Accumulation of the inositol phosphates in thrombin-stimulated, washed rabbit platelets in the presence of lithium.

Experiments with washed rabbit platelets demonstrate that stimulation with a low concentration of thrombin (0.1 unit/ml), that causes maximal aggregation and partial release of amine granule contents, also causes increased accumulation of [3H]inositol-labelled inositol trisphosphate (InsP3) in the presence of 20 mM-Li+. This concentration of Li+ was found to inhibit the degradation of inositol phosphates by phosphomonoesterases. This result indicates that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is degraded early after platelet stimulation with thrombin, although in a previous study we had found no decrease in amount. In the absence of Li+, the labelling of inositol bisphosphate (InsP2) increased more rapidly than that of InsP3, consistent with rapid degradation of InsP3 by phosphomonoesterase. After 30s the increase in InsP2 was augmented by Li+. This increase in InsP2 could have been due to increased degradation of phosphatidylinositol 4-phosphate or inhibition of breakdown of InsP2 to InsP with a lesser inhibition of breakdown of InsP3 to InsP2. The effect on InsP3 and InsP2 of stimulation of the platelets with 1.0 unit of thrombin/ml was comparable with the effect of the lower concentration of thrombin. Inositol phosphate (InsP) labelling did not increase in response to 0.1 unit of thrombin/ml, but increased when the platelets were stimulated with 1.0 unit of thrombin/ml. Whether the increase in InsP was due to increased degradation of phosphatidylinositol or a greater rate of breakdown of InsP2 to InsP than InsP to inositol cannot be determined in these experiments. These results indicate that degradation of PtdIns(4,5)P2 is an early event in platelet activation by thrombin and that formation of inositol phosphates and 1,2-diacylglycerol rather than a decrease in PtdIns(4,5)P2 may be the important change.     

The effects of lithium on platelet phosphoinositide metabolism.

The effects on phosphoinositide metabolism of preincubation of platelets for 90 min with 10 mM-Li+ were studied. Measurements were made of [32P]phosphate-labelled phosphoinositides and of [3H]inositol-labelled inositol mono-, bis- and tris-phosphate (InsP, InsP2 and InsP3). Li+ had no effect on the basal radioactivity in the phosphoinositides or in InsP2 or InsP3, but it caused a 1.8-fold increase in the basal radioactivity in InsP. Li+ caused a 4-, 3- and 2-fold enhanced thrombin-induced accumulation of label in InsP, InsP2 and InsP3 respectively. Although the elevated labelling of InsP2 and InsP3 returned to near-basal values within 30-60 min, the high labelling of InsP did not decline over a period of 60 min after addition of thrombin to Li+-treated platelets, consistent with inhibition of InsP phosphatase by Li+. The effect of Li+ was not due to a shift in the thrombin dose-response relationship; increasing concentrations of thrombin enhanced the initial rate of production of radiolabelled inositol phosphates, whereas Li+ affected either a secondary production or the rate of their removal. The only observed effect of Li+ on phosphoinositide metabolism was a thrombin-induced decrease (P less than 0.05) in labelled phosphatidylinositol 4-phosphate in Li+-treated platelets; this suggests an effect on phospholipase C. Li+ enhanced (P less than 0.05) the thrombin-induced increase in labelled lysophosphatidylinositol, suggesting an effect on phospholipase A2. It is concluded that Li+ inhibits InsP phosphatase and has other effects on phosphoinositide metabolism in activated platelets. The observed effects occur too slowly to be the mechanism by which Li+ potentiates agonist-induced platelet activation.     

Stimulation, by vasopressin and other agonists, of inositol-lipid breakdown and inositol phosphate accumulation in WRK 1 cells.

