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© The Rockefeller University Press, 0021-9525/1999//241 $5.00
The Journal of Cell Biology, Volume 144, Number 2, , 1999 241-254

Regular Articles

Ca2+-induced Ca2+ Release in Chromaffin Cells Seen from inside the ER with Targeted Aequorin



Maria Teresa Alonso*, Maria José Barrero*, Pedro Michelena{ddagger}, Estela Carnicero*, Inmaculada Cuchillo{ddagger}, Antonio G. García{ddagger}, Javier García-Sancho*, Mayte Montero*, and Javier Alvarez*

* Instituto de Biología y Genética Molecular, Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina, Universidad de Valladolid y Consejo Superior de Investigaciones Científicas, E-47005 Valladolid, Spain; and {ddagger} Instituto de Farmacología Teófilo Hernando, Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, E-28029 Madrid, Spain

The presence and physiological role of Ca2+-induced Ca2+ release (CICR) in nonmuscle excitable cells has been investigated only indirectly through measurements of cytosolic [Ca2+] ([Ca2+]c). Using targeted aequorin, we have directly monitored [Ca2+] changes inside the ER ([Ca2+]ER) in bovine adrenal chromaffin cells. Ca2+ entry induced by cell depolarization triggered a transient Ca2+ release from the ER that was highly dependent on [Ca2+]ER and sensitized by low concentrations of caffeine. Caffeine-induced Ca2+ release was quantal in nature due to modulation by [Ca2+]ER. Whereas caffeine released essentially all the Ca2+ from the ER, inositol 1,4,5-trisphosphate (InsP3)- producing agonists released only 60–80%. Both InsP3 and caffeine emptied completely the ER in digitonin-permeabilized cells whereas cyclic ADP-ribose had no effect. Ryanodine induced permanent emptying of the Ca2+ stores in a use-dependent manner after activation by caffeine. Fast confocal [Ca2+]c measurements showed that the wave of [Ca2+]c induced by 100-ms depolarizing pulses in voltage-clamped cells was delayed and reduced in intensity in ryanodine-treated cells. Our results indicate that the ER of chromaffin cells behaves mostly as a single homogeneous thapsigargin-sensitive Ca2+ pool that can release Ca2+ both via InsP3 receptors or CICR.

Key Words: endoplasmic reticulum • aequorin • chromaffin cells • calcium • ryanodine

Abbreviations used in this paper: [Ca2+]c, cytosolic [Ca2+]; [Ca2+]ER, ER [Ca2+]; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; cADPR, cyclic adenosine diphosphate ribose; CICR, Ca2+-induced Ca2+ release; CPA, ciclopiazonic acid; DMPP, 1,1-dimethyl-4-phenyl- piperazinium iodide; HSV-1, herpes simplex virus type 1; InsP3R, InsP3 receptor; InsP3, inositol 1,4,5-trisphosphate; ivu, infectious virus units; RyR, ryanodine receptor.

M.J. Barrero was supported by a predoctoral fellowship from the University of Valladolid and I. Cuchillo is a fellow from the Fundación Teófilo Hernando. Financial support from Fondo de Investigaciones Sanitarias to J. Alvarez (96/0456) and to M.T. Alonso (96/1443), Junta de Castilla y León to J. García-Sancho (VA 87/96), Dirección General de Enseñanza Superior (PB97/0474) to J. García-Sancho and Dirección General de Investigación Científica y Técnica (PB94/0150) to A.G. García is gratefully acknowledged.

M.T. Alonso and M.J. Barrero contributed equally to this work.


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