Delayed "all-or-none" activation of inositol 1,4,5-trisphosphate-dependent calcium signaling in single rat hepatocytes.
When single rat hepatocytes were stimulated with the phospholipase C-activating hormone, vasopressin (from 300 pM to 1 microM), the [Ca2+]i signals were always "all-or-none" responses. At low concentrations of vasopressin, Ca2+ release was maximal because liberation of additional inositol 1,4,5-trisphosphate (IP3) by photolysis of its caged precursor at the top of the [Ca2+]i spike failed to increase [Ca2+]i further. However, if IP3 was generated by photolysis of caged IP3 in previously unstimulated cells, [Ca2+]i increased immediately, and the magnitude of the response was a graded function of the quantity of IP3 released. We also analyzed the kinetics of activation of intracellular IP3 receptor/Ca2+ channels by monitoring the quench of sequestered dye by the entry of cytoplasmic Mn2+ into fura-2-loaded intracellular IP3-sensitive organelles. This Mn(2+)-induced quench was precipitous and always preceded by a delay inversely related to the vasopressin concentration. In hepatocytes stimulated with 10 nM vasopressin, IP3 increased slowly, and the half-time of the IP3 rise was comparable with the latency for the release of intracellular calcium. The slow rise in IP3 would be predicted to produce accelerating Ca2+ release. This is consistent with the results of the Mn2+ quench experiments, which revealed accelerating activation of intracellular IP3-regulated calcium channels. We conclude that this accelerating release of Ca2+, which does not occur with instantaneous increases in IP3 due to flash photolysis, is likely to be important for generating the all-or-none Ca2+ mobilization that initiates the processes of intracellular [Ca2+]i oscillations.
Chiavaroli
C
C
Laboratory of Cellular and Molecular Pharmacology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709.
Bird
G
G
Putney
J W
JW
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D002451:Cell Compartmentation; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D016257:Fura-2 / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007473:Ion Channels / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D008297:Male; D008345:Manganese / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D010738:Type C Phospholipases / Q000378:metabolism; D014667:Vasopressins / Q000494:pharmacology*
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007473:Ion Channels; D018160:Receptors, Cytoplasmic and Nuclear; D014667:Vasopressins; D008345:Manganese; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; D002118:Calcium; D016257:Fura-2