Lithium treatment of affective disorders: effects of lithium on the inositol phospholipid and cyclic AMP signalling pathways.

The effects of lithium (Li+) on the adenylyl cyclase and inositol phospholipid receptor signalling pathways were compared directly in noradrenergic and carbachol stimulated rat brain cortical tissue slices. Li+ was a comparatively weak inhibitor of noradrenaline-stimulated cyclic AMP accumulation with an IC50 of approx. 20 mM. By contrast, half-maximal effects of Li+ on inositol monophosphate (InsP) accumulation in [3H]inositol labelled tissue slices occurred at about 1 mM. A similar IC50 for Li+ of about 1 mM was also obtained for noradrenaline-stimulated accumulation of CMP-phosphatidate (CMPPA), a sensitive indicator of intracellular inositol depletion, in tissue slices that had been prelabelled with [3H]cytidine. The effect of myo-inositol (inositol) depletion on the prolonged activity of phosphoinositidase C (PIC) was examined in carbachol-stimulated cortical slices using a novel mass assay for InsP. Exposure to a maximal dose of carbachol for 30 min in the presence of 5 mM Li+ caused a 10-fold increase in the level of radioactivity associated with the InsP fraction, but only a 2-fold increase in InsP mass. During prolonged incubations in the presence of both carbachol and Li+ the accumulation of InsP mass was enhanced if 30 mM inositol was included in the medium. The results are compatible with the inositol depletion hypothesis of Li+ action but do not support the concept that adenylyl cyclase or guanine nucleotide dependent proteins represent therapeutically relevant targets of this drug.     

Radio-label and mass determinations of inositol 1,3,4,5-tetrakisphosphate formation in rat cerebral cortical slices: Differential effects ofmyo-inositol

To investigate the effects of increasing concentrations ofmyo-inositol (inositol) on receptor stimulated [³H]inositol polyphosphate formation in the absence of lithium, slices of rat cerebral cortex were incubated with various concentrations of [³H]inositol (1 to 30 M). Carbachol stimulated formation of [³H]inositol trisphosphate (InsP₃) and [³H]inositol 1,3,4,5-tetrakisphosphate {Ins(1,3,4,5)P₄} increased several fold when the inositol concentration was increased reaching a plateau at approximately 12 M inositol. Time course studies revealed that in the presence of low concentrations of inositol (1 M), [³H]InsP₃ and [³H]Ins(1,3,4,5)P₄ formation in response to carbachol stimulation increased slowly over a 10 to 20 min time period, whereas in the presence of 4 and 12 M inositol, carbachol stimulated [³H]InsP₃ and [³H]Ins(1,3,4,5)P₄ formation was rapid and essentially complete within 3 to 5 min after carbachol addition. Although the carbachol dose response in 12 M inositol had a much greater maximal efficacy, there was no change in potency. Similar to the effects of carbachol on [³H]Ins(1,3,4,5)P₄ formation from prelabeled phosphoinositides, muscarinic receptor stimulation increased Ins(1,3,4,5)P₄ mass formation by seven fold. Furthermore, Li⁺ (8 mM) completely inhibited carbachol stimulated increases in Ins(1,3,4,5)P₄ mass formation. In contrast to the effects of increasing inositol on carbachol stimulated formation of radiolabeled inositol phosphates, increasing inositol had no effect upon mass formation of Ins(1,3,4,5)P₄. These results show that when measuring inositol polyphosphate formation by the radiolabeling technique in the absence of Li⁺, increasing the inositol concentration greatly increases the stimulated component of [³H]InsP₃ and [³H]Ins(1,3,4,5)P₄ formation. However, this inositol induced increase in agonist stimulated Ins(1,3,4,5)P₄ formation is not reflected as an increase in mass formation.     

Inhibition by veratridine of carbachol-stimulated inositol tetrakisphosphate accumulation in rat brain cortical slices

The present studies examined the inhibitory effect of veratridine (a Na⁺ channel activator) on carbachol (a cholinergic agonist) stimulated inositol 1,3,4,5-tetrakisphosphate accumulation in rat brain cortical slices. Veratridine inhibited carbachol stimulation of inositol 1,3,4,5-tetrakisphosphate formation (after a delay of about 30 seconds) at 60 or 120 seconds when there was little inhibition of inositol 1,4,5 trisphophate accumulation. The inhibitory effect of veratridine on carbachol stimulated inositol 1,3,4,5-tetrakisphosphate accumulation was abolished in the presence of ouabain or tetrodotoxin but was unaffected in low calcium conditions. Veratridine reduced the total ATP content and this effect was abolished by tetrodotoxin. The inhibitory effect of 10 but not 30 M veratridine on inositol 1,3,4,5-tetrakisphosphate accumulation in the presence of carbachol was reversed by the presence of exogenous 8-bromo cyclic AMP or forskolin which activates adenylylcyclase. However, the decrease in brain slice ATP seen in the presence of veratridine was unaffected by forskolin. Our results are compatible with the hypothesis that veratridine inhibition of carbachol-stimulated inositol 1,3,4,5-tetrakisphosphate formation is due to depletion of ATP at the site of Ins 1,3,4,5-P₄ formation from Ins 1,4,5-P₃.Abbreviations used Ins 1,4,5-P₃ inositol 1,4,5 trisphosphate - Ins 1,3,4,5-P₄ inositol 1,3,4,5-tetrakisphosphate - PMA phorbol 12-myristate 13-acetate     

