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 Ca(2+)-sequestering organelles (> 5 microM 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 microns 2) 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 microns, higher than within suspensions of permeabilized cells. The action of InsP3 was competitively blocked by 10 micrograms/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 Ca(2+)-pumping organelles with identical spatial distribution as before Ca2+ release. InsP3 released Ca2+ from all areas of high Ca(2+)-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.
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Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201.
Klein
M G
MG
Schneider
M F
MF
Gill
D L
DL
D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D002460:Cell Line; D016257:Fura-2; D006493:Heparin / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology*; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009130:Muscle, Smooth; D015388:Organelles / Q000187:drug effects / Q000378:metabolism*
D008345:Manganese; D015544:Inositol 1,4,5-Trisphosphate; D006493:Heparin; D002118:Calcium; D016257:Fura-2