Elsevier

Brain Research

Volume 830, Issue 2, 5 June 1999, Pages 218-225
Brain Research

Research report
Characterization of the neuroprotective and toxic effects of α7 nicotinic receptor activation in PC12 cells

https://doi.org/10.1016/S0006-8993(99)01372-4Get rights and content

Abstract

The α7 nicotinic receptor partial agonist DMXB protected differentiated PC12 cells from NGF+serum deprivation over a concentration range (1–10 μM) that correlated with activation of protein kinase C. Increased toxicity was observed at a higher concentration of DMXB (30 μM) that did not elevate protein kinase C activity, but did increase tyrosine protein kinase activity. Neuroprotection was blocked with the protein kinase C-inhibitor bis-indolemaleimide, while toxicity was attenuated with the tyrosine protein kinase-antagonists herbimycin and genistein. The α7-selective antagonist methyllyconitine attenuated both the protective and toxic actions of DMXB, but in temporally distinct manners. Methyllyconitine (1 μM) attenuated toxicity when added 10 s before, but not 10 s after, 30 μM DMXB. In contrast, it blocked neuroprotection when added 10 min post-agonist addition. This temporal difference in receptor-activation that was necessary for protection vs. toxicity reflected the time courses for agonist-induced desensitization of the receptor expressed in Xenopus oocytes. These results indicate that α7 nicotinic receptors act through different intracellular transduction processes to protect or kill cells. Further, they suggest that the transduction processes may be differentially activated depending on the amplitude and duration of calcium signals.

Introduction

The α7 nicotinic receptor functions as an homo-oligomeric channel that is as permeant to calcium ions as excitotoxic NMDA receptors, but is much more rapidly desensitizing 5, 25. This receptor is found in a variety of brain regions, and is particularly concentrated in hippocampus and other telencephalic regions. It has been targeted therapeutically for Alzheimer's disease, based on studies with specific agonists such as 2,4-dimethoxybenzylideine anabaseine (DMXB, also known as GTS-21) that protect brain neurons as well as improve memory-related behaviors 18, 19, 27. α7 receptors are activated by the transmitter acetylcholine (ACh) as well as the membrane phospholipid-breakdown product choline; this choline-activation is not observed with other nicotinic receptor subtypes [21]. The model systems in which selective activation of α7 nicotinic receptors prevents neuronal death are many, including focal ischemia [26], excitotoxic insults 1, 27, trophic factor-deprivation 16, 18, 19, amyloid-exposure [12], and axotomy [16]. While information remains sparse about the intracellular processes underlying α7 nicotinic receptor-mediated neuroprotection, they are likely to be triggered by the receptor-mediated calcium-influx.

Two calcium-sensitive transduction systems that are associated with changes in cell viability are protein kinase C (PKC) and tyrosine protein kinase (TPK) [31]. PKC-activation has been associated with the anti-apoptotic effects of non-selective nicotinic receptor activation [32], so we investigated whether it may be involved in the DMXB-induced neuroprotection as well. Changes in TPK activity can also affect cell viability, though its role in nicotinic receptor-mediated neuroprotection has not been described 13, 33. This study used rat pheochromocytoma (PC12) cells that express α7 receptors [3]and are sensitive to the protective effects of DMXB during trophic factor deprivation 9, 16, 18, 19.

The possibility that over-activation of α7 nicotinic receptors may be neurotoxic was also considered. A previous study involving the expression of a mutated, non-desensitizing form of this receptor found reduced cell viability, apparently due to calcium-overloading [29]. Unpublished observations from our laboratory suggested that even cells expressing normal α7 receptors could be killed by acute application of high concentrations of nicotinic agonists. We therefore investigated the possible cytotoxic effect of high-dose DMXB in PC12 cells, as well as the roles of PKC and TPK activities in this toxicity.

The duration of receptor-activation necessary for cell loss was then compared to that required for neuroprotection using a selective receptor antagonist, methyllyconitine (MLA) [2]. Finally, the dose–response and temporal characteristics of α7 nicotinic receptor activation relative to cell viability and second messenger systems were compared to those seen in the Xenopus laevis oocyte expression system. This system effectively predicts the properties of α7 nicotinic receptors found in both peripheral and CNS neurons, e.g., with respect to rapid desensitization, high calcium permeability, pharmacological properties, and inward rectification 2, 36. Our results indicate that these receptor-properties are also consistent with the differential cellular actions of low- vs. high-dose DMXB in PC12 cells.