WRK 1 cells were labelled to equilibrium with 2-myo-[3H]inositol and stimulated with vasopressin. Within 3 s of hormone stimulation there was a marked accumulation of 3H-labelled InsP2 and InsP3 (inositol bis- and tris-phosphate), but not of InsP (inositol monophosphate). There was an associated, and rapid, depletion of 3H-labelled PtdInsP and PtdInsP2 (phosphatidylinositol mono- and bis-phosphates), but not of PtdIns (phosphatidylinositol), in these cells. Some 4% of the radioactivity in the total inositol lipid pool of WRK 1 cells was recovered in InsP2 and InsP3 after 10 s stimulation with the hormone. The selectivity of the vasopressin receptors of WRK 1 cells for a variety of vasopressin agonists and antagonists revealed these to be of the V1a subtype. There was no receptor reserve for vasopressin-stimulated inositol phosphate accumulation in WRK 1 cells. The accumulation of inositol phosphates was enhanced in the presence of Li+ions. Half-maximal accumulation of InsP, InsP2 and InsP3 in vasopressin-stimulated cells was observed with 0.9, 3.0 and 3.6 mM-Li+ respectively. Bradykinin and 5-hydroxytryptamine also provoked inositol phosphate accumulation in WRK 1 cells. The effects of sub-optimal concentrations of bradykinin and vasopressin upon inositol phosphate accumulation were additive, but those of optimal concentrations of the hormones were not.     

Inositol phospholipid metabolism in human platelets stimulated by ADP

ADP-induced changes in inositol phospholipids, phosphatidic acid and inositol phosphates of human platelets have been studied in detail, using not only 32P labelling, but also by examining changes in amounts of the phospholipids, their labelling with [3H]glycerol and their specific radioactivities; changes in the labelling of inositol phosphates in platelets prelabelled with [3H]inositol were also measured. During the early (10 s) stage of reversible ADP-induced primary aggregation in a medium containing fibrinogen and with a concentration of Ca2+ in the physiological range (2 mM), the amounts of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) and phosphatidylinositol 4-phosphate (PtdInsP) decreased (by 11.2 +/- 4.9% and 11.3 +/- 5.3%, respectively) while the labelling, but not the amount, of phosphatidic acid increased. The decreases do not appear to be attributable to the action of phospholipase C because the specific radioactivity of phosphatidic acid labelling with [3H]glycerol was not significantly increased at 10 s (although the initial specific radioactivities of the inositol phospholipids and PtdCho were more than double that of phosphatidic acid), and no increases in the labelling of inositol trisphosphate (InsP3), inositol bisphosphate (InsP2) or inositol phosphate (InsP) were detectable at 10 s. Shifts in the interconversions between PtdInsP2 and PtdInsP, and PtdInsP and PtdIns may occur. By 30 to 60 s, when deaggregation was beginning, the amounts of PtdInsP2, PtdInsP and phosphatidic acid were not different from those in unstimulated platelets, but large increases in the 32P-labelling and [3H]glycerol labelling of phosphatidic acid were observed. Formation of [3H]inositol-labelled InsP3 was not detectable at any time in association with ADP-induced primary aggregation, indicating that degradation of PtdInsP2 by phospholipase C is not appreciably stimulated by ADP. These findings were compared with those obtained when platelets were aggregated by ADP in a medium without added of Ca2+ in which secondary aggregation associated with thromboxane A2 (TXA2) formation and release of granule contents occurs. At 10 s (during primary aggregation) the changes were similar in the two media. At 30 s and 60 s (during secondary aggregation in the low-Ca2+ medium), the increases in PtdInsP2, PtdInsP and phosphatidic acid in platelets suspended in the absence of added Ca2+ were larger than those in platelets suspended in the presence of 2 mM Ca2+. In the absence of added Ca2+, ADP-induced increases in the labelling of InsP3, InsP2 and InsP which were probably due to the effects of TXA2 since they were abolished by aspirin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of guanosine 5'-[gamma-thio]triphosphate and thrombin on the phosphoinositide metabolism of electropermeabilized human platelets

