Threshold for Inositol 1,4,5-Trisphosphate Action

Abstract

We developed a unidirectional 45 Ca2؉ efflux technique in which 60 cumulative doses of inositol 1,4,5-trisphosphate (InsP 3 ), each lasting 6 s, were subsequently added to permeabilized A7r5 cells. This technique allowed an accurate determination of the threshold for InsP 3 action, which was around 32 nM InsP 3 under control conditions. The InsP 3 -induced Ca 2؉ release was characterized by an initial rapid phase, after which the normalized rate progressively decreased. The slowing of the release was associated with a shift of the threshold to higher InsP 3 concentrations. Stimulatory concentrations of thimerosal (10 M) shifted the threshold to 4.5 nM InsP 3 and increased both the cooperativity and the maximal normalized rate of Ca 2؉ release. This low threshold was maintained when the thimerosal concentration was increased to inhibitory levels (100 M) but then the effects on the cooperativity and on the normalized rate of Ca 2؉ release disappeared. Oxidized glutathione (5 mM) was much less effective in stimulating the release and did not have an effect on the threshold or on the cooperativity. ATP (5 mM) stimulated the release despite a shift in threshold toward higher InsP 3 concentrations. Luminal Ca 2؉ did not affect the threshold for InsP 3 action but stimulated the normalized release at each InsP 3 concentration. The inhibitory effect of 10 M free cytosolic Ca 2؉ was associated with a shift in threshold to higher InsP 3 concentrations and a decreased cooperativity of the release process. We conclude that this novel technique of accurately measuring the threshold for InsP 3 action under various experimental conditions has allowed us to refine the analysis of the kinetic parameters involved in the regulation of the InsP 3 receptor.Many hormones, neurotransmitters, and growth factors induce the hydrolysis of phosphatidylinositol 4,5-bisphosphate and thereby produce inositol 1,4,5-trisphosphate (InsP 3 ) 1 as an intracellular messenger (1 MATERIALS AND METHODSA7r5 cells, an established cell line derived from embryonic rat aorta, were used between the 7th and the 17th passage after receipt from the American Type Culture Collection (Bethesda, MD) and subcultured weekly by trypsinization. The cells were cultured at 37°C in a 9% CO 2 incubator in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 3.8 mM L-glutamine, 0.9% (v/v) nonessential amino acids, 85 IU ml Ϫ1 penicillin, and 85 g ml Ϫ1 streptomycin. The cells were seeded in 12-well dishes (4 cm 2 , Costar, MA) at a density of approximately 10 4 cells cm Ϫ2 . 45Ca 2ϩ fluxes on permeabilized cells were done on a thermostated plate at 25°C. The culture medium was aspirated and replaced by 1 ml of permeabilization medium containing 120 mM KCl, 30 mM imidazole HCl (pH 6.8), 2 mM MgCl 2 , 1 mM ATP, 1 mM EGTA, and 20 g ml Ϫ1 saponin. The saponin-containing solution was removed after 10 min, and the cells were washed once with a similar saponin-free solution. 45 Ca 2ϩ uptake into the nonmitochondrial Ca 2ϩ stores was accomplished by incubati...

Molecular and Functional Evidence for Multiple Ca2+-binding Domains in the Type 1 Inositol 1,4,5-Trisphosphate Receptor

“…2B, and a 45 Ca 2ϩ overlay of a blot of a similar gel was carried out. In contrast, the release rate curves became less steep as the free [Ca 2ϩ ] increased from 5 nM to 1 and 4 M. The Hill coefficients for these traces, and also for the traces measured at intermediate Ca 2ϩ concentrations (data not shown), were calculated as described (22) and plotted by the open symbols in Fig. 6A.…”

“…The experiment was therefore repeated at 1 M free Ca 2ϩ , which is an optimal effective [Ca 2ϩ ] for stimulating the Ca 2ϩ release in A7r5 cells (11). Fig.…”

“…The effects of Ca 2ϩ on the V max of InsP 3 -induced Ca 2ϩ release were not determined in the present experiments because our previous work on fully loaded stores already indicated that the Ca 2ϩ release in response to a supramaximal [InsP 3 ] (320 M) was not affected when the cytosolic [Ca 2ϩ ] was varied between 10 nM and 10 M (11,23). The stimulation of the release by cytosolic Ca 2ϩ was due to a shift in EC 50 toward lower InsP 3 concentrations.…”

“…In the second part of the study we accurately determined the effects of Ca 2ϩ and Sr 2ϩ on the cooperativity and on the EC 50 of the InsP 3 -induced Ca 2ϩ release in permeabilized A7r5 cells using our recently developed rapid 45 Ca 2ϩ efflux technique (22). The effects of Ca 2ϩ on the V max of InsP 3 -induced Ca 2ϩ release were not determined in the present experiments because our previous work on fully loaded stores already indicated that the Ca 2ϩ release in response to a supramaximal [InsP 3 ] (320 M) was not affected when the cytosolic [Ca 2ϩ ] was varied between 10 nM and 10 M (11,23).…”

“…Additions of InsP 3 or Ca 2ϩ are indicated in the figures. This efflux medium was replaced every 6 s as described (22). At the end of the experiment the 45 Ca 2ϩ remaining in the stores was released by incubation in 1 ml of 2% (w/v) SDS for 30 min.…”