Lithium Enhances Muscarinic Receptor–Stimulated CDP-Diacylglycerol Formation in Inositol-Depleted SK-N-SH Neuroblastoma Cells

Abstract: The psychotherapeutic action of Li⁺ in brain has been proposed to result from the depletion of cellular inositol secondary to its block of inositol monophosphatase. This action is thought to slow phosphoinositide resynthesis, thereby attenuating stimulated phosphoinositidase-mediated signal transduction in affected cells. In the present study, the effect of Li⁺ on muscarinic receptor–stimulated formation of the immediate precursor of phosphatidylinositol, CDP-diacylglycerol (CDP-DAG), has been examined in human SK-N-SH neuroblastoma cells that have been cultured under conditions that alter the cellular content of myo-inositol. Resting neuroblastoma cells, like brain cells in vivo, were found to concentrate inositol from the culture medium, achieving an intracellular level of 60.0 ± 4 nmol/mg of protein. The addition of carbachol to [³H]cytidine-prelabeled cells elicited a four- to fivefold increase in the accumulation of labeled CDP-DAG. This stimulated formation of [³H]CDP-DAG was completely blocked by the addition of 10 μM atropine, was not dependent on the presence of Li⁺, nor was it affected by co-incubation with myo-inositol. This result was in sharp contrast to findings in rat brain slices, in which carbachol-stimulated formation of [³H]CDP-DAG was potentiated ～ 10-fold by Li⁺ and substantially reduced by coincubation with inositol. The formation of [³H]CDP-DAG in labeled SK-N-SH cells by carbachol was both concentration and time dependent. The order of efficacy of muscarinic ligands in stimulating [³H]-CDP-DAG accumulation paralleled that established in these cells for inositol phosphate accumulation, i.e., carbachol ≥ oxotremorine-M > bethanecol ≥ arecoline > oxotremorine > pilocarpine. Extended culture of the SK-N-SH cells in an inositol-free chemically defined growth medium progressively reduced the intracellular inositol content to <5 nmol/mg of protein, a level comparable with that seen in cortical slices. In these inositol-depleted cells, Li⁺ potentiated carbachol-stimulated [³H]CDP-DAG formation, and this effect was completely reversed by coincubation with inositol (EC₅₀ 0.2 mM). The present study thus demonstrates, in the same cultured cell line, the effects of normal and reduced intracellular inositol levels on the ability of Li⁺ to attenuate phosphoinositide resynthesis, as inferred from [³H]CDP-DAG accumulation. The results indicate that Li⁺ can lead to a slowing of stimulated phosphoinositide turnover in neuroblastoma cells, provided that the intracellular inositol content has been significantly reduced.     

Lithium Selectively Inhibits Muscarinic Receptor-Stimulated Inositol Tetrakisphosphate Accumulation in Mouse Cerebral Cortex Slices

The in vitro effects of Li on agonist- and depolarization-stimulated accumulation of inositol phosphates were determined in mouse cerebral cortex slices. Of the agents examined, only the cholinergic agonist carbachol produced a significant accumulation of inositol tetrakisphosphate (InsP4) in the absence of Li. Lithium at 5 mM enhanced the accumulation of inositol monophosphate (InsP1) and inositol bisphosphate (InsP2) due to all the stimuli used and potentiated inositol trisphosphate (InsP3) accumulation due to histamine and noradrenaline, although at lower Li concentrations, carbachol-stimulated InsP3 accumulation was reduced. Li also enhanced InsP4 accumulation in the presence of noradrenaline, histamine, and elevated KCl level but, in marked contrast, reduced carbachol-stimulated InsP4 accumulation with an IC50 of 100 microM. There was a significant time delay between the initiation of carbachol stimulation and the beginning of the InsP4 inhibition due to Li. The phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate did not mimic the effects of Li. The results suggest that muscarinic receptor-mediated InsP4 production might be one of the targets for the therapeutic action of Li.     

