Simultaneous Monitoring of Cytosolic and Reticular Ca Indicates Heterogeneity of Ca Store

Transduction of many agonists involves mobilization of intracellular Ca. The dynamics and shape of intracellular Ca signals are determined by Ca fluxes across the plasma membrane and membranes of intracellular organelles, primarily the endoplasmic reticulum (ER). Although traditionally, intracellular Ca signaling has been studied using chemical fluorescent Ca probes, the advent of genetically encoded Ca sensors with different intracellular localizations has significantly expanded the instrumental capabilities. In the present work, synchronous monitoring of cytosolic Ca in HEK-293 cells using Fluo-8 and of reticular Ca using the genetically encoded ER-localized Ca sensor R-CEPIA1er was performed. Upon stepwise stimulation of cells with acetylcholine (ACh), a coordinated increase in cytosolic Ca and a decrease in ER Ca were observed in the front phase of the Ca response, the relaxation of which was often accompanied by superimposed oscillations. A greater detailing of the correlated behavior of the cytosolic Ca (C) and reticular Ca (C) concentrations could be provided by representing cellular responses in the phase plane (C, C). Since C and C are not measurable directly but evaluated through Ca-dependent fluorescence of Ca probes, the experimental data were presented in the (F, F) plane, where F and F are ΔF/F for R-CEPIA1er and Fluo-8, respectively. Qualitatively, this is equivalent to the presentation of data in the (C, C) plane. The phase trajectories indicated that Ca responses to ACh could not be generated solely by Ca exchange between the homogeneous Ca store and the cytosol, since in approximately 30% of cases the phase trajectory contained a loop, wherein a simultaneous increase in C and C took place. The loop was observed in a calcium-free medium with negligible Ca influx into the cell, indicating the involvement of an intracellular Ca source that was not accessible to monitoring by R-CEPIA1er or did not significantly contribute to its fluorescence. The inhibitory analysis showed that the suggested source was not acidic endosomes and lysosomes containing two-pore channels, the Golgi apparatus, and vesicles loaded with Ca by SPCA-type ATPase, organelles releasing Ca through ryanodine receptors, and/or mitochondria releasing Ca into the cytosol through the Na/Ca exchanger. The results obtained pointed out that a system mediating agonist-induced Ca signals is more complex than one in the paradigm with one homogeneous pool of stored Ca.