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Li+ increases accumulation of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in cholinergically stimulated brain cortex slices in guinea pig, mouse and rat. The increases require inositol supplementation in mouse and rat but not in guinea pig.

ABSTRACT: Li+, beginning at a concentration as low as 1 mM, produced a time- and dose-dependent increase in accumulation of [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4,5)P4 in acetylcholine (ACh)-stimulated guinea-pig brain cortex slices prelabelled with [3H]inositol and containing 1 mM-inositol in the final incubation period. Similar results were obtained by mass measurement of samples incubated with 10 mM-Li+ by using a receptor-binding assay, although the percentage stimulation of Ins(1,4,5)P3 accumulation by Li+ was somewhat less by this assay. The increase in accumulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 by Li+ was absolutely dependent on the presence of ACh. In the absence of added inositol, 1-5 mM-Li+ produced smaller increases in Ins(1,4,5)P3, but the Li(+)-dependent increase in Ins(1,3,4,5)P4 was not as affected by inositol omission. In previous studies with cholinergically stimulated rat and mouse brain cortex slices, Li+ inhibited accumulation of Ins(1,4,5)P3 in rat and inhibited Ins(1,3,4,5)P4 accumulation in rat and mouse [Batty & Nahorski (1987) Biochem. J. 247, 797-800; Whitworth & Kendall (1988) J. Neurochem. 51, 258-265]. We found that Li+ inhibited both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 accumulation in these species, but we could reverse this inhibition by adding 10-30 mM-inositol; we then observed a Li(+)-induced increase in Ins(1,4,5)P3 and Ins(1,3,4,5)P4. The species differences observed in the absence of supplemented inositol were explained by the fact that a much higher concentration of inositol was required to bring the Li(+)-elevated levels of CDP-diacylglycerol (CDPDG) down to baseline in the rat and mouse. These data suggest that inositol is more rate-limiting for phosphatidylinositol synthesis in the presence of Li+ in rat and mouse, which can account for the previous reports of inhibition of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 accumulation by this ion in these species. Thus, in all species examined. Li+ could be shown to increase accumulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 in cholinergically stimulated brain cortex slices if the slices were supplemented with sufficient inositol to bring the Li(+)-elevated level of CDPDG down to near baseline, as seen in the absence of Li+. In guinea-pig brain cortex slices, increases in Ins(1,4,5)P3 and Ins(1,3,4,5)P4 accumulation could then be seen at Li+ concentrations as low as 1 mM, which falls within the therapeutic range of plasma concentrations in the treatment of manic-depressive disorders. These observations may have therapeutic implications.

