Positive allosteric modulation of alpha7 neuronal nicotinic acetylcholine receptors: lack of cytotoxicity in PC12 cells and rat primary cortical neurons

Abstract

Background and purpose: alpha7-Nicotinic acetylcholine receptors (alpha7 nAChRs) play an important role in cognitive function. Positive allosteric modulators (PAMs) amplify effects of alpha7 nAChR agonist and could provide an approach for treatment of cognitive deficits in neuropsychiatric diseases. PAMs can either predominantly affect the apparent peak current response (type I) or increase both the apparent peak current response and duration of channel opening, due to prolonged desensitization (type II). The delay of receptor desensitization by type II PAMs raises the possibility of Ca2+-induced toxicity through prolonged activation of alpha7 nAChRs. The present study addresses whether type I and II PAMs exhibit different cytotoxicity profiles.

Experimental approach: The present studies evaluated cytotoxic effects of type I PAM [N-(4-chlorophenyl)]-alpha-[(4-chlorophenyl)-aminomethylene]-3-methyl-5-isoxazoleacet-amide (CCMI) and type II PAM 1-[5-chloro-2,4-dimethoxy-phenyl]-3-[5-methyl-isoxazol-3-yl]-urea (PNU-120596), or 4-[5-(4chloro-phenyl)-2-methyl-3-propionyl-pyrrol-1-yl]-benzenesulphonamide (A-867744). The studies used cultures of PC12 cells and primary cultures of rat cortical neuronal cells.

Key results: Our results showed that neither type I nor type II PAMs had any detrimental effect on cell integrity or cell viability. In particular, type II PAMs did not affect neuron number and neurite outgrowth under conditions when alpha7 nAChR activity was measured by Ca2+ influx and extracellular signal-regulated kinases 1 and 2 phosphorylation, following exposure to alpha7 nAChR agonists.

Conclusions and implications: This study demonstrated that both type I and type II alpha7 nAChR selective PAMs, although exhibiting differential electrophysiological profiles, did not exert cytotoxic effects in cells endogenously expressing alpha7 nAChRs.

Figure 1

Effect of α7 nAChR type I and type II PAMs on intracellular Ca²⁺ levels in PC12 cells and rat cortical neuronal cells in culture. PC12 cells (A) or rat cortical neuronal cells (B) were treated with a range of concentrations of PNU-120596, A-867744 or CCMI for 10 min before the addition of 10 µM PNU-282987 for 5 min. Ca²⁺ influx was measured by FLIPR calcium 4 assay and expressed as relative fluorescence units (RFUs). Each point represents the mean ± SEM of three independent experiments.

Figure 2

Effect of α7 nAChR type I and type II PAMs on ERK1/2 phosphorylation in PC12 cells. (A) Representative images of ERK1/2 phosphorylation measured by InCell Western in a 96-well format. PC12 cells were treated with varying concentrations of PNU-120596, A-867744 or CCMI for 10 min at 37°C prior to the addition of 10 µM PNU-282987 for 5 min. Cells were then imaged for ERK1/2 phosphorylation signals. The superimposed image indicates co-staining of pERK (green) and tERK (red). (B) ERK1/2 phosphorylation. tERK and pERK signals were quantified and pERK/tERK ratio were normalized to the control response to 10 µM PNU-120596 + 10 µM PNU-282987. Each point represents the mean ± SEM of three independent experiments.

Figure 3

α7 nAChR type I and type II PAMs did not show cytotoxic effect on cell integrity or cell viability of PC12 cells. PC12 cells were cultured for 3 days and then treated with a range of concentrations of PNU-120596, A-867744 or CCMI alone or in the presence of 10 µM PNU-282987 for 24 h followed by measurements of cell integrity determined by the release of adenylate kinas and cell viability determined by mitochondrial dehydrogenase enzyme activity. (A) Cell integrity: PAMs alone or in combination with agonist did not increase the release of adenylate kinase, whereas thapsigargin reduced cell integrity in a concentration-dependant manner. (B) Cell viability: PAMs alone or in combination with the agonist did not affect mitochondrial dehydrogenase enzyme activity, whereas thapsigargin significantly reduced cell viability in a concentration-dependent manner. The values are shown as the percentage of untreated cells. Each point represents the mean ± SEM of 5 independent experiments. **P < 0.01 versus the untreated group.

Figure 4

α7 nAChR type I and type II PAMs did not show cytotoxic effect on integrity or viability of rat cortical neuronal cells. Rat primary cortical neuronal cells were cultured for 6 days and then treated with a range of concentrations of PNU-120596 or CCMI alone or in the presence of 10 µM PNU-282987 for 24 h. Cell integrity was determined by the release of adenylate kinase and cell viability was determined by mitochondrial dehydrogenase enzyme activity. (A) Cell integrity: PAMs alone or in combination with the agonist did not increase the release of adenylate kinase, whereas thapsigargin reduced cell integrity in a concentration-dependent manner. (B) Cell viability: PAMs alone or in combination with the agonist did not affect mitochondrial dehydrogenase enzyme activity, whereas thapsigargin significantly reduced cell viability in a concentration-dependent manner. The values are shown as the percentage of the untreated group. Each point represents the mean ± SEM of four independent experiments. **P < 0.01 versus the untreated group.

Figure 5

α7 nAChR type II PAMs did not show cytotoxic effect on morphology, cell number or neurite outgrowth of rat primary cortical neurons. Rat primary cortical neuronal cells were cultured for 6 days and then treated with a range of concentrations of PNU-120596 or A-867744 in the presence or absence of the α7 nAChR agonist PNU-282987 for 3 days. Neurons were stained with anti-tubulin antibody (green) and nuclei were stained with Hoechst 33342 (blue). (A) Representative superimposed images of neurons and nuclei treated with buffer control, 30 µM PNU-120596 or 30 µM PNU-120596 in combination with 10 µM PNU-282987 respectively. The PAMs alone or in combination with the agonist did not affect neuron morphology (A), neuron number (B), or neurite outgrowth (C). The values are shown as the percentage of the untreated group. Each point represents the mean ± SEM of three independent experiments.