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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

The effect of NNK on α4β2 nAChRs, in the presence of nicotine. (A) Representative current traces elicited at −80 mV by the indicated concentration of nicotine (Nico), in the presence or in the absence of the indicated concentration of NNK. The currents elicited by 10 μM nicotine (top and middle panels) are representative of 11 similar experiments. The bottom panel illustrates the potentiating effect of 100 μM NNK on the current activated by 100 nM nicotine (representative of eight similar experiments). Bars mark the time of application of nicotine and NNK, as indicated. (B) Concentration-response relation for the inhibitory effect of NNK tested on currents activated by 10 μM nicotine, as illustrated in (A). Data points are average steady state currents (n = 11) measured in the presence of a given NNK concentration, divided by the current obtained after NNK was rinsed. Continuous line through the data points is best fitting to Equation 3, giving IC50 > 100 μM (nH = 0.95). (C) Summary of the effects of NNK plus nicotine. All data are normalized to the current measured with 300 μM nicotine. Black circles: concentration-response for nicotine (same as in Figure 3; only concentrations up to 100 μM are shown). Red symbols: average fractional currents in the presence of 1 μM (squares), 10 μM (triangles), or 100 μM (squares) NNK, as a function of [nicotine]. Data were obtained as illustrated in (A). The values obtained with NNK were generally significantly different from those obtained with nicotine alone. Detailed statistics for the potentiation data are given in (D). The red continuous line is the curve best fitting the data points relative to 1 μM NNK, obtained by using Equation 4. The fit parameters were A = 1, B = 0.27, [NNK] = 1 μM, αNIC = 24 μM, αNNK = 5 μM. (D) The peak current measured in the presence of 10 nM nicotine was 119 ± 24 pA, which was brought to 160 ± 30 pA by 1 μM NNK (0.05 < p < 0.01, with t-test for paired samples; n = 11). The effect of 100 μM NNK was tested in a different series of cells, in which the average current elicited by 100 nM nicotine was 89.4 ± 19.4 pA, which was brought to 159.1 ± 25.1 pA by 100 μM NNK (0.05 < p < 0.01, with t-test for paired samples; n = 8). These results are plotted as black and red bars, respectively for nicotine (Nico) and NNK. Statistical significance is indicated by *.
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Figure 5: The effect of NNK on α4β2 nAChRs, in the presence of nicotine. (A) Representative current traces elicited at −80 mV by the indicated concentration of nicotine (Nico), in the presence or in the absence of the indicated concentration of NNK. The currents elicited by 10 μM nicotine (top and middle panels) are representative of 11 similar experiments. The bottom panel illustrates the potentiating effect of 100 μM NNK on the current activated by 100 nM nicotine (representative of eight similar experiments). Bars mark the time of application of nicotine and NNK, as indicated. (B) Concentration-response relation for the inhibitory effect of NNK tested on currents activated by 10 μM nicotine, as illustrated in (A). Data points are average steady state currents (n = 11) measured in the presence of a given NNK concentration, divided by the current obtained after NNK was rinsed. Continuous line through the data points is best fitting to Equation 3, giving IC50 > 100 μM (nH = 0.95). (C) Summary of the effects of NNK plus nicotine. All data are normalized to the current measured with 300 μM nicotine. Black circles: concentration-response for nicotine (same as in Figure 3; only concentrations up to 100 μM are shown). Red symbols: average fractional currents in the presence of 1 μM (squares), 10 μM (triangles), or 100 μM (squares) NNK, as a function of [nicotine]. Data were obtained as illustrated in (A). The values obtained with NNK were generally significantly different from those obtained with nicotine alone. Detailed statistics for the potentiation data are given in (D). The red continuous line is the curve best fitting the data points relative to 1 μM NNK, obtained by using Equation 4. The fit parameters were A = 1, B = 0.27, [NNK] = 1 μM, αNIC = 24 μM, αNNK = 5 μM. (D) The peak current measured in the presence of 10 nM nicotine was 119 ± 24 pA, which was brought to 160 ± 30 pA by 1 μM NNK (0.05 < p < 0.01, with t-test for paired samples; n = 11). The effect of 100 μM NNK was tested in a different series of cells, in which the average current elicited by 100 nM nicotine was 89.4 ± 19.4 pA, which was brought to 159.1 ± 25.1 pA by 100 μM NNK (0.05 < p < 0.01, with t-test for paired samples; n = 8). These results are plotted as black and red bars, respectively for nicotine (Nico) and NNK. Statistical significance is indicated by *.