Incubation of human platelets with myo-[3H]inositol in a low-glucose Tyrode's solution containing MnCl2 enhanced the labelling of phosphoinositides about sevenfold and greatly facilitated the measurement of [3H]inositol phosphates formed by the activation of phospholipase C. Labelled platelets were permeabilized by high-voltage electric discharges and equilibrated at 0 degree C with ATP, Ca2+ buffers and guanine nucleotides, before incubation in the absence or presence of thrombin. Incubation of these platelets with ATP in the presence or absence of Ca2+ ions led to the conversion of [3H]phosphatidylinositol to [3H]phosphatidylinositol 4-phosphate and [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PtdInsP2). At a pCa of 6, addition of 100 microM GTP[gamma S] both prevented this accumulation of [3H]PtdInsP2 and stimulated its breakdown; the formation of [3H]inositol phosphates was increased ninefold. After 5 min these comprised 70% [3H]inositol monophosphate ([3H]InsP), 28% [3H]inositol bisphosphate ([3H]InsP2) and 2% [3H]inositol trisphosphate ([3H]InsP3). In shorter incubations higher percentages of [3H]InsP2 and [3H]InsP3 were found. In the absence of added Ca2+, the formation of [3H]inositol phosphates was decreased by over 90%. Incubation of permeabilized platelets with GTP[gamma S] in the presence of 10 mM Li+ decreased the accumulation of [3H]InsP and increased that of [3H]InsP2, without affecting [3H]InsP3 levels. Addition of unlabelled InsP3 decreased the intracellular hydrolysis of exogenous [32P]InsP3 but did not trap additional [3H]InsP3. These results and the time course of [3H]inositol phosphate formation suggest that GTP[gamma S] stimulated the action of phospholipase C on a pool of [3H]phosphatidylinositol 4-phosphate that was otherwise converted to [3H]PtdInsP2 and that much less hydrolysis of [3H]phosphatidylinositol to [3H]InsP or of [3H]PtdInsP2 to [3H]InsP3 occurred. At a pCa of 6, addition of thrombin (2 units/ml) to permeabilized platelets caused small increases in the formation of [3H]InsP and [3H]InsP2. This action of thrombin was enhanced twofold by 10-100 microM GTP and much more potently by 4-40 microM GTP[gamma S]. In the presence of the latter, thrombin also increased [3H]InsP3. The total formation of [3H]inositol phosphates by permeabilized platelets incubated with thrombin and GTP[gamma S] was comparable with that observed on addition of thrombin alone to intact platelets. However, HPLC of the [3H]inositol phosphates formed indicated that about 75% of the [3H]InsP accumulating in permeabilized platelets was the 4-phosphate, whereas in intact platelets stimulated by thrombin, up to 80% was the 1-phosphate.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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)     

Production of inositol trisphosphates and inositol tetrakisphosphate in stimulated pancreatic islets

Glucose and carbamylcholine caused concentration-dependent increases in the production of total [3H]inositol phosphates in [3H]inositol-labelled rat pancreatic islets. When extracts from islets stimulated with glucose, carbamylcholine or depolarising concentrations of K+ were analysed using anion-exchange high performance liquid chromatography, increased production of [3H]Ins1,4,5-P3 was detected, and in addition, elevated levels of two other labelled compounds which co-chromatographed with Ins1,3,4-P3 and Ins1,3,4,5-P4. In the case of carbamylcholine and high K+, such an effect was apparent within 20 s, whereas glucose appeared to cause a delayed response. In the presence of 5 mM LiCl, the accumulation of Ins1,3,4-P3 was more marked. The presence of LiCl had no major influence on the levels of Ins1,4,5-P3 or Ins1,3,4,5-P4. It is suggested that the stimulation of pancreatic islets with glucose, carbamylcholine or high K+ results in the hydrolysis of inositol lipids with the production of Ins1,4,5-P3 and in addition, Ins1,3,4-P3 and Ins1,3,4,5-P4. The physiological functions of these novel inositol phosphates in islets remain to be established.     

