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

A. D. Short, M. G. Klein, M. F. Schneider, D. L. Gill

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)25887-25893
Number of pages7
JournalJournal of Biological Chemistry
Volume268
Issue number34
StatePublished - Jan 1 1993

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Inositol 1,4,5-Trisphosphate
Fura-2
Organelles
Imaging techniques
Coloring Agents
Suspensions
Heparin Antagonists
Guanosine Triphosphate
Spatial distribution
Muscle
Heparin
Quenching
Cells
Monitoring
Smooth Muscle Myocytes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "Inositol 1,4,5-trisphosphate-mediated quantal Ca2+ release measured by high resolution imaging of Ca2+ within organelles",
abstract = "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.",
author = "Short, {A. D.} and Klein, {M. G.} and Schneider, {M. F.} and Gill, {D. L.}",
year = "1993",
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Inositol 1,4,5-trisphosphate-mediated quantal Ca2+ release measured by high resolution imaging of Ca2+ within organelles. / Short, A. D.; Klein, M. G.; Schneider, M. F.; Gill, D. L.

In: Journal of Biological Chemistry, Vol. 268, No. 34, 01.01.1993, p. 25887-25893.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Inositol 1,4,5-trisphosphate-mediated quantal Ca2+ release measured by high resolution imaging of Ca2+ within organelles

AU - Short, A. D.

AU - Klein, M. G.

AU - Schneider, M. F.

AU - Gill, D. L.

PY - 1993/1/1

Y1 - 1993/1/1

N2 - 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.

AB - 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.

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