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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

NNK, but not NNN, activates α4β2 nAChRs. (A) Consecutive whole-cell current traces, elicited at -80 mV by the indicated compound. Tests were spaced 2–3 min apart (gaps in the continuous current trace). Continuous bars above the current traces indicate the time of agonist application. Nicotine was repeatedly applied during the experiment to check for possible current rundown. (B) Concentration-response relations for nicotine (black squares) and NNK (red circles). Data points are average peak whole-cell currents recorded at −80 mV, normalized to the current elicited by 10 μM nicotine and plotted against the agonist concentration (in Log10 scale). Each point is the average of at least seven determinations. The concentration-response relation for nicotine (continuous line) and that for NNK (dashed line) were fitted with Equation 1, which gave an EC50 = 27.8 ± 5.6 μM (nH = 0.64) for nicotine and 16.7 ± 20 μM (nH = 0.44) for NNK. The estimated maximal current activated by NNK was approximately 16% of the current elicited by 300 μM nicotine. The measured peak current in the presence of 100 μM NNK was 0.14 ± 0.02 (n = 13) of the value measured in the presence of 300 μM nicotine. (C) DHβE (1 μM) strongly blocked the currents elicited by 300 μM ACh, 300 μM nicotine, and 10 μM NNK, as indicated. Data are averages of at least seven determination for each condition, carried out at −80 mV. Bars display the fractional residual currents (i.e., the peak current measured in the presence of the agonist plus DHβE divided by the peak current activated by the agonist alone). In the presence of 10 μM NNK, DHβE brought the average peak current density (pA/pF) from 5.75 ± 1.22 to 0.53 ± 0.199 (p < 0.01 with paired t-test; n = 9). (D) Comparison of the effects of NNN (10 μM) and nicotine (10 μM) on whole-cell currents from α4β2 nAChRs, at −80 mV. NNN never produced nAChR activation in cells in which functional receptors were shown to be present by nicotine application.
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Figure 2: NNK, but not NNN, activates α4β2 nAChRs. (A) Consecutive whole-cell current traces, elicited at -80 mV by the indicated compound. Tests were spaced 2–3 min apart (gaps in the continuous current trace). Continuous bars above the current traces indicate the time of agonist application. Nicotine was repeatedly applied during the experiment to check for possible current rundown. (B) Concentration-response relations for nicotine (black squares) and NNK (red circles). Data points are average peak whole-cell currents recorded at −80 mV, normalized to the current elicited by 10 μM nicotine and plotted against the agonist concentration (in Log10 scale). Each point is the average of at least seven determinations. The concentration-response relation for nicotine (continuous line) and that for NNK (dashed line) were fitted with Equation 1, which gave an EC50 = 27.8 ± 5.6 μM (nH = 0.64) for nicotine and 16.7 ± 20 μM (nH = 0.44) for NNK. The estimated maximal current activated by NNK was approximately 16% of the current elicited by 300 μM nicotine. The measured peak current in the presence of 100 μM NNK was 0.14 ± 0.02 (n = 13) of the value measured in the presence of 300 μM nicotine. (C) DHβE (1 μM) strongly blocked the currents elicited by 300 μM ACh, 300 μM nicotine, and 10 μM NNK, as indicated. Data are averages of at least seven determination for each condition, carried out at −80 mV. Bars display the fractional residual currents (i.e., the peak current measured in the presence of the agonist plus DHβE divided by the peak current activated by the agonist alone). In the presence of 10 μM NNK, DHβE brought the average peak current density (pA/pF) from 5.75 ± 1.22 to 0.53 ± 0.199 (p < 0.01 with paired t-test; n = 9). (D) Comparison of the effects of NNN (10 μM) and nicotine (10 μM) on whole-cell currents from α4β2 nAChRs, at −80 mV. NNN never produced nAChR activation in cells in which functional receptors were shown to be present by nicotine application.

Mentions: We first tested if NNK can activate α4β2 nAChRs, by applying the drug at −80 mV. A representative example is shown in Figure 2A. Consecutive applications of different NNK concentrations were spaced 2–3 min apart, to permit full recovery from nAChR desensitization. NNK concentrations higher than 10 nM elicited inward currents, and the maximal effect was obtained at 100 μM NNK. Nicotine was usually applied at the beginning and at the end of the experiment, to check for possible current rundown. The concentration-response relation for NNK was obtained by plotting the average fractional peak currents obtained at the indicated NNK concentrations (Figure 2B). On average, the current elicited by 100 μM NNK was approximately 14% of the current activated by saturating concentrations of nicotine (300 μM). No significantly higher currents were observed when using 300 μM NNK. For both nicotine and NNK, data points were fitted with Equation 1, which gave apparent EC50's of approximately 28 μM for nicotine and 17 μM for NNK. For all kinetic parameters, detailed statistics are given in the figure legends, which also report the Hill coefficients. For easier consulting, the main results are summarized in Table 1.


