• Cytoprotective Actions of Nicotine: The Increased Expression of a7 Nicotinic Receptors and NGF/TrkA Receptors

      Jonnala, Ramamohana R.; Department of Pharmacology and Toxicology (2001-07)
      Certain epidemiological studies have reported a negative correlation between smoking and neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease, reflecting perhaps the neurotrophic actions of nicotine. In recent years there has been intense interest in the development of new nicotinic acetylcholine receptor (nAChR) agonists. These agents have the potential to be used in the treatment of patients with AD. However, the mechanism for the neuroprotective action of the nicotine is not yet known, indeed, it is not yet clear as to which subtype of nAChR mediates the response. In neuronal cell lines, the induction of cytoprotection often requires exposure to nicotine for up to 24 hr to produce a full neuroprotective effect and this chronic exposure of nicotine is also known to increase nAChR receptors and cell surface nerve growth factor (NGF) receptors. The purpose of this study is to determine which subtype of nAChRs are involved in nicotine’s neuroprotective actions and also to determine whether nicotine’s neuroprotective actions are related to its ability to increase cell surface nAChRs and NGF receptors. Preincubation of differentiated PC 12 cells with nicotine for 24 hr protected the cells from growth factor withdrawal-induced toxicity in a time and concentration-dependent manner. Nicotine’s cytoprotective actions were completely blocked by non-selective nAChR antagonist mecamylamine, and the cc7nAChR preferring antagonist methyllcaconitine (MLA) indicating that the response was primarily mediated by the subtype of a7 nAChR receptors. The acetylcholine precursor, choline is a very selective and full agonist at a7 receptors. Among five choline analogs tested for neuroprotection potential, acetylpyrrolidinecholine and pyrrolidinecholine were found to be more potent and more efficacious than their parent compound, choline. The rank order of the six compounds tested for their cytoprotective ability is as follows: acetylpyrrolidinecholine = pyrrolidinecholine > choline = monoethylcholine = diethylcholine = triethylcholine. Further, to confirm the above structure activity relationships with respect to their binding affinities at a7nAChR, [I25I]a-bungarotoxin (BGT) displacement binding studies were performed using differentiated PC 12 cells. Choline was only 50 fold less potent than nicotine in displacing [ I]a-BGT binding. Pyrrolidinecholine, the most active analog, fully displaced [l25I]a-BGT binding and it exhibited a slightly greater affinity for the site than did choline. Next we compared the ability of seven different nAChR agonists with varying activities at a7 receptors for their ability to produce cytoprotection. Among the seven compounds tested, nicotine was the most effective and the most potent followed in order of potency by 40H-GTS21, epibatidine, methylcarbamylcholine, l,l-Dimethyl-4-phenyl-piperazinum, cytisine and tetraethylammonium. Since, epibatidine and cytisine were less efficacious than nicotine despite their greater affinity for a7 receptors and because short-term exposure of cells to nicotine did not produce cytoprotective actions, we next compared the ability of these compounds to upregulate cell surface a7 receptors. After, incubation of cells for 2 hr with either nicotine or cytisine, the number o f [I25I]a-BGT binding sites on differentiated PC 12 cells were measured. Nicotine, the most efficacious compound increased the [l2SI]a- BGT binding sites by ~40% over the untreated control cells. In contrast, cytisine, the least effective compound failed to do so, indicating that the ability to upregulate a7 receptors may provide one possible mechanism for neuroprotective actions of nicotine. Further, we confirmed that these additional populations of receptors were functional and that they mediate an enhanced neuroprotective response to subsequent nicotine stimulation. Earlier studies had shown that prolonged exposure to nicotine increases NGF receptors on differentiated PC12 cells. In the present study, high affinity NGF receptors (TrkA) were shown to increase with nicotine treatment in a time-and concentrationdependent manner. This effect was blocked by co-treatment with mecamylamine and with MLA, but not a low concentration of dihydro-P-erythroidine (selective for P2 and p4 containing receptors), indicating that the response was mediated by predominantly a7 nAChRs. Next, we measured the TrkA protein levels in rat hippocampal and cortical tissues after nicotine treatment. Chronic nicotine infusion increased TrkA protein levels within the hippocampus, and this effect was blocked by co-treatment with mecamylamine. In contrast, TrkA protein levels in cortical tissues were not altered. Since the majority of nAChRs in hippocampus are of the a7 subtype and whereas in cortex consist largely of the a4p2 subtype, it is reasonable to conclude that the differences observed in TrkA receptor expression in hippocampus and cortex were due to presence of a7nAChR. and non-a7nAChR. To determine whether the neuroprotective actions of nicotine were due to enhanced NGF trophic activity during the drug incubation period, the ability of nicotine to protect cells from trophic factor withdrawal in the presence and in the absence of NGF were compared. Nicotine was found to be effective in both experimental paradigms. However, nicotine was found to be 10 fold more effective when it was incubated with cells in the presence of NGF. When cells were treated with nicotine and k252a or nicotine and anti-TrkA antibody, nicotine was only partially effective as a neuroprotective agent, indicating that mechanisms apart from enhanced NGF mediated trophic activity during drug incubation period were involved in nicotine’s cytoprotective actions. One consistent finding with regard to in AD pathology is the selective loss of basal forebrain cholinergic neurons (BFC). The survival and maintenance of these neurons depended on the availability of NGF from target tissues. Evidence from previous studies suggests that impairment in NGF support could be an initial insult in AD pathology. Previous studies have shown that much of the oxidative damage in AD tissue was mediated by peroxynitrite. Breif exposure of undifferentiated PC 12 cells to 3- morpholinosydnonmine (SIN-1, peroxynitrite generator) was sufficient to inhibit an NGF mediated cellular response by 67% of that measured in control cells. This inhibition of the NGF cellular response by SIN-1 was not related to generalized cellular toxicity. In fact, the peroxynitrite scavenger uric acid significantly attenuated the inhibitory actions of SIN-1. Pretreatment with SIN-1 also resulted in a decrease in the NGF-induced phosphorylation of TrkA protein. Furthermore, SIN-1 treatment reduced the activity of mitogen activated protein kinase, a downstream kinase activated by TrkA receptor stimulation'. These data suggest that SIN-1 treatment inhibits NGF signaling by inactivating TrkA receptors through the formation of nitrotyrosine residues on the receptor. The inactivation of TrkA receptors may contribute to the initial insult that eventually ieads to neuronal cell death.