The perturbation, by aluminium, of receptor-generated 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-kinase

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...

Aluminium Inhibits Muscarinic Agonist‐Induced Inositol 1,4,5‐Trisphosphate Production and Calcium Mobilization in Permeabilized SH‐SY5Y Human Neuroblastoma Cells

“…This has enabled us to investigate the effect of A13+ on Ca2+ release induced by exogenous and endogenous InsP,, as well as agonist-stimulated InsP, production. The close parallel inhibition by A13+ of muscarinic agonist-stimulated InsP, production and subsequent Ca2+ release and the lack of effect of A13+ on Ca2+ release induced by exogenous InsP, largely agree with recent observations made in experiments using rat liver microsomes indicating that the effects of A13+ on InsP,-induced Ca2+ release does not result from perturbation of either InsP, metabolism or InsP,-induced Ca2+ release (Shears et al, 1990). Rather, our data strongly suggest that the inhibitory effect of this ion is proximal to InsP, production.…”

The Structural and Functional Versatility of Inositol Phosphates

“…For example, Ins(l,4,5)P 3 and Ins(l,3,4,5)P 4 metabolism by the 5-phosphatases were proposed to be particularly susceptible to toxic insult by Al (55). However, we could found no evidence that this was the case (56). The HIV gpl20 coat protein has been proposed to target 5-phosphatase activity (57,58), but when we investigated this issue we saw no such effects of gpl20, despite our preparations of this protein being biologically competent in several other respects (59).…”

Effects of aluminium on the hepatic inositol polyphosphate phosphatase

“…When 1-50 ,uM AP3+ was present, InsP6 became a less effective inhibitor of Ins(1,3,4,5)P4 3-phosphatase activity; this effect did not depend on the presence of cellular membranes, contrary to a previous proposal. The latter phenomenon largely explains how, in a cell-free system where Ins (1,3,4,5)P4 3-phosphatase is inhibited by endogenous InsP6, the addition of A13+ can apparently increase the enzyme activity.…”

“…Subsequent reports that A13+ disrupted Ca2+-oscillatory activity in cells [2,3] reinforced the idea that some toxic effects of A13+ may arise from interactions with the phosphoinositide signalling system. However, up to 40 ,uM A13+ has since been shown not to affect either Ins(1,4,5)P3-mediated Ca2+ release or Ins (1,4,5)P3 metabolism by the 5-phosphatase or 3-kinase [4]. On the other hand, the addition of A13+ to permeabilized L1210 cells stimulated 3phosphatase activity towards Ins(1,3,4,5)P4, and the Ins (1,4,5)P3 that was formed then mobilized intracellular Ca2+ stores [5].…”

“…However, up to 40 ,uM A13+ has since been shown not to affect either Ins(1,4,5)P3-mediated Ca2+ release or Ins (1,4,5)P3 metabolism by the 5-phosphatase or 3-kinase [4]. On the other hand, the addition of A13+ to permeabilized L1210 cells stimulated 3phosphatase activity towards Ins(1,3,4,5)P4, and the Ins (1,4,5)P3 that was formed then mobilized intracellular Ca2+ stores [5]. These results suggested that 3-phosphatase activity might be a metabolic target during A13+ intoxication in vivo.…”