Role of the endoplasmic reticulum in shaping calcium dynamics in human lens cells.
OBJECTIVE
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 lens cells.


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 microM) 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 microM) 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 microM), 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 microM 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-microM 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 microM) 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 cataracts.
School of Biological Sciences, University of East Anglia, Norwich, United Kingdom.
M R
MR
Williams
R A
RA
Riach
D J
DJ
Collison
G
G
Duncan
D000255:Adenosine Triphosphate / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016257:Fura-2 / Q000378:metabolism; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism; D008564:Membrane Potentials; D010539:Permeability; D015398:Signal Transduction; D019284:Thapsigargin / Q000494:pharmacology
D002121:Calcium Channel Blockers; D004791:Enzyme Inhibitors; D008345:Manganese; D019284:Thapsigargin; D006632:Histamine; D000255:Adenosine Triphosphate; D002118:Calcium; D016257:Fura-2