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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

Activation and desensitization profiles for α4β2 nAChRs. The activation curve for NNK (red circles) was generated by using experiments analogous to those shown in Figure 2. The average peak current measured at each concentration of NNK was normalized to the current elicited by 300 μM NNK. Data points are averages of at least seven determinations and were fitted by using Equation 2 (continuous line), which gave EC50high = 0.035 ± 0.012 μM (nH2 = 1.2), and EC50low = 12.81 ± 1.58 μM (nH1 = 1.4). The desensitization curve for NNK was generated by plotting average steady state fractional currents (red triangles), as a function of NNK concentration. At each concentration, the current decay in the presence of the drug was fitted with a single exponential function. The steady state current values thus estimated were divided by the corresponding peak current values. Data points are averages of at least 6 determinations and were fitted by using Equation 3 (continuous line), which gave IC50 = 1.7 μM ± 0.2 (nH = 0.9). The nicotine activation (black circles) and desensitization (black triangles) curves were obtained in a similar way. For activation, data points are averages of at least 6 determinations. They were fitted by using Equation 2 (continuous line), giving EC50high = 0.14 ± 1.03 μM (nH2 = 0.62), and EC50low = 7.7 ± 14.7 μM (nH1 = 1.8). For desensitization, data points were fitted with Equation 3 (continuous line), which gave IC50 = 2.64 ± 0.47 μM (nH = 1.84).
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Figure 3: Activation and desensitization profiles for α4β2 nAChRs. The activation curve for NNK (red circles) was generated by using experiments analogous to those shown in Figure 2. The average peak current measured at each concentration of NNK was normalized to the current elicited by 300 μM NNK. Data points are averages of at least seven determinations and were fitted by using Equation 2 (continuous line), which gave EC50high = 0.035 ± 0.012 μM (nH2 = 1.2), and EC50low = 12.81 ± 1.58 μM (nH1 = 1.4). The desensitization curve for NNK was generated by plotting average steady state fractional currents (red triangles), as a function of NNK concentration. At each concentration, the current decay in the presence of the drug was fitted with a single exponential function. The steady state current values thus estimated were divided by the corresponding peak current values. Data points are averages of at least 6 determinations and were fitted by using Equation 3 (continuous line), which gave IC50 = 1.7 μM ± 0.2 (nH = 0.9). The nicotine activation (black circles) and desensitization (black triangles) curves were obtained in a similar way. For activation, data points are averages of at least 6 determinations. They were fitted by using Equation 2 (continuous line), giving EC50high = 0.14 ± 1.03 μM (nH2 = 0.62), and EC50low = 7.7 ± 14.7 μM (nH1 = 1.8). For desensitization, data points were fitted with Equation 3 (continuous line), which gave IC50 = 2.64 ± 0.47 μM (nH = 1.84).

Mentions: A more detailed analysis of the concentration-response relations for nicotine and NNK is shown in Figure 3. Best fitting of the experimental data points was obtained by using the two-terms Hill function (Equation 2). This is usually observed with α4β2 nAChRs (Covernton and Connolly, 2000; Buisson and Bertrand, 2001), and is attributed to the coexistence of two receptor's stoichiometries: (α4)3(β2)2 (with lower affinity) and (α4)2(β2)3 receptors (with higher affinity; Nelson et al., 2003). The fitting parameters were: EC50high = 0.035 μM and EC50low = 12.81 μM, for NNK, and EC50high = 0.14 μM and EC50low = 7.7 μM for nicotine. These results suggest that NNK is particularly effective at stimulating the high affinity nAChR component. Moreover, for both nicotine (e.g., Figure 5A) and NNK (e.g., Figure 2A), the activated current displayed progressive desensitization in the presence of the agonist. To quantify such process, the current decay was fitted with a monoexponential function (e.g., Paradiso and Steinbach, 2003). Next, desensitization curves were generated by plotting the average fractional steady state currents calculated from the fitting procedure, for the indicated agonist concentration (Figure 3). By using Equation 3 (continuous lines through the data points), we estimated IC50 = 1.7 μM for NNK, and IC50 = 2.64 μM for nicotine. In the absence of other nicotinic ligands, the nAChR-dependent biological effect of NNK must depend on the steady state current sustained by this drug. This is proportional to the product of the activation and desensitization curves (sometimes referred to as “window current”), at a given NNK concentration. Figure 3 suggests that the window currents of NNK and nicotine are broadly similar, but that NNK tends to be comparatively more effective at the low concentrations, in line with the range of plasma doses observed in smokers.


