Cellular and molecular mechanisms underlying nicotine induced upregulation of alpha 7 nicotinic acetylcholine receptor expressed in xenopus oocytes: The role of CA2+ and CA2+-dependent signaling pathways.

摘要

Neuronal nicotinic-acetylcholine receptors (nAChRs) (e.g., alpha4beta2, alpha7 Rs) appear to play critical roles in learning, memory, and various neuropathologies including nicotine addiction. Nicotine-upregulation of alpha7 Rs is thought to play a significant role in these phenomena. But whether nicotine-upregulation of alpha7 Rs in fact occurs, and the nature of its underlying mechanism(s), are largely unknown. Previous in vitro studies of alpha7 nAChRs heterologously expressed in Xenopus oocytes failed to observe nicotine-upregulation. These failures might have been due to incomplete removal of nicotine from the recording media, as a result of its intracellular accumulation and subsequent slow release from the oocytes, resulting in desensitization of alpha7 Rs during functional assays. Our GC/MS measurements confirmed that this was likely to be the case. In our experiments, 12-14 hr exposure to nicotine (100 microM), followed by extensive 7 hr washout yielded reliable, statistically-significant ~2-fold increases in macroscopic alpha7 R currents (as determined by two-electrode voltage clamp) and alpha7-protein (by Western blot). Less-extensive washout failed to produce upregulation; instead, desensitization was observed. Nicotine-upregulation was also correlated with the level of surface expression of alpha7 Rs, and did not involve new protein synthesis. Similar to nicotine, methyllycaconitine, a cell-permeable competitive antagonist of alpha7 Rs, as well as carbachol, a membrane-impermeable agonist, also produced upregulation, suggesting that ligand-binding to alpha7 Rs (but not activation of the receptors) was critical. Nicotine-upregulation of alpha7 Rs was unaffected by removal of extracellular Ca2+. However, intracellular Ca2+ chelation completely blocked upregulation. Several Ca2+ -dependent intracellular signaling pathways appeared to be critical for nicotine -upregulation: PP2B/calcineurin (inhibited by cyclosporine A), serine-threonine protein kinase-activity (inhibited by the compound H7), and perhaps one or more PKC isozymes (activated by a phorbol ester). In contrast, although protein tyrosine kinase (PTK) activity (inhibited by genistein) had a great influence on basal alpha7 currents [via positive allosteric modulatory (PAM-) and non-PAM effects], PTKs did not seem to participate in nicotine-produced upregulation. Tests of the potential contribution to nicotine-upregulation of cis-Golgi/ER quality control mechanisms (by the COPI-inhibitor CI-976) and alpha7-GPC signaling (via Gq/11; by a Substance-P analog) were inconclusive. These compounds strongly inhibited basal alpha7 currents.