Having demonstrated the contribution of extracellular Ca
2+ to the abnormally raised [Ca
2+]
i in glaucoma LC cells, we next tested whether the release of Ca
2+ from intracellular stores could also contribute to the abnormally elevated levels of [Ca
2+]
i found in glaucoma LC cells. Therefore, we experimentally separated intracellular Ca
2+ release from extracellular Ca
2+ influx. The first part of the experiment (
Fig. 2) showed that the release of Ca
2+ from intracellular stores also contributed to the defective Ca
2+ homeostasis found in glaucoma LC cells, as removal of the residual external Ca
2+ from the external medium with EGTA followed by TG application resulted in a transient rise of [Ca
2+]
i, with similar amplitudes in glaucoma and normal LC cells. However, in normal LC cells, the [Ca
2+]
i returned to basal levels approximately 2 minutes after TG application, while return to baseline took 12 to 14 minutes in the glaucoma cells, indicating a lack of refilling of intracellular stores in glaucoma LC cells. These results complement our previous work, where an increase in SERCA2 and SERCA3 protein expression levels was observed in glaucoma LC cells.
12 Interestingly, the second part of the experiment (
Fig. 2) showed that the addition of extracellular Ca
2+ to the hypotonic buffer markedly increased the Ca
2+ amplitude of the sustained phase of Ca
2+ elevation more in the glaucoma than in normal LC cells. This suggests that the Ca
2+ entry channels are operating in a high open probability gating mode,
40 thus generating persistent Ca
2+ influx in glaucoma LC cells. Furthermore, and more importantly, the normal LC cells recovered to baseline Ca
2+ levels in contrast to the glaucoma LC cells after changing back to isotonic solution, suggesting a defect of Ca
2+ extrusion from the intracellular space. The key components that maintain stable Ca
2+ gradients in cells are believed to be plasma membrane Ca
2+ entry channels, ATP-driven pumps (PMCAs), electrogenic exchangers Na
+-Ca
2+ (NCX), and SERCA pumps.
40 In our previous study,
12 we also found that the Ca
2+ extrusion system is altered in glaucoma LC cells. Our data showed that NCX expression levels were significantly lower in glaucoma LC cells, suggesting a defect in Ca
2+ extrusion from the intracellular space.
12