Differential effects of chronic drug treatment on alpha3* and alpha7 nicotinic receptor binding sites, in hippocampal neurones and SH-SY5Y cells

Abstract

1. The aim of this study was to compare the effects of chronic treatment (for 4 or 7 days) with nicotinic drugs and 20 mM KCl on numbers of surface alpha7 nicotinic AChR, identified by [(125)I]-alpha bungarotoxin (alpha-Bgt) binding, in primary hippocampal cultures and SH-SY5Y cells. Numbers of alpha3* nicotinic AChR were also examined in SH-SY5Y cells, using [(3)H]-epibatidine, which is predicted to label the total cellular population of predominantly alpha3beta2* nicotinic AChR under the conditions used. 2. All the nicotinic agonists examined, the antagonists d-tubocurarine and methyllycaconitine, and KCl, upregulated [(125)I]-alpha Bgt binding sites by 20 - 60% in hippocampal neurones and, where examined, SH-SY5Y cells. 3. Upregulation of [(125)I]-alpha-Bgt binding sites by KCl was prevented by co-incubation with the L-type Ca2+ channel blocker verapamil or the Ca2+-calmodulin dependent kinase II (CaM-kinase II) inhibitor KN-62. Upregulation of [(125)I]-alpha-Bgt binding sites by nicotine or 3,[(4-dimethylamino) cinnamylidene] anabaseine maleate (DMAC) was insensitive to these agents. 4. [(3)H]-Epibatidine binding sites in SH-SY5Y cells were not affected by KCl but were upregulated in a verapamil-insensitive manner by nicotine and DMAC. KN-62 itself provoked a 2 fold increase in [(3)H]-epibatidine binding. The inactive analogue KN-04 had no effect, suggesting that CaM-kinase II plays a role in regulating numbers of alpha3* nicotinic AChR. 5. These data indicate that numbers of alpha3* and alpha7 nicotinic AChR are modulated differently. Nicotinic agonists and KCl upregulate alpha7 nicotinic AChR through distinct cellular mechanisms, the latter involving L-type Ca2+ channels and CaM-kinase II. In contrast, alpha3* nicotinic AChR are not upregulated by KCl. This difference may reflect the distinct physiological roles proposed for alpha7 nicotinic AChR.

Figure 1

The effect of chronic drug treatment on numbers of [¹²⁵I]-α-Bgt binding sites in hippocampal cultures. Hippocampal cultures from E18 rat foetuses were treated for 7 days with (a) nicotinic agonists or salts, (b) nicotinic antagonists in the presence or absence of agonist, or (c) KCl and agonist separately and in combination. Following extensive washing, as described in Methods, binding assays for surface [¹²⁵I]-α-Bgt binding sites were carried out on cells in situ. Specific [¹²⁵I]-α-Bgt binding to control cells, cultured in parallel in the absence of drug, was taken to be 100%. Results are expressed as mean±s.e.mean of at least four independent cultures, each culture was treated and assayed in triplicate for each drug condition. ^*Significantly different from control, P<0.05, (one way ANOVA). NS, not significantly different.

Figure 2

The effects of verapamil and KN-62 on the upregulation of [¹²⁵I]-α-Bgt binding sites in hippocampal cultures. Cultures were treated for 4 days with 20 mM KCl or 10 μM nicotine, in the presence or absence of (a) 5 μM verapamil or (b) 5 μM KN-62. Controls with no drug treatment were cultured in parallel. Specific [¹²⁵I]-α-Bgt binding to cells in situ was determined as described in Methods, and binding to control cultures was taken to be 100%. Results are expressed as mean±s.e.mean of at least four independent cultures, each culture was treated and assayed in triplicate for each drug condition. ^*Significantly different from control, P<0.05, (one way ANOVA).

Figure 3

The upregulation of [¹²⁵I]-α-Bgt binding sites in SH-SY5Y cells treated with (a) KCl or (b) nicotine, and the effects of verapamil and KN-62. SH-SY5Y cells were cultured for 4 days with 20 mM KCl, 10 μM nicotine or no addition (control), in the presence or absence of 5 μM verapamil or 5 μM KN-62. Specific [¹²⁵I]-α-Bgt binding to cells in situ was determined as described in Methods, and binding to control cultures was taken to be 100%. Results are expressed as mean±s.e.mean of at least four independent cultures, each culture was treated and assayed in triplicate for each drug condition. ^*Significantly different from control, P<0.05, (one way ANOVA).

Figure 4

The upregulation of [³H]-epibatidine binding sites in SH-SY5Y cells treated with (a, d) KCl, (b, e) nicotine and (c, f) DMAC, and the effects of verapamil, KN-62 and KN-04. SH-SY5Y cells were cultured for 4 days with 20 mM KCl, 10 μM nicotine, 10 μM DMAC or no addition (control), in the presence or absence of (a, b, c) 5 μM verapamil or (d, e, f) 5 μM KN-62 or 5 μM KN-04. Specific [³H]-epibatidine binding to cells in situ was determined as described in Methods, and binding to control cultures was taken to be 100%. Results are expressed as mean±s.e.mean of at least four independent cultures, each culture was treated and assayed in triplicate for each drug condition. ^*Significantly different from control, P<0.05, (one way ANOVA).