Chronic exposure to nicotine upregulates the human (alpha)4((beta)2 nicotinic acetylcholine receptor function

Abstract

Widely expressed in the brain, the alpha4beta2 nicotinic acetylcholine receptor (nAChR) is proposed to play a major role in the mechanisms that lead to and maintain nicotine addiction. Using the patch-clamp technique and pharmacological protocols, we examined the consequences of long-term exposure to 0.1-10 micrometer nicotine in K-177 cells expressing the major human brain alpha4beta2 receptor. The acetylcholine dose-response curves are biphasic and revealed both a high- and a low-affinity component with apparent EC(50) values of 1.6 and 62 micrometer. Ratios of receptors in the high- and low-affinity components are 25 and 75%, respectively. Chronic exposure to nicotine or nicotinic antagonists [dihydro-beta-erytroidine (DHbetaE) or methyllycaconitine (MLA)] increases the fraction of high-affinity receptors up to 70%. Upregulated acetylcholine-evoked currents increase by twofold or more and are less sensitive to desensitization. Functional upregulation is independent of protein synthesis as shown by the lack of effect of 20 micrometer cycloheximide. Single-channel currents recorded with 100 nm acetylcholine show predominantly high conductances (38.8 and 43.4 pS), whereas additional smaller conductances (16.7 and 23.5 pS) were observed with 30 micrometer acetylcholine. In addition, long-term exposure to dihydro-beta-erytroidine increases up to three times the frequency of channel openings. These data indicate, in contrast to previous studies, that human alpha4beta2 nAChRs are functionally upregulated by chronic nicotine exposure.

Fig. 1.

Two-component dose–response curves of human α4β2 nAChRs. A, Increasing ACh concentrations (200 msec pulses) were delivered every 15 sec. Typical traces of ACh-evoked currents are presented at the top. Horizontal bars indicate the ACh pulses with the concentration values (in micromolar). The fast desensitization and the current rebound observed at the end of the application pulse (1000 μm ACh) might indicate an open-channel block by the high ACh concentration. Mean current amplitudes were plotted as a function of the ACh concentration on a semilogarithmic scale (n = 12). Thedashed line corresponds to the best fit that can be drawn using a single Hill equation (EC₅₀ = 30 μm and nH = 0.8). A better fit is obtained with the sum of two Hill equations (continuous line) yielding high-affinity coefficients of EC₅₀H = 1.60 μm and nH1 = 0.92, whereas the low-affinity values are EC₅₀L = 68 μm and nH2 = 1.60. The fraction of high- and low-affinity states are 25 and 75%, respectively. B, The same protocol as inA was repeated for the determination of the α4β2 nAChR sensitivity toward nicotine. Currents were then elicited with increasing concentrations of nicotine (top).Horizontal bars indicate the nicotine pulses with the concentration values (in micromolar). Mean current amplitudes were plotted as a function of the nicotine concentration on a semilogarithmic scale (n = 11). The dashed line corresponds to the best fit that can be drawn using a single Hill equation (EC₅₀ = 10 μm and nH = 1.30). A better fit is obtained with the sum of two Hill equations (thick line) yielding high-affinity coefficients of EC₅₀H = 2.4 μm and nH1 = 0.91, whereas the low-affinity values are EC₅₀L = 14.5 μm and nH2 = 1.53. The fractions of high- and low-affinity states are 25 and 75%, respectively. For comparison, the ACh dose–response profile is scaled up to the maximal nicotine-evoked current (gray line). Note that the low-affinity component is much more sensitive to nicotine than to ACh.

Fig. 2.

Effects on human α4β2 nAChRs of a long-term exposure to nicotine. A, Chronic nicotine exposure failed to suppress ACh-evoked currents. Cells were incubated overnight in a culture medium containing 100 nm nicotine. In this particular experiment, the culture medium was not washed out before the recording. The cell was still superfused with the saline solution containing 100 nm nicotine until the establishment of the whole-cell configuration. First, ACh-evoked currents could be recorded even in the continuous presence of 100 nm nicotine between ACh pulses (top traces). Second, immediately after nicotine removal from the perfusion medium, the same protocol evoked currents of larger amplitudes (bottom traces). Thehorizontal bars indicate the duration of the ACh applications with the concentration values. B, The ACh dose–response relationship was determined in a series of cells (n = 7) either under a 100 nmnicotine-containing solution (squares) or immediately (2 min) after nicotine removal (circles). In the presence of 100 nm nicotine, high-affinity coefficients are EC₅₀H = 3.48 μm and nH1 = 0.98, whereas the low-affinity values are EC₅₀L = 127 μm and nH2 = 1.44; the fractions of high- and low-affinity states are 21 and 79%, respectively. After nicotine removal, high-affinity coefficients were EC₅₀H = 2.3 μm and nH1 = 0.82, whereas the low-affinity values were EC₅₀L = 91.4 μm and nH2 = 1.37; the fractions of high- and low-affinity states were 33 and 67%, respectively. C, The ACh dose–response profile was determined either for control cells or for cells exposed to 0.1 or 1 μm nicotine. ACh-evoked currents recorded in a typical control cell (top traces) are presented in comparison to currents elicited in another cell incubated for 10 hr in 100 nm nicotine with an extensive wash before recording (bottom traces). Horizontal bars indicate the ACh applications. D, ACh dose–response curves measured in control (○, n = 11) and after chronic nicotine incubation (100 nm, ▪, n = 8; 1 μm, ▿, n = 5; parameters for the fit are given in Table 1).