The effect of ischaemia and reperfusion on sarcolemmal inositol phospholipid and cytosolic inositol phosphate metabolism in the isolated perfused rat heart

In this study the mass of polyphosphoinositides as well as the turnover of [³H]inositol phospholipids and [³H]inositol phosphates during ischaemia and short periods of reperfusion were studied in the isolated perfused rat heart. Since the phosphoinositides located within the sarcolemma are precursors for release of inositoltrisphosphate (InsP₃) and diacylglycerol, sarcolemmal membranes (rather than whole tissue) isolated at the end of the experimental procedure, were used. Hearts were prelabelled with [³H]inositol and subsequently perfused with 10 mM LiCI to block the phosphatidylinositol (PI) pathway. The results showed that 20 min of global ischaemia depressed the amount of [³H]inositol present in both sarcolemmal phosphatidylinositol-4-phosphate (PI-4-P) and phosphatidylinositol-4,5-bisphosphate (PI-4,5-P₂), as well as in the cytosolic [³H]inositol phosphates, [³H]InsP₂ and [³H]InsP₃. The mass of the sarcolemmal inositol phospholipids remained unchanged during ischaemia. Reperfusion caused an immediate (within 30 sec) increase in the amount of [³H]inositol in sarcolemmal PI, PI-4-P and PI-4,5-P₂. PI-4-P levels showed a transient increase after 30 seconds postischaemic reperfusion, while the mass of the other sarcolemmal inositol phospholipids, PI and PI-4,5-P₂, remained unchanged. [³H]Insp, [³H]InsP₂ and [³H]InsP₃ also increased significantly in comparison to ischaemic hearts after only 30 sec postischaemic reperfusion.In summary, the results obtained indicate inhibition of the PI pathway during ischaemia with an immediate significant stimulation upon reperfusion. In view of the capacity of InsP3 to mobilize Ca²⁺ the possibility exists that stimulation of this pathway during reperfusion may play a role in the intracellular Ca²⁺ overload, characteristic of postischaemic reperfusion.     

Potassium ions potentiate the muscarinic receptor-stimulated phosphoinositide metabolism in cerebral cortex slices: A comparison of neonatal and adult rats

Activation of cholinergic muscarinic receptors results in an increased turnover of membrane inositol phospholipids. In rat cerebral cortex slices, carbachol- and acetylcholine-induced inositol phosphates ([³H]InsPs) accumulation is maximal in 7 day-old rats and lowest in adults, while the density of muscarinic binding sites increases gradually with age, suggesting the presence of a more effective receptor-effector coupling during neonatal life. In the process of investigating the nature of such differential stimulation, we have studied the effects of potassium ions on muscarinic receptor-stimulated phosphoinositide metabolism during development. Increasing the concentration of K⁺ from 6 to 12 mM potentiated the stimulating effect of carbachol by 80–100% in adult animals, as previously shown, but only 10–20% in 7 day-old animals, without altering its EC₅₀ values. The differential potentiation by K⁺ at these two ages was specific for muscarinic receptors, since norepinephrine-stimulated accumulation was potentiated only 18% and 12% in adult and 7 day-old rats, respectively. Two other monovalent cations, rubidium and cesium, had the same effect as K⁺ on carbachol-stimulated [³H]-InsPs accumulation. The effect of K⁺ was not antagonized by the K⁺ channel blocker 4-aminopyridine, but was antagonized by tetraethylammonium (TEA). TEA, however, also interacted with muscarinic binding sites. Omission of calcium from the incubation medium did not influence the potentiating effect of 12 mM K⁺. However, when EDTA (1 mM) was added, the stimulating effect of carbachol alone or carbachol + K⁺ was almost completely prevented. The potentiating effect of K⁺ during development was inversely proportional to the stimulation of phosphoinositide metabolism induced by carbachol. These results suggest that the mechanism responsible for the potentiating effect of K⁺ in adult rats might be already operating in neonatal animals.     

Characterization of agonist-stimulated incorporation of myo-[3H]inositol into inositol phospholipids and [3H]inositol phosphate formation in tracheal smooth muscle.

