Measurement of sr free ca2+ and mg2+ in saponin-permeabilized
smooth muscle cells using furaptra.
Sugiyama, Takao, and William F. Goldman.
Departments of Physiology and Medicine, University of Maryland
School of Medicine and the Geriatric Research, Education and Clinical
Center, Baltimore Veterans Administration Medical Center, Baltimore,
MD 21201
APStracts 2:0178C, 1995.
The concentrations of intra sarcoplasmic reticulum (SR) free Ca2+
([Ca2+]SR) and Mg2+ ([Mg2+]SR) were measured in furaptra-loaded,
saponin-permeabilized, cultured aortic smooth muscle cells (A7r5).
Ca2+-independent fluorescence emitted by furaptra trapped within
organelles, excited at 346 nm (isosbestic point), decreased with a
T1/2 = 30 min. All Ca2+ measurements appeared to be from SR because
the apparent Ca2+ distribution within permeabilized cells was uniform
and therefore inconsistent with furaptra loading into mitochondria.
Moreover, thapsigargin-induced SR Ca2+-ATPase inhibition caused near
total depletion of Ca2+, and the metabolic poisons, oligomycin and
rotenone had no effect. Calibration curves relating 370 nm/346 nm
ratios to [Ca2+] and to [Mg2+] were calculated in situ; Kd values for
Ca2+ and Mg2+ binding were 49 M and 6.8 mM, respectively. Resting
[Ca2+]SR ranged from 75-130 M with a mean of 97.2 2.2 M (n = 376),
while [Mg2+]SR, estimated in the absence of Ca2+, was 1.0 mM.
Stimulation with inositol 1,4,5-trisphosphate (InsP3) resulted in
time-dependent declines in [Ca2+]SR, and pretreatment with GTP caused
a large increase in the rate of InsP3-evoked SR Ca2+ release,
although GTP had no effect by itself. These observations indicate
that furaptra will be a valuable tool with which to directly study
[Ca2+]SR and SR function.
Received 26 October 1994; accepted in final form 19 April 1995.
APS Manuscript Number C639-4.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 2 May 1995.