On the use of EGTA to assess the effect of Ca2+ on liver microsomal glucose-6-phosphatase

Abstract

The perturbation, by aluminium, of receptorgenerated calcium transients in hepatocytes is not due to effects of Ins(1,4,5)P3-stimulated Ca2+ release or Ins(1,4,5)P3 metabolism by the 5-phosphatase and 3-kinaseThe pursuit of the mechanism underlying toxic effects of Al3+ has led to the demonstration that A13+ may compete for Mg2+-binding sites on certain enzymes even when the free Mg2+ concentration is as much as 108-fold in excess (Miller et al., 1989). Moreover, a recent publication in this journal has highlighted the possibility of an interaction between aluminium and the phosphoinositide-mediated calcium signalling pathway (Schofl et al., 1990); in hepatocytes, hormone-stimulated repetitive Ca2+ transients (Woods et al., 1986), were perturbed by superfusion of the cells with as little as 5-10 FuM-Al3+ (as A13+ citrate). Amongst possible explanations for these effects are the proposals that Ins(1,4,5)P3 might bind A13+ particularly avidly, thereby affecting Ins(1,4,5)P3 metabolism or Ca2+ mobilization (Birchall & Chappell, 1988; Sch6fl et al., 1990). Therefore we decided to look at possible effects of aluminium on (i) calcium movements in isolated rat liver microsomes, and (ii) Ins(1,4,5)P3 metabolism by the 5-phosphatase and 3-kinase. It should be emphasized that in view of the uncertainties surrounding the kinetics of Al3+ exchange between various chelators (Miller et al., 1989), the media used in our experiments were pre-equilibrated for several hours in plastic containers.The preparation of rat liver microsomes and the measurements of Ca2+ uptake and release using a Ca2+-sensitive electrode were as described by Dawson et al. (1987). Aluminium was solubilized by mixing AICl3 (10 mM) and sodium citrate (50 mM) for at least 2 h prior to use. Rat liver microsomes were preincubated for 2 h at 0-4 'C with 300 /uM-Al3+/1.5 mM-citrate (to give 10 /LM-Al3+ in the final assay medium) and, separately, 0.5 mM-Ins(1,4,5)P3 with 5 mM-Al3+/25 mM-citrate for up to 5 h. Al3+-pretreated microsomes behaved exactly like control microsomes (i.e. plus citrate alone) in terms of Ca2+ uptake, GTP-enhancement of Ins(1,4,5)P3 sensitivity and Ins(1,4,5)P3-stimulated Ca2+ release. Similarly, Al3+-pretreated Ins(1,4,5,)P3 behaved just like control Ins(1,4,5)P3.For the studies of Ins(1,4,5)P3 metabolism, 1 mM-AlC13 was stirred with 25 mM-sodium citrate for 3-4 h. Ins(1,4,5)P3 5-phosphatase was assayed in 0.5 ml aliquots of buffer containing 2 /tM-[3H]Ins(1,4,5)P3 (1 nCi/ml), 100 mM-KCl, 1 mM-MgSO4, 10 mM-Hepes (pH 7.2 with KOH) and saponin (0.2 mg/ml). Ins(1,4,5)P3 3-kinase was assayed in similar buffer, except for the addition of 5 mM-Na2ATP plus 5 mM-MgSO4. Either 40 ,tM-Al3+ (or the equivalent citrate concentration as the control) was added to each of the buffers, which were preincubated overnight at 0-4 IC before being brought to 37°C and mixed with 25 ,ul of a 1 % (w/v) liver homogenate prepared in 0.25 M-sucrose/5 mmHepes (pH 7.2 with KOH). Ins(1,4,5)P3 metabolism was then analysed as described by Shears (1989). There was no sign...

Histone II-A stimulates glucose-6-phosphatase and reveals mannose-6-phosphatase activities without permeabilization of liver microsomes

“…Presumably, mannose produced from M6P by histone II-A-treated microsomes is released directly in the extravesicular medium. Figure 3 shows that increasing concentrations of EGTA (0.1-25 mM) have no effect on G6Pase or M6Pase activities in untreated microsomes, as we have shown previously [17,18], but almost completely reversed the stimulatory effect of histone 11-A on both G6Pase and M6Pase. Similar results were obtained when EDTA was used instead of EGTA (not shown).…”

Liver glucose-6-phosphatase activity is not modulated by physiological intracellular Ca2+ concentrations

“…In particular, G-6-P enters the vesicles through a transporter (TI), is hydrolysed intravesicularly by the phosphohydrolase component, and Pi and glucose produced are exported from the vesicles in the medium by two transporters, T2 and T3 respectively [16]. For this, the possibility exists that the rate of hydrolysis of G-6-P would be affected by the level of intravesicular Ca2+, as suggested by others [17]. This possibility, however, is unlikely, since liver microsomes which have released the accumulated Ca2+, after treatment with the Ca2+ ionophore A23 187 (2 #m; 2 min), exhibit the same G-6-Pase activity (2.61 + 0.41 nmol of glucose/min per mg of protein; mean+ S.E.M., n = 3) as those loaded with Ca2+ up to their maximal capacity (2.65 + 0.53 nmol of glucose/min per mg of protein; mean + S.E.M., n = 3; experimental conditions as in Fig.…”