Observing Spontaneous Calcium Release
In many cells transient releases of local cytosolic calcium have been shown to originate from the endoplasmic reticulum via ryanodine receptors (sparks) and inositol triphosphate receptors (puffs).
In dendrites of chick retinal amacrine cells, we have also seen transient increases of calcium, including a type that persists after endoplasmic reticulum stores have been depleted of calcium following incubation with thapsigargin. These spontaneous events (blips) can be visualized (below) by fast scanning a line along a portion of dendrite using a confocal microscope.
The cultured amacrine cells are loaded with a fluorescent dye (Oregon Green Bapta-1, AM) that is sensitive to the concentration of calcium within the dendrite (color bar indicates concentration). The thapsigargin-resistant blips have an average duration of about 700 milliseconds and an average spread of about 9 µm. The manually drawn line along the dendrite directs the laser to scan the line rapidly over a set time. The observed blips are transient increases in calcium occurring in hot spots along the dendrite.
Removal of external calcium completely and immediately eliminated blips suggesting that they originate from external calcium entry. Application of nifedipine or Bay-K which are regulators of L-type calcium channels have no effect on the duration, spread or frequency of the blips, nor do agonists or antagonists for acetylcholine, glutamate or GABA receptors.
Sphingosine and several related lipids significantly increased the frequency of blips in addition to causing a generalized increase in calcium concentration in the cell. However blip frequency is not a result of the generalized increase in calcium since ionomycin, a calcium ionophore, also similarly raises calcium but does not increase the frequency of blips. Blocking mitochondrial calcium release with CGP-37157 had no effect on sphingosine evoked blips. Lanthanum at 20-50 µm blocked all blips, including those evoked by sphingosine.