INOSITOL 1,4,5-TRISPHOSPHATE-MEDIATED QUANTAL CA2-RESOLUTION IMAGING OF CA2+ WITHIN ORGANELLES( RELEASE MEASURED BY HIGH)

The distribution and operation of Ca2+ pools within cells has been dir ectly studied in situ by monitoring the Ca2+ inside Ca2+ dye-loaded or ganelles using high resolution imaging procedures. Using DDT1MF-2 smoo th muscle cells, loaded with fura-2 under conditions favoring dye entr y into organelles and subjected to carefully controlled permeabilizati on still attached to coverslips, the Ca2+ within organelles was analyz ed by high resolution, z axis-controlled imaging, and deblurring metho ds. Saturation analysis of entrapped fura-2 indicated that the dye rep orted Ca2+ identically to fura-2 in solution. Areas containing high Ca 2+-sequestering organelles (>5 muM free Ca2+) were observed to predomi nate around the nucleus and close to the periphery of the cell. Analys is of the actions of inositol 1,4,5-trisphosphate (InsP3) within small (3 mum2) selected intracellular areas, revealed a ''quantal' release phenomenon, with rapid attainment of limited stable release at submaxi mal InsPs levels. The apparent EC50 for InsP3 Was approximately 3 muM, higher than within suspensions of permeabilized cells. The action of InsP3 was competitively blocked by 10 mug/ml of the InsP3 antagonist, heparin. Applied after maximal InsP3-mediated Ca2+ release, heparin re versed InsP3-induced Ca2+ release resulting in reuptake of Ca2+ into C a2+-pumping organelles with identical spatial distribution as before C a2+ release. InsP3 released Ca2+ from all areas of high Ca2+-pumping o rganelles; extensive areas of high fura-2-loading, but low intraorgane lle Ca2+, were unchanged by InsP3. GTP induced no alteration in Ca2+ r elease (in contrast to suspensions of permeabilized cells), suggesting that the InsP3-sensitive Ca2+ pool was functioning as a single homoge neous 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 rele ase, with slightly increased apparent InsP3 sensitivity. The results p rovide the first high resolution in situ localization of Ca2+ signalin g organelles and demonstrate the quantal operation of InsP3-sensitive Ca2+ pools within highly discrete subcellular loci.