The effects of the muscarinic agonist carbachol, histamine and bradykinin on incorporation of [3H]inositol into the phosphoinositides and the formation of [3H]InsPs were examined in bovine tracheal smooth-muscle (BTSM) slices labelled with [3H]inositol. These agonists result in substantial and dose-related increases in the incorporation of [3H]inositol into the phospholipids. Carbachol and histamine stimulated the incorporation of [3H]inositol into the phospholipids to the same degree, despite histamine being only 35% as effective as carbachol on [3H]InsP accumulation. Histamine and carbachol, at maximal concentrations, were non-additive with respect to both the stimulated incorporation of [3H]inositol and [3H]InsP formation. For carbachol this effect on incorporation was found to occur to a similar extent in PtdInsP and PtdInsP2 as well as PtdIns. The initial effect of carbachol on [3H]inositol incorporation was rapid (maximal by 10 min); however, with prolonged stimulation large secondary declines in PtdInsP and PtdInsP2 labelling were observed, with depletion of the much larger PtdIns pool only evident in the presence of Li+. Lowering buffer [Ca2+] increased the incorporation of [3H]inositol under basal conditions, but did not attenuate the subsequent agonist-stimulated incorporation effect. The large changes in specific radioactivity of the phosphoinositides, and consequently the [3H]InsP products, after carbachol stimulation resulted in the apparent failure of atropine to reverse the [3H]InsP response completely. Labelling muscle slices with [3H]inositol in the presence of carbachol or labelling for longer periods (greater than 6 h) prevented subsequent carbachol-stimulated effects on incorporation without significantly altering the dose-response relationship for carbachol-stimulated [3H]InsP formation and resulted in steady-state labelling conditions confirmed by the ability of atropine to reverse fully the [3H]InsP response to carbachol. This study demonstrates the profound effects of a number of agonists on [3H]inositol incorporation into the phospho- and polyphosphoinositides in BTSM with important consequent changes in the specific radioactivity of these lipids and the resulting [3H]InsP products. In addition, a selective depletion of PtdInsP and PtdInsP2 over PtdIns has been demonstrated with prolonged muscarinic-receptor stimulation.     

The Mechanism of Muscarinic Receptor-Stimulated Phosphatidylinositol Resynthesis in 1321N1 Astrocytoma Cells and Its Inhibition by Li+

Abstract: The coupling of muscarinic receptor-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis by phospholipase C to resynthesis of phosphatidylinositol (PtdIns) and the ability of Li⁺ to inhibit this after cellular inositol depletion were studied in 1321N1 astrocytoma cells cultured in medium ± inositol (40 µM). In inositol-replete cells, 1 mM carbachol/10 mM LiCl evoked an initial (0–30 min) ～≥20-fold activation of phospholipase C, whereas prolonged (>60 min) stimulation turned over Ptdlns equal to the cellular total mass, involving ～80% of the cellular Ptdlns pool without reducing PtdIns concentrations significantly. PtdIns resynthesis was achieved by a similar, initial agonist activation of PtdIns synthase. The dose dependency for carbachol stimulation of PtdIns synthase and phospholipase C was similar (EC₅₀～ 20 µM) as was the relative intrinsic activity of muscarinic receptor partial agonists. This demonstrates the tight coupling of phosphoinositide hydrolysis to resynthesis and suggests this is achieved by a direct mechanism. In inositol-replete or depleted cells basal concentrations of inositol and CMP-phosphatidate were respectively ～20 mM or ≤100–500 µM and ～0.1 or ～≥1–10 pmol/mg of protein. Comparison of the effects of agonist ± Li⁺ on the concentrations of these cosubstrates for PtdIns synthase suggest that accelerated activity of this enzyme is differentially driven by stimulated increases in the amounts of CMP-phosphatidate or inositol in inositol-replete or depleted cells, respectively. Thus, the preferential capacity of Li⁺ to impair stimulated phosphoinositide turnover in systems expressing low cellular inositol can be attributed to its ability to attenuate the stimulated rise in inositol concentrations on which such systems selectively depend to trigger accelerated PtdIns resynthesis.     

Stimulatory and Inhibitory Effects of N-Methyl-D-Aspartate on 3H-Inositol Polyphosphate Accumulation in Rat Cortical Slices

The actions of the excitatory amino acid N-methyl-D-aspartate (NMDA) on the accumulation of 3H-inositol polyphosphate isomers in rat cerebral cortex slices have been examined over short (less than 5 min) incubation periods. NMDA caused the dose-dependent accumulation of only [3H]inositol monophosphate and [3H]inositol bisphosphate (maximal effect between 0.3 and 1 mM), with no increase in [3H]inositol trisphosphate ([3H]InsP3) and [3H]inositol tetrakisphosphate ([3H]InsP4). HPLC analysis confirmed this, showing no increases in the breakdown products of [3H]Ins(1,3,4,5)P4. When present with the muscarinic agonist carbachol (1 mM), high concentrations of NMDA (1 mM) could almost totally inhibit carbachol-induced accumulation of 3H-inositol polyphosphates. In contrast, at lower concentrations of NMDA (10 microM), the inhibitory effect was replaced with a synergistic accumulation of inositol polyphosphates, especially [3H]InsP4 and [3H]InsP3. The inhibitory effects of NMDA were only apparent when extracellular Ca2+ was present, although incubation in media with no added Ca2+ resulted in somewhat reduced stimulatory responses to NMDA alone, but suppressed totally the inhibitory effects of 1 mM NMDA and reduced the synergistic effects of 10 microM NMDA on carbachol responses. These studies, therefore, reveal Ca(2+)-dependent effects of NMDA indicative of indirect mechanisms of action and show that care must be made in interpreting the effects of NMDA on phosphoinositide metabolism unless the inositol polyphosphate composition has been fully characterised.     

