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. 1995 Jun;68(6):2601–2607. doi: 10.1016/S0006-3495(95)80444-3

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.

J F Wootton 1, J E Corrie 1, T Capiod 1, J Feeney 1, D R Trentham 1, D C Ogden 1
PMCID: PMC1282170  PMID: 7647263

Abstract

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.

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Selected References

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