Inositol 1,4,5-trisphosphate-mediated quantal Ca²⁺ release measured by high resolution imaging of Ca²⁺ within organelles

The distribution and operation of Ca²⁺ pools within cells has been directly studied in situ by monitoring the Ca²⁺ inside Ca²⁺ dye-loaded organelles using high resolution imaging procedures. Using DDT₁MF-2 smooth muscle cells, loaded with fura-2 under conditions favoring dye entry into organelles and subjected to carefully controlled permeabilization still attached to coverslips, the Ca²⁺ within organelles was analyzed by high resolution, z axis-controlled imaging, and deblurring methods. Saturation analysis of entrapped fura-2 indicated that the dye reported Ca²⁺ identically to fura-2 in solution. Areas containing high Ca²⁺-sequestering organelles (>5 μM free Ca²⁺) were observed to predominate around the nucleus and close to the periphery of the cell. Analysis of the actions of inositol 1,4,5-trisphosphate (InsP3) within small (3 μm²) selected intracellular areas, revealed a "quantal" release phenomenon, with rapid attainment of limited stable release at submaximal InsP₃ levels. The apparent EC₅₀ for InsP₃ was approximately 3 μM, higher than within suspensions of permeabilized cells. The action of InsP₃ was competitively blocked by 10 μg/ml of the InsP₃ antagonist, heparin. Applied after maximal InsP₃-mediated Ca²⁺ release, heparin reversed InsP₃-induced Ca²⁺ release resulting in reuptake of Ca²⁺ into Ca²⁺-pumping organelles with identical spatial distribution as before Ca²⁺ release. InsP₃ released Ca²⁺ from all areas of high Ca²⁺-PumPing organelles; extensive areas of high fura-2-loading, but low intraorganelle Ca²⁺, were unchanged by InsP₃. GTP induced no alteration in Ca²⁺ release (in contrast to suspensions of permeabilized cells), suggesting that the InsP₃-sensitive Ca²⁺ pool was functioning as a single homogeneous pool. Opening of InsP₃-sensitive channels was also monitored by assessing InsP₃-activated channel-mediated Mn²⁺ quenching of organelle-loaded fura-2; the results revealed a similar pattern of quantal release, with slightly increased apparent InsP₃ sensitivity. The results provide the first high resolution in situ localization of Ca²⁺ signaling organelles and demonstrate the quantal operation of InsP₃-sensitive Ca²⁺ pools within highly discrete subcellular loci.