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)

Activation and desensitization profiles for α4β2 nAChRs. The activation curve for NNK (red circles) was generated by using experiments analogous to those shown in Figure 2. The average peak current measured at each concentration of NNK was normalized to the current elicited by 300 μM NNK. Data points are averages of at least seven determinations and were fitted by using Equation 2 (continuous line), which gave EC50high = 0.035 ± 0.012 μM (nH2 = 1.2), and EC50low = 12.81 ± 1.58 μM (nH1 = 1.4). The desensitization curve for NNK was generated by plotting average steady state fractional currents (red triangles), as a function of NNK concentration. At each concentration, the current decay in the presence of the drug was fitted with a single exponential function. The steady state current values thus estimated were divided by the corresponding peak current values. Data points are averages of at least 6 determinations and were fitted by using Equation 3 (continuous line), which gave IC50 = 1.7 μM ± 0.2 (nH = 0.9). The nicotine activation (black circles) and desensitization (black triangles) curves were obtained in a similar way. For activation, data points are averages of at least 6 determinations. They were fitted by using Equation 2 (continuous line), giving EC50high = 0.14 ± 1.03 μM (nH2 = 0.62), and EC50low = 7.7 ± 14.7 μM (nH1 = 1.8). For desensitization, data points were fitted with Equation 3 (continuous line), which gave IC50 = 2.64 ± 0.47 μM (nH = 1.84).
© Copyright Policy
Related In: Results  -  Collection

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Figure 3: Activation and desensitization profiles for α4β2 nAChRs. The activation curve for NNK (red circles) was generated by using experiments analogous to those shown in Figure 2. The average peak current measured at each concentration of NNK was normalized to the current elicited by 300 μM NNK. Data points are averages of at least seven determinations and were fitted by using Equation 2 (continuous line), which gave EC50high = 0.035 ± 0.012 μM (nH2 = 1.2), and EC50low = 12.81 ± 1.58 μM (nH1 = 1.4). The desensitization curve for NNK was generated by plotting average steady state fractional currents (red triangles), as a function of NNK concentration. At each concentration, the current decay in the presence of the drug was fitted with a single exponential function. The steady state current values thus estimated were divided by the corresponding peak current values. Data points are averages of at least 6 determinations and were fitted by using Equation 3 (continuous line), which gave IC50 = 1.7 μM ± 0.2 (nH = 0.9). The nicotine activation (black circles) and desensitization (black triangles) curves were obtained in a similar way. For activation, data points are averages of at least 6 determinations. They were fitted by using Equation 2 (continuous line), giving EC50high = 0.14 ± 1.03 μM (nH2 = 0.62), and EC50low = 7.7 ± 14.7 μM (nH1 = 1.8). For desensitization, data points were fitted with Equation 3 (continuous line), which gave IC50 = 2.64 ± 0.47 μM (nH = 1.84).
Mentions: A more detailed analysis of the concentration-response relations for nicotine and NNK is shown in Figure 3. Best fitting of the experimental data points was obtained by using the two-terms Hill function (Equation 2). This is usually observed with α4β2 nAChRs (Covernton and Connolly, 2000; Buisson and Bertrand, 2001), and is attributed to the coexistence of two receptor's stoichiometries: (α4)3(β2)2 (with lower affinity) and (α4)2(β2)3 receptors (with higher affinity; Nelson et al., 2003). The fitting parameters were: EC50high = 0.035 μM and EC50low = 12.81 μM, for NNK, and EC50high = 0.14 μM and EC50low = 7.7 μM for nicotine. These results suggest that NNK is particularly effective at stimulating the high affinity nAChR component. Moreover, for both nicotine (e.g., Figure 5A) and NNK (e.g., Figure 2A), the activated current displayed progressive desensitization in the presence of the agonist. To quantify such process, the current decay was fitted with a monoexponential function (e.g., Paradiso and Steinbach, 2003). Next, desensitization curves were generated by plotting the average fractional steady state currents calculated from the fitting procedure, for the indicated agonist concentration (Figure 3). By using Equation 3 (continuous lines through the data points), we estimated IC50 = 1.7 μM for NNK, and IC50 = 2.64 μM for nicotine. In the absence of other nicotinic ligands, the nAChR-dependent biological effect of NNK must depend on the steady state current sustained by this drug. This is proportional to the product of the activation and desensitization curves (sometimes referred to as “window current”), at a given NNK concentration. Figure 3 suggests that the window currents of NNK and nicotine are broadly similar, but that NNK tends to be comparatively more effective at the low concentrations, in line with the range of plasma doses observed in smokers.

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