However, the functional effects of these drugs on specific nAChR subtypes are largely unknown. By using patch-clamp methods, we tested 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) on human α4β2 nAChRs. These latter are widely distributed in the mammalian brain and are also frequently expressed outside the nervous system. NNK behaved as a partial agonist, with an apparent EC50 of 16.7 μM. At 100 μM, it activated 16% of the maximal current activated by nicotine. When NNK was co-applied with nicotine, it potentiated the currents elicited by nicotine concentrations ≤ 100 nM. At higher concentrations of nicotine, NNK always inhibited the α4β2 nAChR. In contrast, NNN was a pure inhibitor of this nAChR subtype, with IC50 of approximately 1 nM in the presence of 10 μM nicotine. The effects of both NNK and NNN were mainly competitive and largely independent of Vm. The different actions of NNN and NNK must be taken into account when interpreting their biological effects in vitro and in vivo.

No MeSH data available.


Related in: MedlinePlus