Prostaglandins and muscarinic agonists induce cyclic AMP attenuation by two distinct mechanisms in the pregnant-rat myometrium. Interaction between cyclic AMP and Ca2+ signals.

In pregnant-rat myometrium (day 21 of gestation), isoprenaline-induced cyclic AMP accumulation, resulting from receptor-mediated activation of adenylate cyclase, was negatively regulated by prostaglandins [PGE2, PGF2 alpha; EC50 (concn. giving 50% of maximal response) = 2 nM] and by the muscarinic agonist carbachol (EC50 = 2 microM). PG-induced inhibition was prevented by pertussis-toxin treatment, supporting the idea that it was mediated by the inhibitory G-protein Gi through the inhibitory pathway of the adenylate cyclase. Both isoprenaline-induced stimulation and PG-evoked inhibition of cyclic AMP were insensitive to Ca2+ depletion. By contrast, carbachol-evoked attenuation of cyclic AMP accumulation was dependent on Ca2+ and was insensitive to pertussis toxin. The inhibitory effect of carbachol was mimicked by ionomycin. Indirect evidence was thus provided for the enhancement of cyclic AMP degradation by a Ca2(+)-dependent phosphodiesterase activity in the muscarinic-mediated effect. The attenuation of cyclic AMP elicited by carbachol coincided with carbachol-stimulated inositol phosphate (InsP3, InsP2 and InsP) generation, which displayed an almost identical EC50 (3 microM) and was similarly unaffected by pertussis toxin. Both carbachol effects were reproduced by oxotremorine, whereas pilocarpine (a partial muscarinic agonist) failed to induce any decrease in cyclic AMP accumulation and concurrently was unable to stimulate the generation of inositol phosphates. These data support our proposal for a carbachol-mediated enhancement of a Ca2(+)-dependent phosphodiesterase activity, compatible with the rises in Ca2+ associated with muscarinic-induced increased generation of inositol phosphates. They further illustrate that a cross-talk between the two major transmembrane signalling systems contributed to an ultimate decrease in cyclic AMP in the pregnant-rat myometrium near term.     

Calcium channel involvement in potassium depolarization-induced phosphoinositide hydrolysis in rat cortical slices

The stimulation of production of inositol phosphates in rat cortical slices by KCl depolarization and the effects of calcium channel active drugs were investigated. Elevation of K⁺ in the medium up to 48 mM KCl caused a linear concentration-dependent increase in [³H]inositol phosphate accumulation. The KCl stimulated response was not significantly inhibited in the presence of muscarinic or ₁-adrenergic antagonists. KCl stimulated the production of inositol trisphosphate at 60 min but not 10 min. Addition of peptidase inhibitors did not significantly affect KCl-stimulated PI hydrolysis. The KCl-stimulated response was still observed in the absence of extracellular calcium, although the net accumulation of inositol phosphates was greater in the presence of 0.1 or 0.5 mM calcium. KCl (48 mM) inhibited [³H]inositol uptake into phospholipids of cortical slices. The dihydropyridine calcium channel agonist BAY K 8644 stimulated PI hydrolysis in cortical slices in a concentration dependent manner in the presence of 19 mM KCl. The BAY K 8644-stimulated PI response was partially inhibited by 1M atropine but not by 1M prazosin. Calcium channel blockers nitrendipine, verapamil, flunarizine, and nifedipine slightly inhibited the PI response stimulated by 19 mM KCl in the presence or absence of BAY K 8644. The effects of the calcium channel antagonists were attenuated in the presence of 1 M atropine. The peptide calcium channel blocker -conotoxin did not affect KCl-stimulated PI hydrolysis. These results suggest that endogenous muscarinic or adrenergic neurotransmitters are not involved in KCl-stimulated PI hydrolysis in cortical slices. Although extracellular calcium is necessary for optimal KCl-stimulated PI hydrolysis, it is not required for the expression of the KCl-evoked response suggesting that depolarization is the primary trigger for this stimulant.     

Carbachol causes rapid phosphodiesteratic cleavage of phosphatidylinositol 4,5-bisphosphate and accumulation of inositol phosphates in rabbit iris smooth muscle; prazosin inhibits noradrenaline- and ionophore A23187-stimulated accumulation of inositol phosphates.

