Mitochondrial DNA-depleted neuroblastoma (Rho³) cells exhibit altered
calcium signaling
Todd B. Sherer
aYb
, Patricia A. Trimmer
aYcY
*, Janice K. Parks
aYc
, Jeremy B. Tuttle
aYb
a
Center for the Study of Neurodegenerative Diseases, University of Virginia Health Science Center, Charlottesville, VA 22908, USA
b
Department of Neuroscience, University of Virginia Health Science Center, Charlottesville, VA 22908, USA
c
Department of Neurology, University of Virginia Health Science Center, Box 394, Charlottesville, VA 22908, USA
Received 5 August 1999; received in revised form 1 December 1999; accepted 3 February 2000
Abstract
To investigate the role of chronic mitochondrial dysfunction on intracellular calcium signaling, we studied basal and
stimulated cytosolic calcium levels in SH-SY5Y cells and a derived cell line devoid of mitochondrial DNA (Rho³). Basal
cytosolic calcium levels were slightly but significantly reduced in Rho³ cells. The impact of chronic depletion of
mitochondrial DNA was more evident following exposure of cells to carbachol, a calcium mobilizing agent. Calcium
transients generated in Rho³ cells following application of carbachol were more rapid than those in SH-SY5Y cells. A
plateau phase of calcium recovery during calcium transients was present in SH-SY5Y cells but absent in Rho³ cells. The rapid
calcium transients in Rho³ cells were due, in part, to increased reliance on Na
/Ca
2
exchange activity at the plasma
membrane and the plateau phase in calcium recovery in SH-SY5Y cells was dependent on the presence of extracellular
calcium. We also examined whether mitochondrial DNA depletion influenced calcium responses to release of intracellular
calcium stores. Rho³ cells showed reduced responses to the uncoupler, FCCP, and the sarcoplasmic reticulum calcium
ATPase inhibitor, thapsigargin. Acute exposure of SH-SY5Y cells to mitochondrial inhibitors did not mimic the results seen
in Rho³ cells. These results suggest that cytosolic calcium homeostasis in this neuron-like cell line is significantly altered as a
consequence of chronic depletion of mitochondrial DNA. ß 2000 Elsevier Science B.V. All rights reserved.
Keywords: Parkinson's disease; Alzheimer's disease ; Na
/Ca
2
exchange; Mitochondrion; Membrane potential; Calcium in£ux;
Endoplasmic reticulum
1. Introduction
Fluctuations in cytosolic calcium in£uence neuro-
nal function including the regulation of gene expres-
sion, neurotransmitter release, and apoptosis. Neu-
rons regulate cytosolic calcium by controlling
calcium £ux across the plasma membrane and by
intracellular storage. Cytosolic calcium is elevated
by calcium in£ux or by release of calcium from intra-
cellular stores. Calcium removal involves calcium ef-
£ux across the plasma membrane, uptake by intra-
cellular stores and bu¡ering by calcium binding
proteins [1].
Mitochondria shape intracellular calcium re-
sponses in multiple cell types including smooth
muscle cells and gonadotropes [2,3]. Mitochondria
may regulate the pattern of calcium transients fol-
lowing calcium in£ux and release of calcium from
0167-4889 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved.
PII: S0167-4889(00)00027-6
* Corresponding author. Fax: +1-804-982-1726 ;
E-mail: pat5q@virginia.edu
BBAMCR 14615 5-4-00
Cyaan Magenta Geel Zwart
Biochimica et Biophysica Acta 1496 (2000) 341^355
www.elsevier.com/locate/bba