Choline and nicotine increase glioblastoma cell proliferation by binding and activating α7- and α9- containing nicotinic receptors.

Glioblastomas (GBMs), the most frequent and aggressive human primary brain tumours, have altered cell metabolism, and one of the strongest indicators of malignancy is an increase in choline compounds. Choline is also a selective agonist of some neuronal nicotinic acetylcholine receptor (nAChR) subtypes. As little is known concerning the expression of nAChR in glioblastoma cells, we analysed in U87MG human grade-IV astrocytoma cell line and GBM5 temozolomide-resistant glioblastoma cells selected from a cancer stem cell-enriched culture, molecularly, pharmacologically and functionally which nAChR subtypes are expressed and,whether choline and nicotine can affect GBM cell proliferation. We found that U87MG and GBM5 cells express similar nAChR subtypes, and choline and nicotine increase their proliferation rate and activate the anti-apoptotic AKT and pro-proliferative ERK pathways. These effects are blocked by the presence of non-cell-permeable peptide antagonists selective for α7- and α9-containing nicotinic receptors. siRNA-mediated silencing of α7 or α9 subunit expression also selectively prevents the effects of nicotine and choline on GBM cell proliferation. Our findings indicate that nicotine and choline activate the signalling pathways involved in the proliferation of GBM cells, and that these effects are mediated by α7 and α9-containing nAChRs. This suggests that these nicotinic receptors may contribute to the aggressive behaviour of this tumor and may indicate new therapeutic strategies against high-grade human brain tumours.

Expression of α3ß2ß4 nicotinic acetylcholine receptors by rat adrenal chromaffin cells determined using novel conopeptide antagonists.

Adrenal chromaffin cells release neurotransmitters in response to stress and may be involved in conditions such as post-traumatic stress and anxiety disorders. Neurotransmitter release is triggered, in part, by activation of nicotinic acetylcholine receptors (nAChRs). However, despite decades of use as a model system for studying exocytosis, the nAChR subtypes involved have not been pharmacologically identified. Quantitative real-time PCR of rat adrenal medulla revealed an abundance of mRNAs for α3, α7, ß2, and ß4 subunits. Whole-cell patch-clamp electrophysiology of chromaffin cells and subtype-selective ligands were used to probe for nAChRs derived from the mRNAs found in adrenal medulla. A novel conopeptide antagonist, PeIA-5469, was created that is highly selective for α3ß2 over other nAChR subtypes heterologously expressed in Xenopus laevis oocytes. Experiments using PeIA-5469 and the α3ß4-selective α-conotoxin TxID revealed that rat adrenal medulla contain two populations of chromaffin cells that express either α3ß4 nAChRs alone or α3ß4 together with the α3ß2ß4 subtype. Conclusions were derived from observations that acetylcholine-gated currents in some cells were sensitive to inhibition by PeIA-5469 and TxID, while in other cells, currents were sensitive only to TxID. Expression of functional α7 nAChRs was determined using three α7-selective ligands: the agonist PNU282987, the positive allosteric modulator PNU120596, and the antagonist α-conotoxin [V11L,V16D]ArIB. The results of these studies identify for the first time the expression of α3ß2ß4 nAChRs as well as functional α7 nAChRs by rat adrenal chromaffin cells.

Cy3-RgIA-5727 Labels and Inhibits α9-Containing nAChRs of Cochlear Hair Cells.

Efferent cholinergic neurons inhibit sensory hair cells of the vertebrate inner ear through the combined action of calcium-permeable α9α10-containing nicotinic acetylcholine receptors (nAChRs) and associated calcium-dependent potassium channels. The venom of cone snails is a rich repository of bioactive peptides, many with channel blocking activities. The conopeptide analog, RgIA-5474, is a specific and potent antagonist of α9α10-containing nAChRs. We added an alkyl functional group to the N-terminus of the RgIA-5474, to enable click chemistry addition of the fluorescent cyanine dye, Cy3. The resulting peptide, Cy3-RgIA-5727, potently blocked mouse α9α10 nAChRs expressed in Xenopus oocytes (IC50 23 pM), with 290-fold less activity on α7 nAChRs and 40,000-fold less activity on all other tested nAChR subtypes. The tight binding of Cy3-RgIA-5727 provided robust visualization of hair cell nAChRs juxtaposed to cholinergic efferent terminals in excised, unfixed cochlear tissue from mice. Presumptive postsynaptic sites on outer hair cells (OHCs) were labeled, but absent from inner hair cells (IHCs) and from OHCs in cochlear tissue from α9-null mice and in cochlear tissue pre-incubated with non-Cy3-conjugated RgIA-5474. In cochlear tissue from younger (postnatal day 10) mice, Cy3-RgIA-5727 also labeled IHCs, corresponding to transient efferent innervation at that age. Cy3 puncta in Kölliker's organ remained in the α9-null tissue. Pre-exposure with non-Cy3-conjugated RgIA-5474 or bovine serum albumin reduced this non-specific labeling to variable extents in different preparations. Cy3-RgIA-5727 and RgIA-5474 blocked the native hair cell nAChRs, within the constraints of application to the excised cochlear tissue. Cy3-RgIA-5727 or RgIA-5474 block of efferent synaptic currents in young IHCs was not relieved after 50 min washing, so effectively irreversible.