Rabbit iris smooth muscle was prelabelled with myo-[3H]inositol for 90 min and the effect of carbachol on the accumulation of inositol phosphates from phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol (PtdIns) was monitored with anion-exchange chromatography. Carbachol stimulated the accumulation of inositol phosphates and this was blocked by atropine, a muscarinic antagonist, and it was unaffected by 2-deoxyglucose. The data presented demonstrate that, in the iris, carbachol (50 microM) stimulates the rapid breakdown of PtdIns(4,5)P2 into [3H]inositol trisphosphate (InsP3) and diacylglycerol, measured as phosphatidate, and that the accumulation of InsP3 precedes that of [3H]inositol bisphosphate (InsP2) and [3H]inositol phosphate (InsP). This conclusion is based on the following findings. Time course experiments with myo-[3H]inositol revealed that carbachol increased the accumulation of InsP3 by 12% in 15s and by 23% in 30s; in contrast, a significant increase in InsP release was not observed until about 2 min. Time-course experiments with 32P revealed a 10% loss of radioactivity from PtdIns(4,5)P2 and a corresponding 10% increase in phosphatidate labelling by carbachol in 15s; in contrast a significant increase in PtdIns labelling occurred in 5 min. Dose-response studies revealed that 5 microM-carbachol significantly increased (16%) the accumulation of InsP3 whereas a significant increase in accumulation of InsP2 and InsP was observed only at agonist concentrations greater than 10 microM. Studies on the involvement of Ca2+ in the agonist-stimulated breakdown of PtdIns(4,5)P2 in the iris revealed the following. Marked stimulation (58-78%) of inositol phosphates accumulation by carbachol in 10 min was observed in the absence of extracellular Ca2+. Like the stimulatory effect of noradrenaline, the ionophore A23187-stimulated accumulation of InsP3 was inhibited by prazosin, an alpha 1-adrenergic blocker, thus suggesting that the ionophore stimulation of PtdIns(4,5)P2 breakdown we reported previously [Akhtar & Abdel-Latif (1978) J. Pharmacol. Exp. Ther. 204, 655-688; Akhtar & Abdel-Latif (1980) Biochem. J. 192, 783-791] was secondary to the release of noradrenaline by the ionophore. The carbachol-stimulated accumulation of inositol phosphates was inhibited by EGTA (0.25 mM) and this inhibition was reversed by excess Ca2+ (1.5 mM), suggesting that EGTA treatment of the tissue chelates extracellular Ca2+ required for polyphosphoinositide phosphodiesterase activity. K+ depolarization, which causes influx of extracellular Ca2+ in smooth muscle, did not change the level of InsP3.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparative Effects of Lithium on the Phosphoinositide Cycle in Rat Cerebral Cortex, Hippocampus, and Striatum

Abstract: The effects of lithium on muscarinic cholinoceptor-stimulated phosphoinositide turnover have been investigated in rat hippocampal, striatal, and cerebral cortical slices using [³H]inositol or [³H]cytidine prelabelling and inositol 1,4,5-trisphosphate [lns(1,4,5)P₃] and inositol 1,3,4,5-tetrakisphosphate [lns(1,3,4,5)P₄] mass determination methods. Carbachol addition resulted in maintained increases in lns(1,4,5)P₃ and lns(1,3,4,5)P₄ mass levels in hippocampus and cerebral cortex, whereas in striatal slices these responses declined significantly over a 30-min incubation period. Carbachol-stimulated lns(1,4,5)P₃ and lns(1,3,4,5)P₄ accumulations were inhibited by lithium in all brain regions studied in a time-and concentration-dependent manner. For example, in hippocampal slices significant inhibitory effects of LiCl were observed at times > 10 min after agonist challenge; IC₅₀ values for inhibition of agonist-stimulated lns(1,4,5)P₃ and lns(1,3,4,5)P₄ accumulations by lithium were 0.22 ± 0.09 and 0.33 ± 0.13 mM, respectively. [³H]CMP-phosphatidate accumulation increased in all brain regions when slices were stimulated by agonist and lithium. The ability of myo-inositol to reverse these effects, as well as lithium-suppressed lns(1,4,5)P₃ accumulation, implicates myo-inositol depletion in the action of lithium in the hippocampus and cortex at least. The results of this study suggest that although significant differences in the magnitude and time courses of changes in inositol (poly)phosphate metabolites occur in different brain regions, lithium evokes qualitatively similar enhancements of [³H]inositol monophosphate and [³H]CMP-phosphatidate levels and inhibitions of lns(1,4,5)P₃ and lns(1,3,4,5)P₄ accumulations. However, the inability of striatal slices to sustain carbachol-stimulated inositol polyphosphate accumulation in the absence of lithium and the inability to reverse effects with myo-inositol may indicate differences in phosphoinositide signalling in this brain region.     

Lithium inhibits muscarinic-receptor-stimulated inositol tetrakisphosphate accumulation in rat cerebral cortex.

