Human and rodent bronchial epithelial cells express functional nicotinic acetylcholine receptors.

We demonstrated previously that human skin keratinocytes express acetylcholine receptors (AChRs) sensitive to acetylcholine and nicotine, which regulate cell adhesion and motility. We demonstrate here that human and rodent bronchial epithelial cells (BECs) express AChRs similar to those expressed by keratinocytes and by some neurons. Patch-clamp experiments demonstrated that the BEC AChRs are functional, and they are activated by acetylcholine and nicotine. They are blocked by kappa-bungarotoxin, a specific antagonist of the AChR isotypes expressed by neurons in ganglia. Their ion-gating properties are consistent with those of AChR isotypes expressed in ganglia, formed by alpha3, alpha5, and beta2 or beta4 subunits. Reverse transcription-polymerase chain reaction and in situ hybridization experiments demonstrated the presence in BECs of mRNA transcripts for all those AChR subunits, both in cell cultures and in tissue sections, whereas we could not detect transcripts for the alpha2, alpha4, alpha6, and beta3 AChR subunits. The expression of alpha3 and alpha5 proteins in BEC in vivo was verified by the binding of subunit-specific antibodies to sections of trachea. Mecamylamine and kappa-bungarotoxin, which are cholinergic antagonists able to block the ganglionic alpha3 AChRs, caused a reversible change of the cell shape of cultured, confluent human BECs. This resulted in a reduction of the area covered by the cell and in cell/cell detachment. The presence of AChRs sensitive to nicotine on the lining of the airways raises the possibility that the high concentrations of nicotine resulting from tobacco smoking will cause an abnormal activation, a desensitization, or both of the bronchial AChRs. This may mediate or facilitate some of the toxic effects of cigarette smoking in the respiratory system.

TCR V beta usage by acetylcholine receptor-specific CD4+ T cells in myasthenia gravis.

In myasthenia gravis the muscle acetylcholine receptor (AChR) is the target of an autoimmune response. AChR epitopes recognized by CD4+ T cells in myasthenic patients have been identified. AChR-specific CD4+ cell lines can be propagated by stimulation of blood lymphocytes with synthetic or biosynthetic AChR sequences. We analysed, using a semi-quantitative PCR assay, the T cell receptor (TCR) V beta usage of 16 anti-AChR polyclonal CD4+ T cell lines of known epitope specificity, propagated from myasthenic patients using pools of overlapping peptides corresponding to the sequence of an AChR subunit, or individual synthetic AChR sequences. Twelve lines had been propagated for less than 2 months, four lines for 3.5-5 months. Most lines had limited V beta usage, but in most cases different V beta regions were used for different epitopes in the same patient, and for the same epitope in different patients. In a few patients, the same V beta regions were used for recognition of different epitopes. The V beta 4 and V beta 6 regions were used most frequently. These findings suggest that the potentially autoimmune T cells that survive clonal deletion have a limited TCR repertoire. Although the present data do allow conclusions on the role of a superantigen in triggering the anti-AChR autoimmune response, the finding that different V beta regions were used in different patients does not support an important role of a superantigen in the maintenance of the CD4+ response in myasthenia gravis.

Activation of keratinocyte nicotinic cholinergic receptors stimulates calcium influx and enhances cell differentiation.

Human epidermal keratinocytes synthesize, secrete, and degrade acetylcholine and use their cell-surface nicotinic and muscarinic cholinergic receptors to mediate the autocrine and paracrine effects of acetyl-choline. Because acetylcholine modulates transmembrane Ca2+ transport and intracellular metabolism in several types of cells, we hypothesized that cholinergic agents might have similar effects on keratinocytes. Nicotine increased in a concentration-dependent manner the amount of 45Ca2+ taken up by keratinocytes isolated from human neonatal fore-skins. This effect was abolished in the presence of the specific nicotinic antagonist mecamylamine, indicating that it was mediated by keratinocyte nicotinic acetylcholine receptor(s). The sequences encoding the alpha 5 and alpha 7 nicotinic receptor subunits were amplified from cDNA isolated from cultured keratinocytes. These subunits, as well as the alpha 3, beta 2, and beta 4 subunits previously found in keratinocytes, can be components of Ca(2+)-permeable nicotinic receptor channels. To learn how activation of keratinocyte nicotinic receptors affected the rate of cell differentiation, we measured the nicotinic cholinergic effects on the expression of differentiation markers by cultured keratinocytes. Long-term incubations with micromolar concentrations of nicotine markedly increased the number of cells forming cornified envelopes and the number of cells staining with antibodies to suprabasal keratin 10, transglutaminase type I, involucrin, and filaggrin. The increased production of these differentiation-associated proteins was verified by Western blotting. Because nicotinic cholinergic stimulation causes transmembrane Ca2+ transport into keratinocytes, and because changes in concentrations of intracellular Ca2+ are known to alter various keratinocyte functions, including differentiation, the subcellular mechanisms mediating the autocrine and paracrine actions of epidermal acetylcholine on keratinocytes may involve Ca2+ as a second messenger.

Myasthenia gravis: recognition of a human autoantigen at the molecular level.

The symptoms of myasthenia gravis are primarily or exclusively due to an autoimmune response against the muscle nicotinic acetylcholine receptor (AChR) and this has been the object of intensive investigations for almost 20 years. A detailed picture at the molecular level of the interaction of this autoantigen with the key elements involved in the autoimmune response, such as anti-AChR antibodies, the T-cell receptor and restricting major histocompatibility complex molecules, is now emerging for both human myasthenia gravis and its experimental model, experimental autoimmune myasthenia gravis. Here, Maria Pia Protti and colleagues focus on the molecular interactions occurring in human myasthenia gravis and summarize recent information on pathogenic mechanisms of the autoimmune response, and the structure of epitopes recognized by B cells and CD4+ T cells of myasthenic patients on the AChR molecule.

The 'embryonic' gamma subunit of the nicotinic acetylcholine receptor is expressed in adult extraocular muscle.

We have PCR-amplified cDNA sequence of the "embryonic" gamma subunit of muscle acetylcholine receptor (AChR) from adult bovine extraocular muscle (EOM). We cloned and sequenced this product and used it to probe Northern blots. We detected the gamma subunit in EOM mRNA, but not in control skeletal muscle. The presence of gamma subunit in EOMs may explain their preferential involvement in myasthenia gravis, where an autoimmune response to the gamma subunit is generally present.