Fast kinetics of calcium release induced by myo-inositol trisphosphate in permeabilized rat hepatocytes.
We used a stopped-flow method for determining the kinetic properties (between 10 ms and 10 s) of the Ca2+ release induced by inositol 1,4,5-trisphosphate (InsP3) in saponin-treated rat hepatocytes. Preliminary experiments ensured that the indicator was able to monitor rapid changes in free Ca2+ reliably. At 20 degrees C, a maximally efficient concentration of 10 microM InsP3 released Ca2+ with a half-time of 150-300 ms, the initial rate being about 1-2 nmol of Ca2+/mg of cell protein/s. The delay between the addition of 10 microM InsP3 and the onset of Ca2+ release was shorter than 20 ms, suggesting that the opening process of Ca2+ channels after binding of InsP3 to receptors is completed within a few milliseconds. Half-maximal initial rates for Ca2+ release occurred at about 1 microM InsP3 (Hill index was 1.6). The resulting Ca2+ efflux had a moderate temperature dependence. It could not be fitted to a single exponential. After low speed centrifugation of saponin-treated cells (1000 x g for 1 min), part of the InsP3-sensitive Ca2+ pool was recovered in the cell-free supernatant fraction, suggesting that the response to InsP3 arises from a vesicular fraction which may diffuse from the saponin-treated cells into the medium.
Champeil
P
P
Unité de Recherche Institut National de la Santé et de la Recherche Médicale, Université Paris Sud, Orsay, France.
Combettes
L
L
Berthon
B
B
Doucet
E
E
Orlowski
S
S
Claret
M
M
D000634:Aminoquinolines; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D005260:Female; D005456:Fluorescent Dyes; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology*; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D012503:Saponins; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D000634:Aminoquinolines; D005456:Fluorescent Dyes; D012503:Saponins; D015544:Inositol 1,4,5-Trisphosphate; C035739:Quin2; D002118:Calcium