The effects of Li+ on carbachol-stimulated phosphoinositide metabolism were examined in rat cerebral-cortex slices labelled with myo-[2-3H]inositol. The muscarinic agonist carbachol evoked an enhanced steady-state accumulation of [3H]inositol monophosphate ([3H]InsP1), [3H]inositol bisphosphate ([3H]InsP2), [3H]inositol 1,3,4-trisphosphate ([3H]Ins(1,3,4)P3), [3H]inositol 1,4,5-trisphosphate ([3H]Ins(1,4,5)P3) and [3H]inositol tetrakisphosphate ([3H]InsP4). Li+ (5 mM), after a 10 min lag, severely attenuated carbachol-stimulated [3H]InsP4 accumulation while simultaneously potentiating accumulation of both [3H]InsP1 and [3H]InsP2 and, at least initially, of [3H]Ins(1,3,4)P3. These data are consistent with inhibition of inositol mono-, bis- and 1,3,4-tris-phosphate phosphatases to different degrees by Li+ in brain, but are not considered to be completely accounted for in this way. Potential direct and indirect mechanisms of the inhibitory action of Li+ on [3H]InsP4 accumulation are considered. The present results stress the complex action of Li+ on cerebral inositol metabolism and indicate that more complex mechanisms than are yet evident may regulate this process.     

Reduced inositol polyphosphate accumulation and inositol supply induced by lithium in stimulated cerebral cortex slices.

The ability of lithium to interfere with phosphoinositide metabolism in rat cerebral cortex slices has been examined by monitoring the accumulation of CMP-phosphatidate (CMP-PtdOH) and the reduction in Ins(1,4,5)P3 and Ins(1,3,4,5)P4 levels. A small accumulation of [14C]CMP-PtdOH was seen in slices prelabelled with [14C]cytidine and stimulated with carbachol (1 mM) or Li+ (1 mM). However, simultaneous addition of both agents for 30 min produced a 22-fold accumulation, with Li+ producing a half-maximal effect at a concentration of 0.61 +/- 0.19 mM. Kinetic studies revealed that the effects of carbachol and Li+ on CMP-PtdOH accumulation occurred with no initial lag apparent under these conditions and that preincubation with myo-inositol (10 or 30 mM) dramatically attenuated CMP-PtdOH accumulation. myo-Inositol could also attenuate the rate of accumulation of CMP-PtdOH when added 20 min after carbachol and Li+; these effects were not observed when equimolar concentrations of scyllo-inositol were added. Use of specific radioreceptor assays allowed the mass accumulations of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 to be monitored. Following a lag of 5-10 min, Li+ resulted in a marked reduction in the accumulation of both inositol polyphosphates resulting from muscarinic-cholinergic stimulation. Preincubation of cerebral cortex slices with myo- (but not scyllo-) inositol delayed, but did not prevent, the reduction in the accumulation of Ins(1,4,5)P3 or Ins(1,3,4,5)P4. The results suggest that cerebral cortex, at least in vitro, is very sensitive to myo-inositol depletion under conditions of muscarinic receptor stimulation. The relationship of such depletion to the generation of inositol polyphosphate second messengers is discussed.     

Role of calcium ions in rapid effects of L-thyroxine on phosphoinositide metabolism in rat liver cells

The role of calcium ions in the L-thyroxine-induced initiation of hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP₂) and also the course of releasing individual fractions of inositol phosphates and diacylglycerides (DAG) were studied in liver cells during early stages of the hormone effect. L-Thyroxine stimulated a rapid hydrolysis in hepatocytes of PtdInsP₂ labeled with [¹⁴C]linoleic acid and [³H]inositol mediated by phosphoinositide-specific phospholipase C. This was associated with accumulation of [¹⁴C]DAG, total inositol phosphates, [³H]inositol 1,4,5-trisphosphate (Ins1,4,5P₃) and [³H]inositol 1,4-bisphosphate (Ins1,4P₂). Elimination of calcium ions from the incubation medium of hepatocytes did not abolish the effect of thyroxine on the accumulation of [¹⁴C]DAG and total [³H]inositol phosphates. Preincubation of liver cells with TMB-8 increased the stimulatory effect of L-thyroxine on the accumulation of [¹⁴C]DAG. During the incubation of hepatocytes in the presence of the hormone the content of ¹⁴C-labeled fatty acids did not change. The L-thyroxineinduced accumulation of [³H]Ins1,4,5P₃ and [³H]Ins1,4P₂ did not depend on the presence of calcium ions in the incubation medium of the cells.     

Functional identification of sll1383 from<Emphasis Type="Italic"> Synechocystis</Emphasis> sp PCC 6803 as L-<Emphasis Type="Italic">myo</Emphasis>-inositol 1-phosphate phosphatase (EC 3.1.3.25): molecular cloning,expression and characterization

