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Mark R. Williams, Robert A. Riach, David J. Collison, George Duncan; Role of the Endoplasmic Reticulum in Shaping Calcium Dynamics in Human Lens Cells. Invest. Ophthalmol. Vis. Sci. 2001;42(5):1009-1017.
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purpose. Localized cortical cataracts in the human lens have been shown to
involve a selective increase in calcium with no change in sodium
content. Recent studies in the rat lens in vitro have shown that the
store-operated channel is highly selective for calcium over
sodium, and therefore this channel was characterized further in human
methods. Human primary cultures were initiated from epithelial explants and
passaged onto coverslips. After incorporating Fura-2, agonist- or
thapsigargin-induced changes in cytosolic calcium were monitored and
calibrated using fluorometric digital imaging techniques.
results. Histamine and adenosine triphosphate (ATP; 10 μM) induced a large
transient increase in cytosolic calcium followed by a maintained lower
plateau phase in the continued presence of the calcium-signaling
agonist. The second phase was abolished by removing external calcium
and represented the contribution from the store-operated influx. The
store-operated pathway was blocked by inorganic agents such as zinc and
nickel (100 μM) but was insensitive to the voltage-sensitive calcium
channel blocker, nifedipine (1 mM). Depolarizing the membrane voltage
by raising the external potassium (75 mM) also blocked the influx.
Similar results were obtained if the store was first emptied directly
using thapsigargin (1 μM), and with this agent it was also possible
to observe the very slow activation and inactivation kinetics (>10
seconds) of the channel. Addition of manganese to the bathing medium
initiated a quench of Fura-2 isobestic fluorescence that was enhanced
2.9 ± 0.3-fold after 10 μM ATP addition. There was a delay of
82 ± 16 seconds between initiation of the calcium spike and the
Mn2+ quench rate, indicating the presence of a delayed
entry pathway. In the resting state, removal of, or increasing
extracellular calcium concentration 10-fold did not perturb the level
of cytosolic Ca2+. Similar maneuvers performed after
agonist- or thapsigargin-induced store depletion of intracellular
stores brought about dramatic changes in cytosolic Ca2+ consistent with the activation of a Ca2+ entry pathway.
Lower concentrations of agonist induced oscillations of
Ca2+ that continued for a short time in Ca-free solution.
No increase in Mn2+ quench rate was associated with
oscillations. A 100-μM zinc- and KCl-induced blockade of
Ca2+ entry had no effect on the form of agonist-induced
oscillations. Inhibition of Ca2+ influx by zinc (100 μM)
converted a sustained Ca2+ response to a train of
repetitive Ca2+ spikes.
conclusions. Human lens cells normally have very low Ca2+ permeability.
Depletion of intracellular stores by agonists or thapsigargin initiates
a Ca2+ entry pathway that is not required for the
Ca2+ oscillations induced by low concentrations of agonist.
This potentially provides a signal transduction mechanism with minimal
risk of Ca2+ overload to the lens, whereas overactivation
of the store-operated channel is a possible way of increasing calcium
in the lens and could explain the distribution found in localized
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