Lithium-induced accumulation of inositol 1-phosphate during cholecystokinin octapeptide- and acetylcholine-stimulated phosphatidylinositol breakdown in dispersed mouse pancreas acinar cells

Dispersed mouse pancreas acinar cells were prepared in which phosphatidylinositol had been labeled with myo[2-3H]inositol. During incubation with 0.3 microM cholecystokinin octapeptide (CCK-8) for 15 min, there was a loss of [3H]phosphatidylinositol radioactivity (23%) and a 3-fold gain in trichloroacetic acid-soluble radioactivity. Replacement of NaCl by up to 58 mM LiCl did not significantly affect the amount of CCK-8-stimulated [3H]phosphatidylinositol breakdown or the gain in acid-soluble radioactivity. However, in normal medium, the product of phosphatidylinositol breakdown was almost all inositol, whereas in Li+-containing medium, the product was almost all inositol 1-phosphate. Similar results were obtained with acetylcholine which, in the presence of Li+, gave a dose-responsive increase in inositol 1-phosphate over the concentration range of 0.1 to 10 microM. No increased accumulation of [3H]inositol diphosphate or [3H]inositol triphosphate was detected in stimulated cells. Time courses in the presence of Li+ indicated that the formation of inositol 1-phosphate preceded the formation of inositol. Addition of up to 50 mM myoinositol to the incubation medium showed no diluting effect on the amount of [3H]inositol 1-phosphate found. The accumulation of inositol 1-phosphate is presumably due to the known ability of Li+ to inhibit myoinositol 1-phosphatase. The results provide clear evidence that stimulated phosphatidylinositol breakdown involves a phospholipase C type of phosphodiesterase activity. 1.25 mM Li+ gave half-maximal inositol 1-phosphate accumulation. This is close to the range of plasma Li+ levels which is used therapeutically in psychiatric disorders. In unstimulated cells, [3H]inositol 1-phosphate accumulation in the presence of Li+ corresponded to a breakdown rate for [3H]phosphatidylinositol of 2 to 3%/h.     

Phosphoinositide hydrolysis by guanosine 5''-[gamma-thio]triphosphate-activated phospholipase C of turkey erythrocyte membranes.

Phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] of turkey erythrocytes were labelled by using either [32P]Pi or [3H]inositol. Although there was little basal release of inositol phosphates from membranes purified from labelled cells, in the presence of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) the rate of accumulation of inositol bis-, tris- and tetrakis-phosphate (InsP2, InsP3 and InsP4) was increased 20-50-fold. The enhanced rate of accumulation of 3H-labelled inositol phosphates was linear for up to 20 min; owing to decreases in 32P specific radioactivity of phosphoinositides during incubation of membranes with unlabelled ATP, the accumulation of 32P-labelled inositol phosphates was linear for only 5 min. In the absence of ATP and a nucleotide-regenerating system, no InsP4 was formed, and the overall inositol phosphate response to GTP[S] was decreased. Analyses of phosphoinositides during incubation with ATP indicated that interconversions of PtdIns to PtdIns4P and PtdIns4P to PtdIns(4,5)P2 occurred to maintain PtdIns(4,5)P2 concentrations; GTP[S]-induced inositol phosphate formation was accompanied by a corresponding decrease in 32P- and 3H-labelled PtdIns, PtdIns4P and PtdIns(4,5)P2. In the absence of ATP, only GTP[S]-induced decreases in PtdIns(4,5)P2 occurred. Since inositol monophosphate was not formed under any condition, PtdIns is not a substrate for the phospholipase C. The production of InsP2 was decreased markedly, but not blocked, under conditions where Ins(1,4,5)P3 5-phosphomonoesterase activity in the preparation was inhibited. Thus the predominant substrate of the GTP[S]-activated phospholipase C of turkey erythrocyte membranes is PtdIns(4,5)P2. Ins(1,4,5)P3 was the major product of this reaction; only a small amount of Ins(1:2-cyclic, 4,5)P3 was released. The effects of ATP on inositol phosphate formation apparently involve the contributions of two phenomena. First, the P2-receptor agonist 2-methylthioadenosine triphosphate (2MeSATP) greatly increased inositol phosphate formation and decreased [3H]PtdIns4P and [3H]PtdIns(4,5)P2 in the presence of a low (0.1 microM) concentration of GTP[S]. ATP over the concentration range 0-100 microM produced effects in the presence of 0.1 microM-GTP[S] essentially identical with those observed with 2MeSATP, suggesting that the effects of low concentrations of ATP are also explained by a stimulation of P2-receptors. Higher concentrations of ATP also increase inositol phosphate formation, apparently by supporting the synthesis of substrate phospholipids.(ABSTRACT TRUNCATED AT 400 WORDS)     

Formation of inositol phosphate isomers in GH3 pituitary tumour cells stimulated with thyrotropin-releasing hormone. Acute effects of lithium ions.

