Fast kinetics of calcium release induced by myo-inositol trisphosphate in permeabilized rat hepatocytes

• Published in: The Journal of biological chemistry 1989-10, Vol.264 (30), p.17665-17673
• Main Authors: Champeil, P, Combettes, L, Berthon, B, Doucet, E, Orlowski, S, Claret, M
• Format: Article
• Language: English
• Subjects: Aminoquinolines; Animals; Calcium - metabolism; Cell Membrane Permeability; Cells, Cultured; Female; Fluorescent Dyes; hepatocytes; Inositol 1,4,5-Trisphosphate - pharmacology; Kinetics; Liver - drug effects; Liver - metabolism; phosphoinositides; Rats; Rats, Inbred Strains; saponin; Saponins; Spectrometry, Fluorescence; Thermodynamics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)84623-9

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 °C, a maximally efficient concentration of 10 µM 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 µM 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 µM 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 × 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.