Limits...
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 effects of NNN and NNK are not voltage-dependent. (A) Typical current traces obtained as illustrated in Figure 3, except that trials were carried out at different Vm's. For briefness, only the traces obtained at −80 and +80 mV are displayed. Bars mark time of application of 10 μM nicotine (Nico, continuous) and 10 μM NNN (dashed). (B). Top panel: current-voltage relations obtained in the absence (squares) and in the presence (circles) of NNN. Data points are average steady state current densities measured at the indicated Vm and normalized to the value obtained at -80 mV (with a reversed sign, for consistency with the usual convention of displaying inward current as negative). Data summarize the results of nine independent experiments. Continuous lines are polynomial curves best fitting the data points. No correction was applied for junction potentials. Bottom panel: the fractional block produced by NNN is plotted as a function of Vm. Data points are average steady state currents in the presence of NNN (INNN), divided by the current in the absence of NNN (INico). The data points around Vrev were omitted (see the main text). No significant difference was observed among the results obtained at different Vm's. (C) Top panel: same as in (C), for NNK (1 μM, in the presence of 100 nM nicotine). Bottom panel: the fractional block produced by NNK as a function of Vm was calculated as illustrated for NNN in (B), except that the tested Vm's were: −120/−80/−40/0/+40. Once again, no significant difference was observed among the results obtained at different Vm's.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4585029&req=5

Figure 6: The effects of NNN and NNK are not voltage-dependent. (A) Typical current traces obtained as illustrated in Figure 3, except that trials were carried out at different Vm's. For briefness, only the traces obtained at −80 and +80 mV are displayed. Bars mark time of application of 10 μM nicotine (Nico, continuous) and 10 μM NNN (dashed). (B). Top panel: current-voltage relations obtained in the absence (squares) and in the presence (circles) of NNN. Data points are average steady state current densities measured at the indicated Vm and normalized to the value obtained at -80 mV (with a reversed sign, for consistency with the usual convention of displaying inward current as negative). Data summarize the results of nine independent experiments. Continuous lines are polynomial curves best fitting the data points. No correction was applied for junction potentials. Bottom panel: the fractional block produced by NNN is plotted as a function of Vm. Data points are average steady state currents in the presence of NNN (INNN), divided by the current in the absence of NNN (INico). The data points around Vrev were omitted (see the main text). No significant difference was observed among the results obtained at different Vm's. (C) Top panel: same as in (C), for NNK (1 μM, in the presence of 100 nM nicotine). Bottom panel: the fractional block produced by NNK as a function of Vm was calculated as illustrated for NNN in (B), except that the tested Vm's were: −120/−80/−40/0/+40. Once again, no significant difference was observed among the results obtained at different Vm's.

Mentions: The voltage dependence of the NNN and NNK effect is illustrated in Figure 6. Current traces (Figure 6A) illustrate typical experiments in which nAChRs were activated by 10 μM nicotine, at the indicated Vm. For briefness, only traces obtained at −80 mV (left) and +80 mV (right) are shown. NNN (10 μM) was applied in the presence of nicotine and removed after inhibition had reached the steady state. These results are summarized in Figure 6B (top panel), showing the current voltage relations in the presence of either nicotine alone (Nico) or nicotine plus NNN (NNN). Data points are average current values normalized to the absolute value of the current measured at −80 mV. The I/V plots displayed the typical inward rectification of α4β2 nAChRs. Analogous results were obtained with NNK (Figure 6C, top panel). The apparent reversal potential (Vrev) was usually between +5 and +20 mV. The fractional block produced by either NNN or NNK at the steady state was independent of the applied Vm. This is better appreciated in the bottom panels of Figures 6B,C, which give the average fractional residual currents as a function of Vm, in the presence of NNN or NNK, respectively. The data points around Vrev were removed as the small current values prevented a reliable measure of the drugs' effect. The quick development and reversal of channel block suggests that the effect was mainly caused by the nitrosamines accessing to the channel from the extracellular milieu (i.e., we assume that intracellular accumulation of the drugs was negligible). Hence, because inhibition was virtually independent of the net direction of ion flow, these nitrosamines are unlikely to exert significant open channel block, at the concentrations we applied (much higher than those observed in vivo).