With a h.p.l.c. system, the inositol mono-, bis- and tris-phosphate isomers found in [3H]inositol-labelled GH3 cells were resolved and identified. These cells possess at least ten distinct [3H]inositol-containing substances when acid-soluble extracts are analysed by anion-exchange h.p.l.c. These substances were identified by their co-elution with known inositol phosphate standards and, to a limited extent, by examining their chemical structure. Two major inositol monophosphate (InsP) isomers were identified, namely Ins1P and Ins4P, both of which accumulate after stimulation with the hypothalamic releasing factor (TRH) (thyrotropin-releasing hormone). Three inositol bisphosphate (InsP2) isomers were resolved, of which two were positively identified, i.e. Ins(1,4)P2 and Ins(3,4)P2. TRH treatment increases both of these isomers, with Ins(1,4)P2 being produced at a faster rate than Ins(3,4)P2. The third InsP2 isomer has yet to be fully identified, although it is co-eluted with an Ins(4,5)P2 standard. This third InsP2 is also increased after TRH stimulation. In common with other cell types, the GH3 cell contains two inositol trisphosphate (InsP3) isomers: Ins(1,4,5)P3, which accumulates rapidly, and Ins(1,3,4)P3, which is formed more slowly. The latter substance appears simultaneously with its precursor, inositol 1,3,4,5-tetrakisphosphate. We also examined the effects of acute Li+ treatment on the rates of accumulation of these isomers, and demonstrated that Li+ augments TRH-mediated accumulation of Ins1P, Ins4P, Ins(1,4)P2, the presumed Ins(4,5)P2 and Ins(1,3,4)P3. These results suggest that the effects of Li+ on inositol phosphate metabolism are more complex than was originally envisaged, and support work carried out by less sophisticated chromatographic analysis.     

Inositol phosphate formation in fMet-Leu-Phe-stimulated human neutrophils does not require an increase in the cytosolic free Ca2+ concentration.

The accumulation of inositol phosphates in myo-[3H]inositol-labelled human neutrophils stimulated with the chemotactic peptide fMet-Leu-Phe was measured. The challenge with the chemotactic peptide caused the generation of inositol monophosphate (InsP), inositol bisphosphate (InsP2) and inositol trisphosphate (InsP3). The formation of the three inositol phosphates followed a differential time course: InsP3 accumulated very rapidly and transiently, whereas InsP increased steadily for more than 2 min. Inositol phosphate formation was only partially decreased by procedures which prevented the fMet-Leu-Phe-dependent increase of cytosolic free Ca2+ concentration.     

Metabolism of inositol phosphates in the protozoan Paramecium. Characterization of a novel inositol-hexakisphosphate-dephosphorylating enzyme.

Basal and stimulated levels of inositol phosphates were determined in the protozoan Paramecium labelled with myo-[3H]inositol. Under resting conditions, intracellular InsP6 (phytic acid), InsP5 and InsP4 concentrations were 140, 10 and 2 microM, respectively. InsP5 was comprised of 56% Ins(1,2,3,4,5)P5 and/or Ins(1,2,3,5,6)P5, 40% Ins(1,2,4,5,6)P5 and/or Ins(2,3,4,5,6)P5 and small amounts of Ins(1,3,4,5,6)P5 and Ins(1,2,3,4,6)P5. InsP4 was mainly Ins(1, 4, 5, 6)P4 and/or Ins(3, 4, 5, 6)P4. Other inositol phosphates were not detected at a detection limit of 50-85 nM. Using various depolarizing and hyperpolarizing stimuli, no significant changes in level of inositol phosphates were observed in vivo, indicating that in the ciliate a contribution of inositol phosphates to signal-transduction mechanisms is unlikely. In homogenates prepared from myo-[3H]inositol-labelled cells, a marked relative increase in InsP3 and InsP4 over the concentrations in vivo was observed. These inositol phosphates were identified as degradation products of endogenous InsP6. A novel separation methodology for inositol phosphates was established to allow unequivocal assignment of phosphate locations of all dephosphorylated InsP6-derived products. The dephosphorylation was catalyzed by a phytase-like enzyme with a molecular mass of 240 kDa, most likely of a hexameric structure. The enzyme had a pH optimum of 7.0 and did not require divalent cations for activity. Substrate concentrations above 300 microM were inhibitory. Dephosphorylation of InsP6 by the Paramecium enzyme differs from that of phytases from plants in that it proceeds via a sequential release of phosphate groups from positions 6, 5, 4 and 3 of the myo-inositol ring or/and positions 4, 5, 6 and 1.     