Section snippets

Assessment of PC12 cell number and viability

PC12 cells were obtained from the original clonal line maintained at American Type Culture Collection (Rockville, MD). They were cultured and maintained as described previously 16, 18. Cultures were split at a 1:4 ratio every 3 days, up to eight passages, until differentiated was initiated with 100 ng/ml NGF. PC12 cell density was measured at 24 h or 4 days post-treatment for toxicity and protection studies, respectively, using the NIH Image 1.55 program. Five random photographs were analyzed

Results

Removal of NGF and serum from differentiated PC12 cells reduced cell density in a manner that was attenuated by 1–10 μM DMXB but exacerbated by 30 μM DMXB (Fig. 1). DMXB also increased PKC-translocation to membrane binding sites in a concentration-dependent manner after a 5 min exposure (Fig. 1). That PKC-activation was important for the neuroprotective activity of DMXB was demonstrated with the cell-permeant PKC antagonist bis-indole maleimide (BIM; [14]) (Table 1). This concentration of BIM

Discussion

The present study demonstrates that the degree of α7 nicotinic receptor activation can modulate cell viability in a dual manner that differentially involves PKC and TPK activities. Neuroprotection is observed with low concentrations of DMXB that increase PKC translocation but that have no effect on TPK at any time point assayed up to 30 min. At least 10 min of low-level channel activation is necessary for neuroprotection conferred by 3 μM DMXB, which is consistent with the ongoing tonic

Acknowledgements

The authors appreciate the expert administrative assistance of Allison Dasta. This research was supported in part by NIA PO1 10485 and Taiho Pharmaceuticals, Japan.

References (36)

  • P.R. Walker et al.

    Relationship between apoptosis and the cell cycle in lymphocytes: roles of protein kinase C, tyrosine phosphorylation, and AP1

    Exp. Cell Res.

    (1993)
  • A. Yamauchi et al.

    Target cell-induced apoptosis of interleukin-2-activated human natural killer cells: roles of cell surface molecules and intracellular events

    Blood

    (1996)
  • Z. Zhang et al.

    Synaptic currents generated by neuronal acetylcholine receptors sensitive to α-bungarotoxin

    Neuron

    (1996)
  • M. Alkondon et al.

    Blockade of nicotinic currents in hippocampal neurons defines methyllycaconitine as a potent and specific receptor antagonist

    Mol. Pharmacol.

    (1992)
  • E.M. Blumenthal et al.

    Detection of functional nicotinic receptors blocked by α-bungarotoxin on PC12 cells and dependence of their expression on post-translational events

    J. Neurosci.

    (1997)
  • C.M. Cagnoli et al.

    Apoptosis induced in neuronal cultures by the phosphatase inhibitor staurosporine is blocked by the isoquinolinesulfonamides H-7, H-8, and H-9

    J. Mol. Neurosci.

    (1996)
  • S. Couturier et al.

    A neuronal nicotinic acetylcholine receptor subunit (α7) is developmentally regulated and forms a homo-oligomeric channel blocked by α-bungarotoxin

    Neuron

    (1990)
  • C.M. de Fiebre et al.

    Characterization of a family of anabaseine-derived compounds reveals that the 3-(4)-dimethylamino-cinnamylideine derivative is a selective agonist at neuronal nicotinic α7/α-bungarotoxin receptor subtypes

    Mol. Pharmacol.

    (1995)
  • Cited by (101)

    • B-973, a novel piperazine positive allosteric modulator of the α7 nicotinic acetylcholine receptor

      2017, European Journal of Pharmacology
      Citation Excerpt :

      Partial agonists induce receptor desensitization similar to full orthosteric agonists, suggesting that chronic exposure may limit efficacy and lead to tachyphalaxis. Other potential limitations of partial agonists include the upregulation of receptor number (Sallette et al., 2005; Whiteaker et al., 1998) and possible cellular toxicity associated with chronic receptor activation (Li et al., 1999). α7 receptor PAMs, which lack intrinsic agonist activity, represent an attractive therapeutic alternative to orthosteric agonists.

    View all citing articles on Scopus
    1

    These two authors contributed equally to this work.

    View full text