Desensitization of neuronal nicotinic receptors of human neuroblastoma SH‐SY5Y cells during short or long exposure to nicotine

Abstract

1 Neuronal nicotinic ACh receptors (nAChRs) readily desensitize in the presence of an agonist. However, when the agonist is applied for minutes, hours or days, it is unclear how extensive desensitization is, how long it persists after agonist removal and whether nAChRs consequently change their pharmacological properties. 2 These issues were explored with electrophysiological studies of native receptors of voltage-clamped human neuroblastoma SH-SY5Y cells. Puffer pulses of nicotine (1 mM)-evoked inward currents partly antagonized by methyllycaconitine (MLA; 10 nM) or a-conotoxin MII (MII; 10 nM), suggesting contribution by a7 and a3 subunit containing receptors, respectively. Nicotine-evoked currents desensitized with 150 ms time constant and fully recovered after a few s washout. 3 Although the current induced by 10 min application of nicotine (10 mM) decayed to baseline indicating complete desensitization, puffer applications of maximally effective doses of nicotine still generated small responses (22% of control). Similar responses to puffer-applied nicotine were observed when nicotine was chronically incubated for 8 or 48 h. On nicotine washout, cells recovered their response amplitude within 5 min and then increased it (about 50% of untreated controls) after 30 min without altering response kinetics or sensitivity to MLA and MII. 4 The present results suggest that native nAChRs of SH-SY5Y cells preserved a degree of responsiveness during chronic application of nicotine, and that they rapidly recovered on washout to generate larger responses without changes in kinetics or pharmacology. These data indicate strong compensatory mechanisms to retain nicotinic receptor function during long-term exposure to nicotine.

Increased nicotinic receptor desensitization in hypoglossal motor neurons following chronic developmental nicotine exposure

“…However, the time course and the extent to which nAChR properties are affected in various experimental preparations are still unclear (for reviews, see Gentry and Lukas 2002;Quick and Lester 2002). Depending on the experimental details (e.g., duration and timing of exposure), chronic activations can increase, decrease, or have no effect on nAChR expression and/or function (Rowell and Wonnacott 1990;Lukas 1991;Marks et al 1993;Peng et al 1994;Yu and Wecker 1994;Buisson and Bertrand 2001;Sokolova et al 2005). Here, we used a thick medullary slice preparation, which preserves nAChR location, density, number, and subtype composition, and tested whether DNE increases the magnitude of receptor desensitization and recovery.…”

“…Last, one or all of the following controls were employed for each cell studied: 1) In 12 cells, nicotine-evoked currents were fully suppressed by nAChR antagonists dihydro-␤-erythoidine hydrobromide (DH-␤-E; 0.2 M) and/or mecamylamine (1 M), as described previously (Chamberlin et al 2002;Quitadamo et al 2005); 2) In all 45 cells, nicotine-mediated currents were eliminated at a holding potential near 0 mV, the approximate reversal potential for these nonspecific cation channels (Sokolova et al 2005); 3) In seven cells, we used a 1-s pulse of nicotine to produce a sustained plateau current, which eventually desensitized and decayed back to baseline (Fig. 1C) (Di Angelantonio and Nistri 2001;Quitadamo et al 2005); 4) In three cells, nicotine (0.5 M) was added to the superfusate for Ն15 min (n ϭ 3) to evoke nAChR desensitization, which was documented by showing that the acute nicotine-evoked current evoked by pressure pulses was reduced (Fig.…”

“…Accordingly, membrane properties of nAChRs, especially activation and desensitization, have been extensively studied using receptors expressed at the neuromuscular junction (Colquhoun and Sakmann 1998;Fatt and Katz 1951;Galzi and Changeux 1995;Katz and Thesleff 1957) and in culture (Buisson and Bertrand 2001;Lukas 1991;Peng et al 1997;Sokolova et al 2005;Vallejo et al 2005). Despite their widespread presence in the CNS, much less is known about nAChR behavior in functionally viable brain tissue where the target neurons maintain their in vivo anatomical location, density, number, and subunit composition (but see Calabresi et al 1989;Guo and Lester 2007;Lester and Dani 1995;Wooltorton et al 2003).…”

A Single Administration of Low-Dose Varenicline Saturates α4β2* Nicotinic Acetylcholine Receptors in the Human Brain

“…How the activation and desensitization properties of these nAChRs are modified in the human smoker is not known; although it has been hypothesized that both receptor properties could influence nicotine addiction and mood (Picciotto et al, 2008). Although some authors have provided evidence to suggest that upregulated high-affinity nAChRs may be nonfunctional (Quick and Lester, 2002), other evidence suggests that upregulated high-affinity nAChRs retain functionality in the presence of chronic nicotine (Sokolova et al, 2005), and under certain circumstances are less sensitive to desensitization (Buisson and Bertrand, 2001). The possible reasons for the discrepancy between the findings have been discussed previously (Quick and Lester, 2002).…”

“…Evidence suggests that A-85380 is more functional at the high-affinity nAChRs that have (a4)(2)(b2)(3) vs the low-affinity site having (a4)(3)(b2)(2) stoichiometry (Moroni et al, 2006). These receptor subtypes can have differing functionality, depending, eg, on the length of nicotine exposure (Sokolova et al, 2005) and the presence or absence of excess a4 subunits (Harpsoe et al, 2011) or accessory subunits (such as a5) (Ramirez-Latorre et al, 1996). Unlabeled A-85830 and nicotine, but less so varenicline, appear to have higher functional efficacy at (a4)(2)(b2)(3) vs (a4)(3) (b2)(2) stoichiometry nAChRs (Anderson et al, 2009;Moroni et al, 2006).…”