Cellular responses to nicotinic receptor activation are decreased after prolonged exposure to galantamine in human neuroblastoma cells

Abstract

In this study, we have examined cellular responses of neuroblastoma SH-SY5Y cells after chronic treatment with galantamine, a drug used to treat Alzheimer's disease that has a dual mechanism of action: inhibition of acetylcholinesterase and allosteric potentiation of nicotinic acetylcholine receptors (nAChR). Acute experiments confirmed that maximum potentiation of nicotinic responses occurs at 1 microM galantamine; hence this concentration was chosen for chronic treatment. Exposure to 1 microM galantamine for 4 days decreased Ca(2+) responses (by 19.8+/-3.6%) or [(3)H]noradrenaline ([(3)H]NA) release (by 23.9+/-3.3%) elicited by acute application of nicotine. KCl-evoked increases in intracellular Ca(2+) were also inhibited by 10.0+/-1.9% after 4 days' treatment with galantamine. These diminished responses are consistent with the downregulation of downstream cellular processes. Ca(2+) responses evoked by activation of muscarinic acetylcholine receptors were unaffected by chronic galantamine treatment. Exposure to the more potent acetylcholinesterase inhibitor rivastigmine (1 microM) for 4 days failed to alter nicotine-, KCl-, or muscarinic receptor-evoked increases in intracellular Ca(2+). These observations support the hypothesis that chronic galantamine exerts its effects through interaction with nAChR in this cell line. Exposure to 10 microM nicotine for 4 days produced decreases in acute nicotine- (18.0+/-3.5%) and KCl-evoked Ca(2+) responses (10.6+/-2.5%) and nicotine-evoked [(3)H]NA release (26.0+/-3.3%) that are comparable to the effects of a corresponding exposure to galantamine. Treatment with 1 microM galantamine did not alter numbers of [(3)H]epibatidine-binding sites in SH-SY5Y cells, in contrast to 62% upregulation of these sites in response to 10 microM nicotine. Thus, chronic galantamine acts at nAChR to decrease subsequent functional responses to acute stimulation with nicotine or KCl. This effect appears to be independent of the upregulation of nAChR-binding sites.

Figure 1

Galantamine potentiates nicotine-evoked Ca²⁺ increases in SH-SY5Y cells. SH-SY5Y cells loaded with fluo-3 AM were incubated for 5 min with a range of concentrations of galantamine and then stimulated by addition of 30 μM nicotine (Nic). Responses were monitored by determining the change in fluorescence after 20 s. Data are expressed as a percentage of the response to nicotine in the absence of galantamine. Values represent the mean±s.e.m. of seven experiments, each performed in quadruplicate. Significantly different from control, ^**P<0.01, one-way ANOVA with post hoc Tukey's test.

Figure 2

Effect of chronic drug treatment on nAChR-mediated increases in Ca²⁺ in SH-SY5Y cells. SH-SY5Y cells were cultured for 4 days in presence or absence of 10 μM nicotine (Nic) or 1 μM galantamine (Gal). After extended washing over 3 h with fresh medium, SH-SY5Y cells were loaded with fluo-3 AM and stimulated with 30 μM nicotine. (a) Representative traces showing the time course of increases in fluorescence over 20 s in cells treated for 4 days with 10 μM nicotine (dotted line), 1 μM galantamine (dashed line) or no drug (control; solid line). (b) Cells were stimulated in the presence or absence of 10 μM mecamylamine (mec) or 200 nM α-conotoxin MII (α-cntxMII). Fluorescence increases were monitored for 20 s (see panel a). Values at 20 s were expressed as a percentage of response of control cells. Data are the mean±s.e.m. of at least five experiments, each carried out in quadruplicate. (c) Representative traces showing the time course of increases in fluorescence over an extended time course (10 min) in cells treated for 4 days with 10 μM nicotine (dotted line), 1 μM galantamine (dashed line) or no drug (control; solid line). (d) Comparison of responses at 20 s, peak and 10 min. Values are expressed as a percentage of peak responses and are the mean±s.e.m. of eight experiments, each performed in quadruplicate. Significantly different from control, ^*P<0.05, ^***P<0.001 one-way ANOVA with post hoc Tukey's test; ^†P<0.05 paired t-test.

Figure 3

Effect of 24 h drug treatment on nAChR-mediated Ca²⁺ responses in SH-SY5Y cells. SH-SY5Y cells were cultured for 4 days in presence of 10 μM nicotine (Nic; black bars), 1 μM galantamine (Gal; hatched bars) or no drug (control; open bars). After extended washing over 3 h with fresh medium, SH-SY5Y cells were loaded with fluo-3 AM and stimulated with 30 μM nicotine in the presence or absence of 10 μM mecamylamine (mec) or 200 nM α-conotoxin MII (α-cntxMII). Changes in fluorescence were monitored for 20 s. Values are expressed as a percentage of control responses. Bars represent the mean±s.e.m. of at least four independent experiments. Significantly different from control ^*P<0.05, one-way ANOVA with post hoc Tukey's test; ^††P<0.01 paired t-test.

Figure 4

Comparison of chronic drug treatments on mAChR-, nicotine- and KCl-evoked responses. SH-SY5Y cells were cultured for 4 days in presence of 10 μM nicotine (Nic; black bars), 1 μM galantamine (Gal; hatched bars); 1 μM rivastigmine (Riva; finely hatched bars) or no drug (Control; open bars). After extended washing over 3 h with fresh medium, SH-SY5Y cells were loaded with fluo-3 AM and stimulated with (a) 1 μM ACh, in the presence or absence of 1 μM atropine; (b) 40 mM KCl or (c) 30 μM nicotine. Fluorescence was measured for 20 s and results are expressed as a percentage of control responses; significantly different from control, ^*P<0.05, ^**P<0.01, one-way ANOVA with post hoc Tukey's test.

Figure 5

Nicotine-evoked [³H]NA release from SH-SY5Y cells after chronic drug treatment. SH-SY5Y cells were cultured for 4 days in presence of 10 μM nicotine (Nic; black bars), 1 μM galantamine (Gal; hatched bars) or no drug (control; open bars). After extended washing over 3 h with fresh medium, SH-SY5Y cells were loaded with [³H]NA and the release of tritium was induced by 5 min incubation with buffer or 30 μM nicotine. Data are expressed as a percentage of buffer-evoked release and represent the mean±s.e.m. of five independent experiments, each conducted with eight replicates. ^†P<0.001, significantly different from control response to nicotine stimulation (one-way ANOVA with post hoc Tukey's test).

Figure 6

Upregulation of [³H]epibatidine-binding sites in SH-SY5Y cells. SH-SY5Y cells were cultured for 4 days in presence of 10 or 50 μM nicotine (Nic; black bars), 1 or 10 μM galantamine (Gal; hatched bars) or no drug (control; open bar). The cells were washed and membranes prepared as described in Methods. The density of nAChR-binding sites was determined by incubation with 500 pM [³H]epibatidine in the absence or presence of 1 mM nicotine, to determine total and nonspecific binding, respectively. Results are expressed as a percentage of control and are the mean±s.e.m. of four experiments, each performed in triplicate. Significantly different from control, ^*P<0.05, Student's paired t-test.