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 effects of NNN and NNK are not voltage-dependent. (A) Typical current traces obtained as illustrated in Figure 3, except that trials were carried out at different Vm's. For briefness, only the traces obtained at −80 and +80 mV are displayed. Bars mark time of application of 10 μM nicotine (Nico, continuous) and 10 μM NNN (dashed). (B). Top panel: current-voltage relations obtained in the absence (squares) and in the presence (circles) of NNN. Data points are average steady state current densities measured at the indicated Vm and normalized to the value obtained at -80 mV (with a reversed sign, for consistency with the usual convention of displaying inward current as negative). Data summarize the results of nine independent experiments. Continuous lines are polynomial curves best fitting the data points. No correction was applied for junction potentials. Bottom panel: the fractional block produced by NNN is plotted as a function of Vm. Data points are average steady state currents in the presence of NNN (INNN), divided by the current in the absence of NNN (INico). The data points around Vrev were omitted (see the main text). No significant difference was observed among the results obtained at different Vm's. (C) Top panel: same as in (C), for NNK (1 μM, in the presence of 100 nM nicotine). Bottom panel: the fractional block produced by NNK as a function of Vm was calculated as illustrated for NNN in (B), except that the tested Vm's were: −120/−80/−40/0/+40. Once again, no significant difference was observed among the results obtained at different Vm's.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585029&req=5

Figure 6: The effects of NNN and NNK are not voltage-dependent. (A) Typical current traces obtained as illustrated in Figure 3, except that trials were carried out at different Vm's. For briefness, only the traces obtained at −80 and +80 mV are displayed. Bars mark time of application of 10 μM nicotine (Nico, continuous) and 10 μM NNN (dashed). (B). Top panel: current-voltage relations obtained in the absence (squares) and in the presence (circles) of NNN. Data points are average steady state current densities measured at the indicated Vm and normalized to the value obtained at -80 mV (with a reversed sign, for consistency with the usual convention of displaying inward current as negative). Data summarize the results of nine independent experiments. Continuous lines are polynomial curves best fitting the data points. No correction was applied for junction potentials. Bottom panel: the fractional block produced by NNN is plotted as a function of Vm. Data points are average steady state currents in the presence of NNN (INNN), divided by the current in the absence of NNN (INico). The data points around Vrev were omitted (see the main text). No significant difference was observed among the results obtained at different Vm's. (C) Top panel: same as in (C), for NNK (1 μM, in the presence of 100 nM nicotine). Bottom panel: the fractional block produced by NNK as a function of Vm was calculated as illustrated for NNN in (B), except that the tested Vm's were: −120/−80/−40/0/+40. Once again, no significant difference was observed among the results obtained at different Vm's.
Mentions: The voltage dependence of the NNN and NNK effect is illustrated in Figure 6. Current traces (Figure 6A) illustrate typical experiments in which nAChRs were activated by 10 μM nicotine, at the indicated Vm. For briefness, only traces obtained at −80 mV (left) and +80 mV (right) are shown. NNN (10 μM) was applied in the presence of nicotine and removed after inhibition had reached the steady state. These results are summarized in Figure 6B (top panel), showing the current voltage relations in the presence of either nicotine alone (Nico) or nicotine plus NNN (NNN). Data points are average current values normalized to the absolute value of the current measured at −80 mV. The I/V plots displayed the typical inward rectification of α4β2 nAChRs. Analogous results were obtained with NNK (Figure 6C, top panel). The apparent reversal potential (Vrev) was usually between +5 and +20 mV. The fractional block produced by either NNN or NNK at the steady state was independent of the applied Vm. This is better appreciated in the bottom panels of Figures 6B,C, which give the average fractional residual currents as a function of Vm, in the presence of NNN or NNK, respectively. The data points around Vrev were removed as the small current values prevented a reliable measure of the drugs' effect. The quick development and reversal of channel block suggests that the effect was mainly caused by the nitrosamines accessing to the channel from the extracellular milieu (i.e., we assume that intracellular accumulation of the drugs was negligible). Hence, because inhibition was virtually independent of the net direction of ion flow, these nitrosamines are unlikely to exert significant open channel block, at the concentrations we applied (much higher than those observed in vivo).

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