Use of intracellular Ca2+ stores from rat basophilic leukemia cells to study the molecular mechanism leading to quantal Ca2+ release by inositol 1,4,5-trisphosphate.
Quantal Ca2+ release is a novel motif for the mediation of signal transduction in which the amplitude of a biological response following multiple stepwise increases in agonist concentration is retained. The release of Ca2+ from permeabilized cells in response to the second messenger inositol 1,4,5-trisphosphate (InsP3) proceeds in this fashion. The mechanisms leading to quantal Ca2+ release are unknown. InsP3 releases 50-90% of the Ca2+ sequestered within the intracellular stores of mammalian cells permeabilized with saponin. However, preparation of microsomes results in the loss of this sensitivity. In this report, functionally intact intracellular Ca2+ stores were isolated from rat basophilic leukemia (RBL) cells by osmotic lysis followed by differential and sucrose density gradient centrifugation. From this preparation, 64% of the stored Ca2+ is released by InsP3. We demonstrate that quantal Ca2+ release is retained by isolated Ca2+ stores and is identical to that observed in permeabilized cells. Addition of a subsaturating (28 nM) concentration of InsP3 to permeabilized cells at 37 degrees C results in the release of only a small fraction of the sequestered Ca2+. When the cells are cooled to 11 degrees C, the remaining Ca2+ is rapidly released. Hence, the mechanism leading to the quantal nature of Ca2+ release is reversible and is thus not likely to be the result of a covalent modification of the channel protein or of the Ca2+ store.(ABSTRACT TRUNCATED AT 250 WORDS)
Kindman
L A
LA
Department of Cell Biology, Stanford University Medical Center, California 94305.
Meyer
T
T
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D003198:Computer Simulation; D004273:DNA, Neoplasm / Q000032:analysis; D015544:Inositol 1,4,5-Trisphosphate / Q000031:analogs & derivatives / Q000494:pharmacology* / Q000502:physiology*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D015471:Leukemia, Basophilic, Acute / Q000378:metabolism*; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D011789:Quantum Theory; D051381:Rats; D015290:Second Messenger Systems; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000378:metabolism; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D000001:Calcimycin; D015544:Inositol 1,4,5-Trisphosphate; D007770:L-Lactate Dehydrogenase; D002118:Calcium