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)

NNK, but not NNN, activates α4β2 nAChRs. (A) Consecutive whole-cell current traces, elicited at -80 mV by the indicated compound. Tests were spaced 2–3 min apart (gaps in the continuous current trace). Continuous bars above the current traces indicate the time of agonist application. Nicotine was repeatedly applied during the experiment to check for possible current rundown. (B) Concentration-response relations for nicotine (black squares) and NNK (red circles). Data points are average peak whole-cell currents recorded at −80 mV, normalized to the current elicited by 10 μM nicotine and plotted against the agonist concentration (in Log10 scale). Each point is the average of at least seven determinations. The concentration-response relation for nicotine (continuous line) and that for NNK (dashed line) were fitted with Equation 1, which gave an EC50 = 27.8 ± 5.6 μM (nH = 0.64) for nicotine and 16.7 ± 20 μM (nH = 0.44) for NNK. The estimated maximal current activated by NNK was approximately 16% of the current elicited by 300 μM nicotine. The measured peak current in the presence of 100 μM NNK was 0.14 ± 0.02 (n = 13) of the value measured in the presence of 300 μM nicotine. (C) DHβE (1 μM) strongly blocked the currents elicited by 300 μM ACh, 300 μM nicotine, and 10 μM NNK, as indicated. Data are averages of at least seven determination for each condition, carried out at −80 mV. Bars display the fractional residual currents (i.e., the peak current measured in the presence of the agonist plus DHβE divided by the peak current activated by the agonist alone). In the presence of 10 μM NNK, DHβE brought the average peak current density (pA/pF) from 5.75 ± 1.22 to 0.53 ± 0.199 (p < 0.01 with paired t-test; n = 9). (D) Comparison of the effects of NNN (10 μM) and nicotine (10 μM) on whole-cell currents from α4β2 nAChRs, at −80 mV. NNN never produced nAChR activation in cells in which functional receptors were shown to be present by nicotine application.
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Related In: Results  -  Collection

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Figure 2: NNK, but not NNN, activates α4β2 nAChRs. (A) Consecutive whole-cell current traces, elicited at -80 mV by the indicated compound. Tests were spaced 2–3 min apart (gaps in the continuous current trace). Continuous bars above the current traces indicate the time of agonist application. Nicotine was repeatedly applied during the experiment to check for possible current rundown. (B) Concentration-response relations for nicotine (black squares) and NNK (red circles). Data points are average peak whole-cell currents recorded at −80 mV, normalized to the current elicited by 10 μM nicotine and plotted against the agonist concentration (in Log10 scale). Each point is the average of at least seven determinations. The concentration-response relation for nicotine (continuous line) and that for NNK (dashed line) were fitted with Equation 1, which gave an EC50 = 27.8 ± 5.6 μM (nH = 0.64) for nicotine and 16.7 ± 20 μM (nH = 0.44) for NNK. The estimated maximal current activated by NNK was approximately 16% of the current elicited by 300 μM nicotine. The measured peak current in the presence of 100 μM NNK was 0.14 ± 0.02 (n = 13) of the value measured in the presence of 300 μM nicotine. (C) DHβE (1 μM) strongly blocked the currents elicited by 300 μM ACh, 300 μM nicotine, and 10 μM NNK, as indicated. Data are averages of at least seven determination for each condition, carried out at −80 mV. Bars display the fractional residual currents (i.e., the peak current measured in the presence of the agonist plus DHβE divided by the peak current activated by the agonist alone). In the presence of 10 μM NNK, DHβE brought the average peak current density (pA/pF) from 5.75 ± 1.22 to 0.53 ± 0.199 (p < 0.01 with paired t-test; n = 9). (D) Comparison of the effects of NNN (10 μM) and nicotine (10 μM) on whole-cell currents from α4β2 nAChRs, at −80 mV. NNN never produced nAChR activation in cells in which functional receptors were shown to be present by nicotine application.
Mentions: We first tested if NNK can activate α4β2 nAChRs, by applying the drug at −80 mV. A representative example is shown in Figure 2A. Consecutive applications of different NNK concentrations were spaced 2–3 min apart, to permit full recovery from nAChR desensitization. NNK concentrations higher than 10 nM elicited inward currents, and the maximal effect was obtained at 100 μM NNK. Nicotine was usually applied at the beginning and at the end of the experiment, to check for possible current rundown. The concentration-response relation for NNK was obtained by plotting the average fractional peak currents obtained at the indicated NNK concentrations (Figure 2B). On average, the current elicited by 100 μM NNK was approximately 14% of the current activated by saturating concentrations of nicotine (300 μM). No significantly higher currents were observed when using 300 μM NNK. For both nicotine and NNK, data points were fitted with Equation 1, which gave apparent EC50's of approximately 28 μM for nicotine and 17 μM for NNK. For all kinetic parameters, detailed statistics are given in the figure legends, which also report the Hill coefficients. For easier consulting, the main results are summarized in Table 1.

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