Kinetics of cytosolic Ca2+ concentration after photolytic release of 1-D-myo-inositol 1,4-bisphosphate 5-phosphorothioate from a caged derivative in guinea pig hepatocytes.
The influence of 1-D-myo-inositol 1,4,5-trisphosphate (InsP3) breakdown by InsP3 5-phosphatase in determining the time course of Ca2+ release from intracellular stores was investigated with flash photolytic release of a stable InsP3 derivative, 5-thio-InsP3, from a photolabile caged precursor. The potency and Ca(2+)-releasing properties of the biologically active D isomers of 5-thio-InsP3 and InsP3 itself were compared by photolytic release in guinea pig hepatocytes. After a light flash, cytosolic free calcium concentration ([Ca2+]i) showed an initial delay before rising quickly to a peak and declining more slowly to resting levels, with time course and amplitude generally similar to those seen with photolytic release of InsP3. Differences were a three- to eightfold lower potency of 5-thio-InsP3 in producing Ca2+ release, much longer delays between photolytic release and Ca2+ efflux with low concentrations of 5-thio-InsP3 than with InsP3, and persistent reactivation of Ca2+ release, producing periodic fluctuations of cytosolic [Ca2+]i with high concentrations of 5-thio-InsP3 but not InsP3 itself. The lower potency of 5-thio-InsP3 may be a result of a lower affinity for closed receptor/channels or a lower open probability of liganded receptor/channels. The longer delays with 5-thio-InsP3 at low concentration suggest that metabolism of InsP3 by 5-phosphatase may reduce the concentration sufficiently to prevent receptor activation and may have a similar effect on InsP3 concentration during hormonal activation. The maximal rate of rise of [Ca2+]i, the duration of the period of high Ca2+ efflux, and the initial decline of [Ca2+]i are similar with5-thio-lnsP3 and lnsP3, indicating that lnsP3 breakdown is not important in terminating Ca2+ release. The second activation ofInsP3 receptors with 5-thio-lnsP3 and particularly the sustained periodic fluctuations of [Ca2+]i indicate persistence of 5-thio-lnsP3,suggesting that InsP3 breakdown prevents reactivation of InsP3 receptors. The photochemical properties of 1-(2-nitrophenyl)-ethyl caged 5-thio-lnsP3 are photolytic quantum yield = 0.57 (cf. 0.65 for caged InsP3) and rate of photolysis = 87 s-I (half-life approximately 8 ms; cf. 3 ms for caged lnsP3; pH7.1; ionic strength, 0.2 M; 21 OC). Caged 5-thio-lnsP3 at concentrations up to 360 pM did not activate lnsP3 receptors to produce Ca2+ release or block Ca2+ release by free 5-thio-lnsP3.
Wootton
J F
JF
National Institute for Medical Research, London, United Kingdom.
Corrie
J E
JE
Capiod
T
T
Feeney
J
J
Trentham
D R
DR
Ogden
D C
DC
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D006168:Guinea Pigs; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D007295:Inositol Phosphates / Q000378:metabolism* / Q000494:pharmacology; D000072181:Inositol Polyphosphate 5-Phosphatases; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000502:physiology*; D008564:Membrane Potentials; D009946:Organothiophosphorus Compounds / Q000378:metabolism* / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010782:Photolysis; D011789:Quantum Theory; D013050:Spectrometry, Fluorescence / Q000379:methods
C095274:2-thiophosphoryloxyinositol 1,4-bis(dihydrogen phosphate); D007295:Inositol Phosphates; D009946:Organothiophosphorus Compounds; C095275:2-(S-(1-(2-nitrophenyl)ethyl)thiophosphoryloxy)inositol 1,4-bis(dihydrogen phosphate); D015544:Inositol 1,4,5-Trisphosphate; D010744:Phosphoric Monoester Hydrolases; D000072181:Inositol Polyphosphate 5-Phosphatases; D002118:Calcium