Expression of the α7 nicotinic acetylcholine receptor in human lung cells

BACKGROUND: We and others have shown that one of the mechanisms of growth regulation of small cell lung cancer cell lines and cultured pulmonary neuroendocrine cells is by the binding of agonists to the α7 neuronal nicotinic acetylcholine receptor. In addition, we have shown that the nicotine-derived carcinogenic nitrosamine, 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a high affinity agonist for the α7 nicotinic acetylcholine receptor. In the present study, our goal was to determine the extent of α7 mRNA and protein expression in the human lung. METHODS: Experiments were done using reverse transcription polymerase chain reaction (RT-PCR), a nuclease protection assay and western blotting using membrane proteins. RESULTS: We detected mRNA for the neuronal nicotinic acetylcholine receptor α7 receptor in seven small cell lung cancer (SCLC) cell lines, in two pulmonary adenocarcinoma cell lines, in cultured normal human small airway epithelial cells (SAEC), one carcinoid cell line, three squamous cell lines and tissue samples from nine patients with various types of lung cancer. A nuclease protection assay showed prominent levels of α7 in the NCI-H82 SCLC cell line while α7 was not detected in SAEC, suggesting that α7 mRNA levels may be higher in SCLC compared to normal cells. Using a specific antibody to the α7 nicotinic receptor, protein expression of α7 was determined. All SCLC cell lines except NCI-H187 expressed protein for the α7 receptor. In the non-SCLC cells and normal cells that express the α7 nAChR mRNA, only in SAEC, A549 and NCI-H226 was expression of the α7 nicotinic receptor protein shown. When NCI-H69 SCLC cell line was exposed to 100 pm NNK, protein expression of the α7 receptor was increased at 60 and 150 min. CONCLUSION: Expression of mRNA for the neuronal nicotinic acetylcholine receptor α7 seems to be ubiquitously expressed in all human lung cancer cell lines tested (except for NCI-H441) as well as normal lung cells. The α7 nicotinic receptor protein is expressed in fewer cell lines, and the tobacco carcinogen NNK increases α7 nicotinic receptor protein levels

Differential effects of chronic drug treatment on α3(*) and α7 nicotinic receptor binding sites, in hippocampal neurones and SH-SY5Y cells

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 α7 nicotinic AChR, identified by [(125)I]-α bungarotoxin (α-Bgt) binding, in primary hippocampal cultures and SH-SY5Y cells. Numbers of α3(*) nicotinic AChR were also examined in SH-SY5Y cells, using [(3)H]-epibatidine, which is predicted to label the total cellular population of predominantly α3β2(*) nicotinic AChR under the conditions used. 2. All the nicotinic agonists examined, the antagonists d-tubocurarine and methyllycaconitine, and KCl, upregulated [(125)I]-α Bgt binding sites by 20–60% in hippocampal neurones and, where examined, SH-SY5Y cells. 3. Upregulation of [(125)I]-α-Bgt binding sites by KCl was prevented by co-incubation with the L-type Ca(2+) channel blocker verapamil or the Ca(2+)-calmodulin dependent kinase II (CaM-kinase II) inhibitor KN-62. Upregulation of [(125)I]-α-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 α3(*) nicotinic AChR. 5. These data indicate that numbers of α3(*) and α7 nicotinic AChR are modulated differently. Nicotinic agonists and KCl upregulate α7 nicotinic AChR through distinct cellular mechanisms, the latter involving L-type Ca(2+) channels and CaM-kinase II. In contrast, α3(*) nicotinic AChR are not upregulated by KCl. This difference may reflect the distinct physiological roles proposed for α7 nicotinic AChR

Effects of chronic drug treatments on increases in intracellular calcium mediated by nicotinic acetylcholine receptors in SH-SY5Y cells