SUBMITTER: Lee CH

PROVIDER: S-EPMC1130789 | biostudies-other | 1992 Mar

REPOSITORIES: biostudies-other

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Project description:1. The properties of specific Ins(1,4,5)P3- and Ins(1,3,4,5)P4-binding sites have been compared in a crude 'P2' cerebellar membrane fraction. 2. A homogeneous population of [3H]Ins(1,4,5)P3-binding sites was present (KD 23.1 +/- 3.6 nM) at high density (Bmax. 11.9 +/- 1.8 pmol/mg of protein); whereas data obtained for [32P]Ins(1,3,4,5)P4 specific binding were best fitted to a two-site model, the high-affinity binding component (KD 2.6 +/- 0.7 nM) constituted 64.2 +/- 4.3% of the total population and was present at relatively low density (Bmax. 187 +/- 27 fmol/mg of protein). 3. The two high-affinity inositol polyphosphate-binding sites exhibited markedly different pH optima for radioligand binding, allowing the two sites to be independently investigated. At pH 8.0, [3H]Ins(1,4,5)P3 binding was maximal, whereas [32P]Ins(1,3,4,5)P4 specific binding was very low; conversely, at pH 5.0, [32P]Ins(1,3,4,5)P4 binding was maximal, whereas [3H]Ins(1,4,5)P3 binding was undetectably low. 4. Both inositol polyphosphate-binding sites exhibited marked positional and stereo-specificity. Of the analogues studied, only phosphorothioate substitution to form inositol 1,4,5-trisphosphorothioate was tolerated at the Ins(1,4,5)P3-binding site, with only a 2-3-fold loss of binding activity. Addition of a glyceroyl moiety at the 1-phosphate position or addition of further phosphate substituents at the 3- or 6-positions caused dramatic losses in displacing activity. Similarly, complete phosphorothioate substitution of Ins(1,3,4,5)P4 caused an approx. 6-fold loss of binding activity at the [32P]Ins(1,3,4,5)P4-binding site, whereas Ins(1,4,5,6)P4, Ins(1,3,4,6)P4, Ins(1,4,5)P3 and Ins(1,3,4,5,6)P5 were bound at least 100-fold weaker at this site. Therefore, only the phosphorothioate derivatives retained high affinity and selectivity for the two inositol polyphosphate-binding sites. 5. Heparin and pentosan polysulphate were potent but non-selective inhibitors at Ins(1,4,5)P3- and Ins(1,3,4,5)P4-binding sites. N-Desulphation (with or without N-reacetylation) of heparin decreased inhibitory activity at the Ins(1,4,5)P3-, but not at the Ins(1,3,4,5)P4-binding site; however, the selectivity of this effect was only about 4-fold. O- and N-desulphated N-reacetylated heparin was essentially inactive at both sites. 6. The results are discussed with respect to the separate identities of the inositol polyphosphate-binding sites.

Project description:L1210 lymphoma cells were permeabilized with digitonin, and the ability of Ins(2,4,5)P3 and Ins(1,3,4,5)P4 to mobilize intracellular Ca2+ was studied. At high doses of Ins(2,4,5)P3 Ca2+ was rapidly released from intracellular stores, and prior or subsequent addition of Ins(1,3,4,5)P4 had no discernible effect. However, the Ca2(+)-mobilizing action of low (threshold or just above) concentrations of Ins(2,4,5)P3 was markedly enhanced by Ins(1,3,4,5)P4, which alone caused no mobilization of Ca2+; this phenomenon was shown not to be due to protection of Ins(2,4,5)P3 by the Ins(1,3,4,5)P4 against hydrolysis. The ability of the pre-addition of Ins(1,3,4,5)P4 to enhance subsequent Ins(2,4,5)P3-induced Ca2+ mobilization was always seen whether or not the free Ca2+ concentration was low (pCa = 7) or high (pCa = 6). However, at low Ca2+, Ins(1,3,4,5)P4 could cause a further mobilization if added after the Ins(2,4,5)P3, whereas at higher Ca2+ values Ins(1,3,4,5)P4 was only able to affect Ca2+ if added before Ins(2,4,5)P3. These effects of Ins(1,3,4,5)P4 were not, at the same concentration, mimicked by a random mixture of InsP4 isomers obtained by partial acid hydrolysis of phytic acid, by Ins(1,3,4)P3 or by Ins(1,3,4,5,6)P5, and they were shown not to be due to enzymic generation of Ins(1,4,5)P3 from Ins(1,3,4,5)P4 by (a) the absence of any detectable production of Ins(1,4,5)P3 if radiolabelled Ins(1,3,4,5)P4 was used, or (b) the observation that Ins(1,3,4,5,6)P5 could mimic Ins(1,3,4,5)P4 provided that higher doses were used; this inositol phosphate, when added radiolabelled, yielded only trace quantities of D/L-Ins(1,4,5,6)P4, which itself does not mobilize Ca2+. We interpret these results overall to mean that in these cells there is a small proportion of the Ins(2,4,5)P3-mobilizable Ca2+ pools which can only be mobilized in the presence of Ins(1,3,4,5)P4 [or at the least, Ins(1,3,4,5)P4 can help Ins(2,4,5)P3 to gain access to them]. The significance of this conclusion is discussed in the light of current concepts of the second messenger function of Ins(1,3,4,5)P4.

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Li+ increases accumulation of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in cholinergically stimulated brain cortex slices in guinea pig, mouse and rat. The increases require inositol supplementation in mouse and rat but not in guinea pig.

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