Dephosphorylation of myo-inositol 1,4,5-trisphosphate and myo-inositol 1,3,4-triphosphate.

We have augmented our previous studies [Storey, Shears, Kirk & Michell (1984) Nature (London) 312, 374-376] on the subcellular location and properties of Ins(1,4,5)P3 (inositol 1,4,5-trisphosphate) phosphatases in rat liver and human erythrocytes. We also investigate Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate) metabolism by rat liver. Membrane-bound and cytosolic Ins(1,4,5)P3 phosphatases both attack the 5-phosphate. The membrane-bound enzyme is located on the inner face of the plasma membrane, and there is little or no activity associated with Golgi apparatus. Cytosolic Ins(1,4,5)P3 5-phosphatase (Mr 77,000) was separated by gel filtration from Ins(1,4)P2 (inositol 1,4-bisphosphate) and inositol 1-phosphate phosphatases (Mr 54,000). Ins(1,4,5)P3 5-phosphatase activity in hepatocytes was unaffected by treatment of the cells with insulin, vasopressin, glucagon or dibutyryl cyclic AMP. Ins(1,4,5)P3 5-phosphatase activity in cell homogenates was unaffected by changes in [Ca2+] from 0.1 to 2 microM. After centrifugation of a liver homogenate at 100,000 g, Ins(1,3,4)P3 phosphatase activity was largely confined to the supernatant. The sum of the activities in the supernatant and the pellet exceeded that in the original homogenate. When these fractions were recombined, Ins(1,3,4)P3 phosphatase activity was restored to that observed in unfractionated homogenate. Ins(1,3,4)P3 was produced from Ins(1,3,4,5)P4 (inositol 1,3,4,5-tetrakisphosphate) and was metabolized to a novel InsP2 that was the 3,4-isomer. Ins(1,3,4)P3 phosphatase activity was not changed by 50 mM-Li+ or 0.07 mM-Ins(1,4)P2 alone, but when added together these agents inhibited Ins(1,3,4)P3 metabolism. In Li+-treated and vasopressin-stimulated hepatocytes, Ins(1,4)P2 may reach concentrations sufficient to inhibit Ins(1,3,4)P3 metabolism, with little effect on Ins(1,4,5)P3 hydrolysis.     

Stimulus-Induced Accumulation of Inositol Tetrakis-, Pentakis-, and Hexakisphosphates in N1E-115 Neuroblastoma Cells

When [3H]inositol-prelabelled N1E-115 cells were stimulated with carbamylcholine (CCh) (100 microM), high K+ (60 mM), and prostaglandin E1 (PGE1) (10 microM), a transient increase in [3H]inositol pentakisphosphate (InsP5) accumulation was observed. The accumulation reached its maximum level at 15 s and had declined to the basal level at 2 min. CCh, high K+, and PGE1 also caused accumulations of [3H]inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], [3H]inositol 1,3,4,6-tetrakisphosphate [Ins(1,3,4,6)P4], and [3H]inositol hexakisphosphate (InsP6). Muscarine and CCh induced accumulations of [3H]Ins(1,4,5)P3, [3H]-Ins(1,3,4,6)P4, [3H]InsP5, and [3H]InsP6 with a similar potency and exerted these maximal effects at 100 microM, whereas nicotine failed to do so at 1 mM. With a slower time course, CCh, high K+, and PGE1 caused accumulations of [3H]-inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] and [3H]inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. In an N1E-115 cell homogenate, [3H]Ins(1,4,5)P3, [3H]Ins(1,3,4,5)P4, and [3H]Ins(1,3,4)P3 were converted to [3H]InsP5 through [3H]-Ins(1,3,4,6)P4. The above results indicate that Ins(1,3,4,6)P4, InsP5, and InsP6 are rapidly formed by several kinds of stimulants in N1E-115 cells.     

