ROLE OF THE ALPHA7, SUBTYPE-SPECIFIC NICOTINIC ACETYLCHOLINE RECEPTOR IN MEDIATING CALCIUM DYSREGULATION DURING ACUTE METHYLMERCURY EXPOSURE IN PC12 CELLS
Monica Rios-Cabanillas1, William Atchison2.
1Michigan State University, East Lansing, MI, 2Michigan State University, College of Veterinary Medicine, East Lansing, MI.
Methylmercury (MeHg) disrupts internal calcium (Ca2+) regulation, potentially leading to cytotoxicity. We previously showed that activation of neuronal nicotinic acetylcholine receptors (nAChRs) may contribute to the increase of Ca2+i by either involvement of internal or external Ca2+ sources. The nAChRs appear to be involved during MeHg-induced cytotoxicity in differentiated sympathetic-neuron like PC12 (dPC12) cells, and application of the nonspecific nAChR antagonist, mecamyllamine (MEC), increases cell viability after acute MeHg exposure. As different nAChR subtypes act in a cell-type specific manner and affect extra- and intracellular [Ca2+] differently, we compared the role of the highly Ca2+ permeable homomeric α7 subtype nAChR with other heteromeric nAChRs in contributing to MeHg-induced [Ca2+]i increase. We used single cell microfluorimetry with fura2AM as the fluorophore. MeHg causes a kinetically distinct biphasic increase in the fura2AM fluorescence ratio. The first phase results from release of Ca2+i stores followed by extracellular Ca2+ influx (P2). The dPC12 cells were exposed to 1, 2, or 5 μM MeHg in the absence and presence of the α7 specific blocker MLA (5µM) or MEC (5µM). MeHg treatment increased fura2AM fluorescence in dPC12 cells. The time to onset was inversely related to the concentration for P1 only. P2 was drastically hastened at high, 5µM MeHg. MEC significantly delayed the time to onset of P1 (46%) and P2 (46%) at 1 µM MeHg. MLA delayed the time to onset of only P2 (43%) and at only 1µM MeHg. At higher [MeHg], neither MEC nor MLA slowed the response to MeHg. (This project was supported by NIH grant R01ES03299 and R25NS065777.)