Fig. 3.

Long-term exposure to nicotine reduces desensitization. A, Mean ACh-evoked currents recorded in control (left traces) and after nicotine treatment (right traces) have been averaged (n= 5 in each condition). Dashed lines through the data points were computed using a mono-exponential (see Materials and Methods). B, Parameter values determined for the fit of current decays (Eq. 2) in control condition and after 8–10 hr exposure to 100 nm nicotine (nic).

Fig. 4.

Human α4β2 nAChR upregulation is induced by dihydro-β-erytroidine (DHβE).A, Typical currents evoked by increasing ACh concentrations are presented for a control cell (top traces) and for a cell exposed to DHβE (bottom traces). Horizontal bars indicate the ACh application with the concentration values (in micromolar). Note the large increase of the amplitude of the currents and the decrease of desensitization after chronic exposure to DHβE. Long-lasting tails of the current observed with DHβE-incubated cells suggest that ACh dissociates more slowly from the receptors than the competitive antagonist. B, Dose–response relationships were determined in control (○, n = 7) or after chronic exposure to 10 nm DHβE (▵, n = 7) or 10 μm DHβE (▪, n = 7).Lines through the data points are the best fits obtained with the sum of two Hill equations (see Table 1 for the values).

Fig. 5.

Human α4β2 nAChR upregulation is independent of protein synthesis and reverses within a few hours. A, Addition in the culture medium of the protein synthesis inhibitor cycloheximide (20 μm) had no effect on the maximal ACh-evoked current (1 mm, 200 msec) recorded in control (n = 10) or after chronic exposure to DHβE (10 μm, n = 11) (Table 1).B, DHβE upregulation was reversible within a few hours. Current amplitudes evoked by saturating ACh concentration (1 mm, 200 msec) were measured in a series of cells in control (n = 22) after overnight exposure to DHβE (n = 22) or after overnight exposure to DHβE (15 hr) with an additional 6–9 hr recovery period after DHβE removal (n = 7) (see Table 1 for the values).C, ACh-evoked currents return back to control amplitude and desensitization profiles after DHβE removal. Representative ACh-evoked currents recorded in control (top traces), after DHβE exposure (middle traces), and after recovery (bottom traces) are illustrated.Horizontal bars correspond to the ACh applications with concentration values (identical for traces in a vertical column).

Fig. 6.

Single-channel currents of human α4β2 display multiple conductance levels at low and high ACh concentrations.A, Portion of 800 msec recordings performed with a single patch have been enlarged to illustrate the different current amplitudes that could be observed. The thick horizontal bars above top traces indicate the applications of ACh. The thin dashed lines correspond to conductance levels of 0, 40, and 80 pS from top tobottom. B, Cumulative all-point amplitude histograms computed from several traces obtained in the same patch (the bin was set at 0.1 pA) were normalized to the total number of events in each recording condition (20 sweeps recorded in 100 nm ACh and 14 recorded in 30 μm ACh). The barscorresponding to the setup noise have been truncated to present the current amplitudes at a higher resolution. Note the increase of the low conductance event number when openings are elicited by a 30 μm ACh concentration. This observation has been repeated in all patches recorded in both ACh concentrations (n = 7).

Fig. 7.

Chronic incubation with DHβE increases the frequency of opening at 100 nm ACh. A, Typical single-channel currents recorded in an outside-out patch pulled from a control cell (left) or pulled from a cell incubated for 22 hr in 10 μm DHβE (right). The horizontal bars indicate pulses of ACh. Each trace is the first record of multiple sweeps (16 for the control cell and 17 for the 10 μm DHβE-treated cell). B, Average single-channel currents confirm the increase observed for whole-cell currents after DHβE-induced upregulation. Unitary currents including those presented inA were average for a control patch (left; mean of 10 sweeps) or for a patch pulled from a cell exposed for 22 hr to 10 μm DHβE (right; mean of 10 sweeps). C, Cumulative all-point amplitude histograms corresponding to multiple sweeps including and after the ones presented in A (16 sweeps in control and 17 sweeps for 10 μm DHβE). Surface areas of the Gaussians for channel openings and setup noise have values of 99 and 1703 (in control) and 288 and 1344 (in 10 μm DHβE). The open time probabilities are of 5.8% (control) and of 21.4% (10 μm DHβE) for these two representative patches.