The genome sequence of the cyanobacterium Synechocystis sp. PCC6803 revealed four Open reading frame (ORF) encoding putative inositol monophosphatase or inositol monophosphatase-like proteins. One of the ORFs, sll1383, is ∼870 base pair long and has been assigned as a probable myo-inositol 1 (or 4) monophosphatase (IMPase; EC 3.1.3.25). IMPase is the second enzyme in the inositol biosynthesis pathway and catalyses the conversion of L-myo-inositol 1-phosphate to free myo-inositol. The present work describes the functional assignment of ORF sll1383 as myo-inositol 1-phosphate phosphatase (IMPase) through molecular cloning, bacterial overexpression, purification and biochemical characterization of the gene product. Affinity (K _m) of the recombinant protein for the substrate DL-myo-inositol 1-phosphate was found to be much higher (0.0034 ± 0.0003 mM) compared to IMPase(s) from other sources but in comparison V _max (∼0.033 μmol Pi/min/mg protein) was low. Li⁺ was found to be an inhibitor (IC₅₀ 6.0 mM) of this enzyme, other monovalent metal ions (e.g. Na⁺, K⁺ NH₄⁺) having no significant effect on the enzyme activity. Like other IMPase(s), the activity of this enzyme was found to be totally Mg²⁺ dependent, which can be substituted partially by Mn²⁺. However, unlike other IMPase(s), the enzyme is optimally active at ∼42°C. To the best of our knowledge, sll1383 encoded IMPase has the highest substrate affinity and specificity amongst the known examples from other prokaryotic sources. A possible application of this recombinant protein in the enzymatic coupled assay of L-myo-inositol 1-phosphate synthase (MIPS) is discussed.     

Ca2+ Influx through Store-operated Calcium Channels Replenishes the Functional Phosphatidylinositol 4,5-Bisphosphate Pool Used by Cysteinyl Leukotriene Type I Receptors

Oscillations in cytoplasmic Ca²⁺ concentration are a universal mode of signaling following physiological levels of stimulation with agonists that engage the phospholipase C pathway. Sustained cytoplasmic Ca²⁺ oscillations require replenishment of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂), the source of the Ca²⁺-releasing second messenger inositol trisphosphate. Here we show that cytoplasmic Ca²⁺ oscillations induced by cysteinyl leukotriene type I receptor activation run down when cells are pretreated with Li⁺, an inhibitor of inositol monophosphatases that prevents PIP₂ resynthesis. In Li⁺-treated cells, cytoplasmic Ca²⁺ signals evoked by an agonist were rescued by addition of exogenous inositol or phosphatidylinositol 4-phosphate (PI4P). Knockdown of the phosphatidylinositol 4-phosphate 5 (PIP5) kinases α and γ resulted in rapid loss of the intracellular Ca²⁺ oscillations and also prevented rescue by PI4P. Knockdown of talin1, a protein that helps regulate PIP5 kinases, accelerated rundown of cytoplasmic Ca²⁺ oscillations, and these could not be rescued by inositol or PI4P. In Li⁺-treated cells, recovery of the cytoplasmic Ca²⁺ oscillations in the presence of inositol or PI4P was suppressed when Ca²⁺ influx through store-operated Ca²⁺ channels was inhibited. After rundown of the Ca²⁺ signals following leukotriene receptor activation, stimulation of P2Y receptors evoked prominent inositol trisphosphate-dependent Ca²⁺ release. Therefore, leukotriene and P2Y receptors utilize distinct membrane PIP₂ pools. Our findings show that store-operated Ca²⁺ entry is needed to sustain cytoplasmic Ca²⁺ signaling following leukotriene receptor activation both by refilling the Ca²⁺ stores and by helping to replenish the PIP₂ pool accessible to leukotriene receptors, ostensibly through control of PIP5 kinase activity.     

Modulation of carbachol-stimulated inositol phospholipid hydrolysis in rat cerebral cortex

Cortical slices from rat brain were used to study carbachol-stimulated inositol phospholipid hydrolysis. Omission of calcium during incubation of slices with [³H]inositol increased its incorporation into receptor-coupled phospholipids. Carbachol-stimulated hydrolysis of [³H]inositol phospholipids in slices was dose-dependent, was affected by the concentrations of calcium and lithium present and resulted in the accumulation of mostly [³H]inositol-l-phosphate. Incubation of slices withN-ethylmaleimide or a phorbol ester reduced the response to carbachol. Membranes prepared from cortical slices labeled with [³H]inositol retained the receptor-stimulated inositol phospholipid hydrolysis reaction. The basal rate of inositol phospholipid hydrolysis was higher than in slices and addition of carbachol further stimulated the process. Addition of GTP stimulated inositol phospholipid hydrolysis, suggesting the presence of a guanine nucleotide-binding protein coupled to phospholipase C. Carbachol and GTP-stimulated inositol phospholipid hydrolysis in membranes was detectable following a 3 min assay period. In contrast to slices, increased levels of inositol bisphosphate and inositol trisphosphate were detected following incubation of membranes with carbachol. These results demonstrate that agonist-responsive receptors are present in cortical membranes, that the receptors may be coupled to phosphatidylinositol 4,5-bisphosphate, rather than phosphatidylinositol, hydrolysis and that a guanine nucleotide-binding protein may mediate the coupling of receptor activation to inositol phospholipid hydrolysis in brain.