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JM Hurtado, E Gleason, M Wilson; Store Operated Ca2+ Entry in Amacrine Cell Dendrites . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4765.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the presence of store operated Ca2+ inflow pathways in the dendrites of amacrine cells. Methods: Cultured amacrine cells from embryonic chick retinae were loaded with the Ca2+ indicator Fura-2AM. Small segments of dendrites were imaged. Thapsigargin (TG) a specific, irreversible, SERCA pump blocker was used to deplete intracellular Ca2+ stores. Cells were depolarized with a 2 sec high K+ puff. Ca2+ entry pathways were examined by measuring the quenching rate of Fura-2 by 1mM extracellular Mn2+. La3+ (25µM) was used as a nonspecific blocker of store operated Ca2+ influx. Results: Ca2+ influx through voltage-gated Ca2+channels in the dendrites of cultured amacrine cells causes release of Ca2+ from internal stores. Some of this released Ca2+ is recaptured by the ER but some is lost, implying that store refilling mechanisms may play a role in the maintenance of normal Ca2+ dynamics. In the present study we describe a Ca2+ influx pathway in dendrites that activates with Ca2+store depletion. After bath application of TG, an increase in dendritic [Ca2+]i was observed within 15 minutes, that reached ≷ 500nM. Subsequent application of 25µM La3+ restored baseline [Ca2+]i levels. Coapplication of TG and La3+ prevented the [Ca2+]i increase, and subsequent removal of La3+ allowed the [Ca2+]i increase. When cells were bathed in 0 [Ca2+]o, [Ca2+]i decreased slightly and when [Ca2+]o was removed for ≷ 5 min we often observed a large [Ca2+]i overshoot when external [Ca2+]o was reestablished. This effect could be abolished or substantially reduced by La3+. Since [Ca2+]i reflects the balance between Ca2+ influx and extrusion pathways we measured the rate of Fura-2 quenching with Mn2+o as an indicator of Ca2+ conductance in the plasma membrane. Both TG treatment and prolonged 0[Ca2+]o induced a substantial increase in Mn2+ quenching relative to baseline that in both cases was blocked by 25µM La3+. To examine whether the store-operated pathway is enabled after depolarizations we compared the rate of quenching in TG untreated cells before and after each of a series of depolarizations. We found that quenching was substantially accelerated after one or more depolarizations. This depolarization-induced quenching was also blocked by La3+. Conclusion: A store-dependent, La3+-suppressible, Ca2+ entry pathway operates in amacrine cell dendrites and its activation is dependent upon the history of Ca2+ elevation in that dendrite.
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