Mentions: NNK is expected to produce effects more complex than those shown by NNN, as partial agonists can produce channel activation or inhibition depending on the concentration of the full agonist (e.g., Hogg and Bertrand, 2007; Rollema et al., 2007). In fact, concentrations of NNK up to 100 nM produced no effect on the currents activated by 10 μM nicotine, whereas higher concentrations progressively inhibited α4β2 nAChRs (Figure 5A). These data were obtained and analyzed as previously illustrated for NNN. Figure 5B plots the average fractional residual currents measured in the presence of 10 μM nicotine, as a function of NNK concentration. Data points were fitted with Equation 3 (continuous line), giving an IC50 > 100 μM. In contrast, the currents activated by low concentrations of nicotine could be potentiated by NNK. An example is given in the bottom panel of Figure 5A, showing that 100 μM NNK increased the current activated by 100 nM nicotine at −80 mV, by approximately 80%. In agreement with the results shown in Figure 3, the current activated by 100 μM NNK also displayed progressive desensitization. The effects of the tested concentrations of NNK on the currents activated by different concentrations of nicotine are summarized in Figure 5C. For comparison, the activation curve for nicotine (black circles) is also reported. With 1 μM NNK (red squares), the potentiation produced on the currents activated by 0.01 μM nicotine was 26%. The concentration of nicotine at which the drug reversed its effect was around 20 nM, as is estimated by fitting the data points with the simplified model expressed by Equation 4 (continuous red line). Saturating concentrations of NNK (100 μM; red circles) potentiated by about 80% the current activated by 0.1 μM nicotine, whereas they inhibited by 40% the current elicited by 10 μM nicotine. In this case, the sign reversal of NNK effect can be estimated to occur at approximately 400 nM nicotine. For clarity, the error bars are not reported in Figure 5C. Instead, the statistics of the current potentiation observed with NNK are reported in detail in Figure 5D and in the figure legend. We conclude that NNK, consistently with its partial agonist nature, can produce either potentiation or inhibition of α4β2 nAChRs, depending on the concomitant concentration of the full agonist.


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)