1. SH-SY5Y cells express α7 and α3* subtypes of nicotinic acetylcholine receptors (AChR). Numbers of these receptors are upregulated by chronic treatment with nicotinic agonists or KCl. In this study we have examined the functional consequences of these drug treatments on nicotine- or KCl-evoked increases in [Ca(2+)](i), in SH-SY5Y cells. 2. In untreated cells, nicotine increased [Ca(2+)](i) (EC(50) 7.5 μM). Responses to 10 μM nicotine were abolished by the non-selective nicotinic antagonist mecamylamine and were partially blocked by α7-selective antagonists, the α3β2*-selective antagonist α-conotoxin-MII, and by cadmium and verapamil. 3. After treatment for 4 days with nicotinic agonists, nicotine-evoked increases in [Ca(2+)](i) were significantly decreased by about 25%. Nicotine-evoked responses were paradoxically increased in the presence of acute methyllycaconitine (MLA; an α7-selective antagonist) although other α7-selective antagonists were without effect, while α-conotoxin-MII gave a partial inhibition. The increase observed with MLA was abolished by mecamylamine but not by α-conotoxin-MII and was still observed 24 h after chronic nicotine treatment. 4. After treatment for 4 days with KCl, nicotine-evoked increases in [Ca(2+)](i) were also decreased by 25%, but acute MLA was without effect. Responses to 20 mM KCl were unchanged by prior treatment with nicotine or KCl. Treatment for 4 days with 5 μM verapamil reduced responses to both nicotine and KCl by about 50%. 5. Multiple nicotinic AChR subtypes contribute to nicotine-evoked increases in [Ca(2+)](i) in SH-SY5Y cells. Responses to acute nicotine are reduced after chronic nicotine or KCl treatment, with loss of the component attributed to the α7 subtype. However, in nicotine-treated cells this effect is reversed when nicotine stimulation is applied in the presence of acute MLA. The antagonist may assist in converting a non-functional α7 nicotinic AChR to a conducting state

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

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 α-conotoxin MII (MII; 10 nM), suggesting contribution by α7 and α3 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 μM) 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

Effect of acute nicotine administration on striatal dopamine output and metabolism in rats kept at different ambient temperatures

1. The effect of ambient temperature on the nicotine-induced (0.3, 0.5 or 0.8 mg kg(−1) s.c.) changes of the striatal concentrations of dopamine (DA) and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) was studied in freely-moving rats by in vivo microdialysis. 2. At the ambient temperature of 30–33°C, but not at 20–23°C, nicotine doses of 0.5 (P<0.01) and 0.8 mg kg(−1) (P<0.05) significantly increased the extracellular DA concentration. The nicotine doses of 0.5 and 0.8 mg kg(−1) increased the DA metabolite levels similarly at both ambient temperatures studied (P⩽0.0001), but the dose of 0.3 mg kg(−1) only at 30–33°C (DOPAC: P<0.05; HVA: P<0.01). 3. At 30–33°C, dihydro-β-erythroidine (DHβE 2.8 mg kg(−1) i.p.) blocked the nicotine-induced (0.5 or 0.8 mg kg(−1)) increases of extracellular DA concentration but only tended to antagonize the increases of DA metabolites. Mecamylamine (5.0 mg kg(−1) i.p.) blocked the increase of DA output induced by 0.5 mg kg(−1) but not that induced by 0.8 mg kg(−1) of nicotine and fully prevented the nicotine-induced elevations of DOPAC and HVA. 4. Elevation of ambient temperature did not affect the cerebral concentration of nicotine or the nicotine-induced elevation of serum corticosteroids. Also, the rectal temperatures of rats given nicotine at either ambient temperature did not significantly change. 5. Our results show that the nicotine-induced output of striatal DA is enhanced at high ambient temperature. Further, our findings suggest that the nicotinic cholinoceptors mediating the effects of nicotine on striatal DA release are different from those mediating nicotine's effects on DA metabolism

Nicotine modulates GABAergic transmission to dopaminergic neurons in substantia nigra pars compacta

AIM: Dopaminergic neurons in the substantia nigra pars compacta (SNc) play important roles in motor control and drug addiction. As the major afferent, GABAergic innervation controls the activity of SNc dopaminergic neurons. Although it is clear that nicotine modulates SNc dopaminergic neurons by activating subtypes of somatodendritic nicotinic acetylcholine receptors (nAChRs), the detailed mechanisms of this activation remain to be addressed. METHODS: In the current study, we recorded GABA(A) receptor-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) from dissociated SNc dopaminergic neurons that were obtained using an enzyme-free procedure. These neurons preserved some functional terminals after isolation, including those that release GABA. RESULTS: We found that both extra- and intra-cellular calcium modulates sIPSCs in these neurons. Furthermore, both nicotine and endogenous acetylcholine enhance the frequency of sIPSCs. Moreover, endogenous acetylcholine tonically facilitates sIPSC frequency, primarily by activating the α4β2* nAChRs on the GABAergic terminals. CONCLUSION: Nicotine facilitates GABA release onto SNc dopaminergic neurons mainly via the activation of presynaptic α4β2* nAChRs