Kinetics of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate generation in dog-thyroid primary cultured cells stimulated by carbachol

The action of carbachol on the generation of inositol trisphosphate and tetrakisphosphate isomers was investigated in dog-thyroid primary cultured cells radiolabelled with [3H]inositol. The separation of the inositol phosphate isomers was performed by reverse-phase high pressure liquid chromatography. The structure of inositol phosphates co-eluting with inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] standards was determined by enzymatic degradation using a purified Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatase. The data indicate that Ins(1,3,4,5)P4 was the only [3H]inositol phosphate which co-eluted with a [32P]Ins(1,3,4,5)P4 standard, whereas 80% of the [3H]InsP3 co-eluting with an Ins(1,4,5)P3 standard was actually this isomer. In the presence of Li+, carbachol led to rapid increases in [3H]Ins(1,4,5)P4. The level of Ins(1,4,5)P3 reached a peak at 200% of the control after 5-10 s of stimulation and fell to a plateau that remained slightly elevated for 2 min. The level of Ins(1,3,4,5)P4 reached its maximum at 20s. The level of inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] increased continuously for 2 min after the addition of carbachol. Inositol-phosphate generation was also investigated under different pharmacological conditions. Li+ largely increased the level of Ins(1,3,4)P3 but had no effect on Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Forskolin, which stimulates dog-thyroid adenylate cyclase and cyclic-AMP accumulation, had no effect on the generation of inositol phosphates. The absence of extracellular Ca2+ largely decreased the level of Ins(1,3,4,5)P4 as expected considering the Ca2(+)-calmodulin sensitivity of the Ins(1,4,5)P3 3-kinase. Staurosporine, an inhibitor of protein kinase C, increased the levels of Ins(1,4,5)P3, Ins(1,3,4,5)P4 and Ins(1,3,4)P3. This supports a negative feedback control of diacyglycerol on Ins(1,4,5)P3 generation.     

Agonist-stimulated inositol phosphate metabolism in avian erythrocytes.

1. A screen for agonists capable of stimulating the formation of inositol phosphates in erythrocytes from 5-day-old chickens revealed the presence of a population of phosphoinositidase C-linked purinergic receptors. 2. If chicken erythrocytes prelabelled with [3H]Ins were exposed to a maximal effective dose of adenosine 5'-[beta-thio]diphosphate for 30 s, the agonist-stimulated increment in total [3H]inositol phosphates was confined to [3H]Ins(1,4,5)P3, Ins(1,3,4,5)P4 and InsP2. After 40 min stimulation, the radiolabelling of nearly all of the [3H]inositol phosphates that have been detected in these extracts [Stephens, Hawkins & Downes (1989) Biochem. J. 262, 727-737] had risen. However, some of these increases [especially those in Ins(3,4,5,6)P4 and Ins(1,3,4,5,6)P5] were accountable for almost entirely by increases in specific radioactivity rather than in mass. 3. The effect of purinergic stimulation on the rate of incorporation of [32P]Pi in the medium into the gamma-phosphate group of ATP and InsP4 and InsP5 was also measured. After 40 min stimulation, the incorporation of 32P into Ins(1,3,4,6)P4, Ins(1,3,4,5)P4, Ins(3,4,5,6)P4 and Ins(1,3,4,5,6)P5 was significantly elevated, whereas the mass of the last two and the specific radioactivity of the gamma-phosphate of ATP were unchanged compared with control erythrocyte suspensions. 4. In control suspensions of avian erythrocytes, the specific radioactivity of the individual phosphate moieties of Ins(1,3,4,6)P4 increased through the series 1, 6, 4 and 3 [Stephens & Downes (1990) Biochem. J. 265, 435-452]. This pattern of 32P incorporation is not the anticipated outcome of 6-hydroxy phosphorylation of Ins(1,3,4)P3 [the assumed route of synthesis of Ins(1,3,4,6)P4]. Although adenosine [beta-thio]diphosphate significantly stimulated the accumulation of [3H]Ins(1,3,4)P3, and despite the fact that avian erythrocyte lysates were shown to possess a chromatographically distinct, soluble, ATP-dependent, Ins(1,3,4)P3 6-hydroxykinase activity, purinergic stimulation of intact cells did not significantly alter the pattern of incorporation of [32P]Pi into the individual phosphate moieties of Ins(1,3,4,6)P4. These results suggest that the route of synthesis of this inositol phosphate species is not changed during the presence of an agonist.     

