Effect of oxidized glutathione and temperature on inositol 1,4,5-trisphosphate binding in permeabilized hepatocytes

Abstract

The effect of oxidized glutathione (GSSG) on inositol 1,4,5-trisphosphate (IP3) binding and the activity of IP3-gated Ca2+ channels was examined in permeabilized hepatocytes. The permeability properties of the channel were measured by using Mn2+ quenching of compartmentalized fura-2 at 37 degrees C and at 4 degrees C for comparison with IP3-binding measurements. GSSG (2 mM) increased the IP3-sensitivity of Mn2+ quenching, consistent with previous studies based on Ca(2+)-release measurements [Renard, Seitz and Thomas (1992) Biochem. J. 284, 507-512]. Measurements of [3H]IP3 binding were made at 4 degrees C after preincubation of permeabilized hepatocytes at 37 degrees C in the absence or presence of GSSG. Under these conditions GSSG stimulated IP3 binding by increasing the number of binding sites without changing the Kd. This effect was observed in the absence or presence of Ca2+, but was abolished when the preincubation with GSSG was carried out at 4 degrees C. Thimerosal also stimulated [3H]IP3 binding, but this effect was mediated both by an increase in the maximum number of binding sites and by a decrease in the Kd. The effects of thimerosal and GSSG were not additive. Further analysis of the effect of GSSG revealed that preincubation of permeabilized hepatocytes at 37 degrees C results in a progressive loss of [3H]IP3-binding sites that can be prevented and reversed by inclusion of GSSG. A parallel loss of IP3-sensitive Mn(2+)-quenchable stores was observed after incubation at 37 degrees C, and this could also be reversed by adding back GSSG. The loss of IP3 binding was not the result of IP3-receptor proteolysis, as judged by Western blotting of immunoreactive protein. The sensitivity of [3H]IP3 binding in permeabilized hepatocytes to varied ratios of GSSG and GSH suggests that the IP3 receptor responds to an oxidized redox environment such as that found in the lumen of the endoplasmic reticulum. GSSG had no direct effect on the ligand-binding activity of detergent-solubilized and partially purified IP3 receptors. We conclude that GSSG exerts an indirect effect on the IP3 receptors in permeabilized hepatocytes by preventing a temperature-dependent loss of IP3-binding sites. We suggest that the hepatic IP3 receptors may interact with a thiol-disulphide oxidoreductase that utilizes GSSG as a substrate and prevents inappropriate unfolding of the ligand-binding domain occurring after incubation of the receptor at 37 degrees C in vitro.

Glutathione depletion induces glycogenolysis dependent ascorbate synthesis in isolated murine hepatocytes

“…However, there is no exact and direct evidence concerning the effect of diamide or buthionine sulfoximine on intracellular Ca 2+ mobilisation and under our experimental circumstances menadione increased glycogenolysis even in Ca 2+ depleted hepatocytes (data not shown). It should be noted, however, that oxidation of GSH increases the sensitivity of IP3 receptors [37]. Thus, a primary role of GSH was supposed in the regulation of glycogenolysis under various GSH deficient conditions.…”

xCT expression reduces the early cell cycle requirement for calcium signaling

“…Activity of the sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA), which is required for progression through G1/S [73], is inhibited by oxidation [74,75] as is the activity of the plasma membrane Ca 2+ ATPase (PMCA) [76]. Similarly, oxidized GSH, (GSSG) has been shown to directly effect the depletion of IP3-sensitive Ca 2+ stores [77] and, the activity of RYR is redox regulated [78,79]. Although this implies that Ca 2+ -dependent processes may not be required for cell cycle progression, it is possible that these processes are activated at lower Ca 2+ concentrations.…”

Threshold for Inositol 1,4,5-Trisphosphate Action

“…Oxidized glutathione (GSSG) can also stimulate the InsP 3 R, but this stimulation has so far only been reported in hepatocytes (20,21,(35)(36)(37). The effect of 5 mM GSSG in A7r5 cells was further characterized.…”