The effect of NNK on α4β2 nAChRs, in the presence of nicotine. (A) Representative current traces elicited at −80 mV by the indicated concentration of nicotine (Nico), in the presence or in the absence of the indicated concentration of NNK. The currents elicited by 10 μM nicotine (top and middle panels) are representative of 11 similar experiments. The bottom panel illustrates the potentiating effect of 100 μM NNK on the current activated by 100 nM nicotine (representative of eight similar experiments). Bars mark the time of application of nicotine and NNK, as indicated. (B) Concentration-response relation for the inhibitory effect of NNK tested on currents activated by 10 μM nicotine, as illustrated in (A). Data points are average steady state currents (n = 11) measured in the presence of a given NNK concentration, divided by the current obtained after NNK was rinsed. Continuous line through the data points is best fitting to Equation 3, giving IC50 > 100 μM (nH = 0.95). (C) Summary of the effects of NNK plus nicotine. All data are normalized to the current measured with 300 μM nicotine. Black circles: concentration-response for nicotine (same as in Figure 3; only concentrations up to 100 μM are shown). Red symbols: average fractional currents in the presence of 1 μM (squares), 10 μM (triangles), or 100 μM (squares) NNK, as a function of [nicotine]. Data were obtained as illustrated in (A). The values obtained with NNK were generally significantly different from those obtained with nicotine alone. Detailed statistics for the potentiation data are given in (D). The red continuous line is the curve best fitting the data points relative to 1 μM NNK, obtained by using Equation 4. The fit parameters were A = 1, B = 0.27, [NNK] = 1 μM, αNIC = 24 μM, αNNK = 5 μM. (D) The peak current measured in the presence of 10 nM nicotine was 119 ± 24 pA, which was brought to 160 ± 30 pA by 1 μM NNK (0.05 < p < 0.01, with t-test for paired samples; n = 11). The effect of 100 μM NNK was tested in a different series of cells, in which the average current elicited by 100 nM nicotine was 89.4 ± 19.4 pA, which was brought to 159.1 ± 25.1 pA by 100 μM NNK (0.05 < p < 0.01, with t-test for paired samples; n = 8). These results are plotted as black and red bars, respectively for nicotine (Nico) and NNK. Statistical significance is indicated by *.
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Figure 5: The effect of NNK on α4β2 nAChRs, in the presence of nicotine. (A) Representative current traces elicited at −80 mV by the indicated concentration of nicotine (Nico), in the presence or in the absence of the indicated concentration of NNK. The currents elicited by 10 μM nicotine (top and middle panels) are representative of 11 similar experiments. The bottom panel illustrates the potentiating effect of 100 μM NNK on the current activated by 100 nM nicotine (representative of eight similar experiments). Bars mark the time of application of nicotine and NNK, as indicated. (B) Concentration-response relation for the inhibitory effect of NNK tested on currents activated by 10 μM nicotine, as illustrated in (A). Data points are average steady state currents (n = 11) measured in the presence of a given NNK concentration, divided by the current obtained after NNK was rinsed. Continuous line through the data points is best fitting to Equation 3, giving IC50 > 100 μM (nH = 0.95). (C) Summary of the effects of NNK plus nicotine. All data are normalized to the current measured with 300 μM nicotine. Black circles: concentration-response for nicotine (same as in Figure 3; only concentrations up to 100 μM are shown). Red symbols: average fractional currents in the presence of 1 μM (squares), 10 μM (triangles), or 100 μM (squares) NNK, as a function of [nicotine]. Data were obtained as illustrated in (A). The values obtained with NNK were generally significantly different from those obtained with nicotine alone. Detailed statistics for the potentiation data are given in (D). The red continuous line is the curve best fitting the data points relative to 1 μM NNK, obtained by using Equation 4. The fit parameters were A = 1, B = 0.27, [NNK] = 1 μM, αNIC = 24 μM, αNNK = 5 μM. (D) The peak current measured in the presence of 10 nM nicotine was 119 ± 24 pA, which was brought to 160 ± 30 pA by 1 μM NNK (0.05 < p < 0.01, with t-test for paired samples; n = 11). The effect of 100 μM NNK was tested in a different series of cells, in which the average current elicited by 100 nM nicotine was 89.4 ± 19.4 pA, which was brought to 159.1 ± 25.1 pA by 100 μM NNK (0.05 < p < 0.01, with t-test for paired samples; n = 8). These results are plotted as black and red bars, respectively for nicotine (Nico) and NNK. Statistical significance is indicated by *.
Mentions: NNK is expected to produce effects more complex than those shown by NNN, as partial agonists can produce channel activation or inhibition depending on the concentration of the full agonist (e.g., Hogg and Bertrand, 2007; Rollema et al., 2007). In fact, concentrations of NNK up to 100 nM produced no effect on the currents activated by 10 μM nicotine, whereas higher concentrations progressively inhibited α4β2 nAChRs (Figure 5A). These data were obtained and analyzed as previously illustrated for NNN. Figure 5B plots the average fractional residual currents measured in the presence of 10 μM nicotine, as a function of NNK concentration. Data points were fitted with Equation 3 (continuous line), giving an IC50 > 100 μM. In contrast, the currents activated by low concentrations of nicotine could be potentiated by NNK. An example is given in the bottom panel of Figure 5A, showing that 100 μM NNK increased the current activated by 100 nM nicotine at −80 mV, by approximately 80%. In agreement with the results shown in Figure 3, the current activated by 100 μM NNK also displayed progressive desensitization. The effects of the tested concentrations of NNK on the currents activated by different concentrations of nicotine are summarized in Figure 5C. For comparison, the activation curve for nicotine (black circles) is also reported. With 1 μM NNK (red squares), the potentiation produced on the currents activated by 0.01 μM nicotine was 26%. The concentration of nicotine at which the drug reversed its effect was around 20 nM, as is estimated by fitting the data points with the simplified model expressed by Equation 4 (continuous red line). Saturating concentrations of NNK (100 μM; red circles) potentiated by about 80% the current activated by 0.1 μM nicotine, whereas they inhibited by 40% the current elicited by 10 μM nicotine. In this case, the sign reversal of NNK effect can be estimated to occur at approximately 400 nM nicotine. For clarity, the error bars are not reported in Figure 5C. Instead, the statistics of the current potentiation observed with NNK are reported in detail in Figure 5D and in the figure legend. We conclude that NNK, consistently with its partial agonist nature, can produce either potentiation or inhibition of α4β2 nAChRs, depending on the concomitant concentration of the full agonist.

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