Gonadotropin releasing hormone stimulates the formation of inositol phosphates in rat anterior pituitary tissue.

The production of inositol phosphates in response to gonadotropin releasing hormone (GnRH) was studied in rat anterior pituitary tissue preincubated with [3H]inositol. Prelabelled paired hemipituitaries from prepubertal female rats were incubated in the presence or absence of GnRH in medium containing 10 mM-Li+ X Li+, which inhibits myo-inositol-1-phosphatase, greatly amplified the stimulation of inositol phosphate production by GnRH (10(-7) M) to 159, 198 and 313% of paired control values for inositol 1-phosphate, inositol bisphosphate and inositol trisphosphate respectively after 20 min. The percentage distribution of [3H]inositol within the phosphoinositides was 91.3, 6.3 and 2.4 for phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate respectively and was unaffected by GnRH. The stimulation of inositol trisphosphate production by GnRH was evident after 5 min incubation, was dose-dependent with a half-maximal effect around 11 nM, and was not inhibited by removal of extracellular Ca2+. Elevation of cytosolic Ca2+ by membrane depolarization with 50 mM-K+ had no significant effect on inositol phosphate production. These findings are consistent with the hypothesis that GnRH action in the anterior pituitary involves the hydrolysis of phosphatidylinositol 4,5-bisphosphate. The resulting elevation of inositol trisphosphate may in turn lead to intracellular Ca2+ mobilization and subsequent stimulation of gonadotropin secretion.     

Changes in Inositol 1,4,5-Trisphosphate and Inositol 1,3,4,5-Tetrakisphosphate Mass Accumulations in Cultured Adrenal Chromaffin Cells in Response to Bradykinin and Histamine

In previous studies it has been shown that both bradykinin and histamine increase the formation of 3H-labeled inositol phosphates in adrenal chromaffin cells prelabelled with [3H]inositol and that both these agonists stimulate release of catecholamines by a mechanism dependent on extracellular calcium. Here, we have used mass assays of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] to investigate changes in levels of these two candidates as second messengers in response to stimulation with bradykinin and histamine. Bradykinin increased the mass of Ins(1,3,4,5)P4 despite the failure in earlier studies with [3H]inositol-labelled cells to observe a bradykinin-mediated increase in content of [3H]InsP4. Bradykinin elicited a very rapid increase in level of Ins(1,4,5)P3, which was maximal at 5-10 s and then rapidly decreased to a small but sustained elevation at 2 min. The bradykinin-elicited Ins(1,3,4,5)P4 response increased to a maximum at 30-60 s and at 2 min was still elevated severalfold above basal levels. Histamine, which produced a larger overall total inositol phosphate response in [3H]inositol-loaded cells, produced significantly smaller Ins(1,4,5)P3 and Ins(1,3,4,5)P4 responses compared with bradykinin. The bradykinin stimulation of Ins(1,4,5)P3 accumulation was partially dependent on a high (1.8 mM) extracellular Ca2+ concentration, whereas the Ins(1,3,4,5)P4 response was almost completely lost when the extracellular Ca2+ concentration was reduced to 100 nM. Changes in the inositol polyphosphate second messengers are compared with the time course of bradykinin-stimulated increases in free intracellular Ca2+ concentrations and noradrenaline release.     

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.