The pathway of myo-inositol 1,3,4-trisphosphate dephosphorylation in liver.

We studied the dephosphorylation pathway for Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate) by liver homogenates and soluble and particulate subfractions incubated in media resembling physiological ionic strength and pH. Ins(1,3,4)P3 was dephosphorylated to two InsP2 (inositol bisphosphate) isomers, one of which is Ins(3,4)P2 [Shears, Parry, Tang, Irvine, Michell & Kirk (1987) Biochem. J. 246, 139-147]. The second InsP2 is the 1,3 isomer. Ins(3,4)P2 is dephosphorylated to inositol 3-phosphate by an enzyme activity located in both soluble and particulate fractions. The phosphatase(s) that attacks Ins(1,3)P2 was largely soluble, but we have not determined which phosphate(s) is removed. When the initial substrate concentration was 1 nM, the rate of dephosphorylation of Ins(1,4)P2 greater than Ins(1,3)P2 greater than Ins(3,4)P2. None of these bisphosphates was phosphorylated when incubated with liver homogenates and 5 mM-ATP, but their rates of dephosphorylation were then decreased.     

The dephosphorylation pathway of D-myo-inositol 1,3,4,5-tetrakisphosphate in rat brain.

Dephosphorylation of inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] was measured in both the soluble and the particulate fractions of rat brain homogenates. Analysis of the hydrolysis of [4,5-32P]Ins(1,3,4,5)P4 showed that for both fractions the 5-phosphate of Ins(1,3,4,5)P4 was removed and inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] was specifically produced. In the soluble fraction, Ins(1,3,4)P3 was further hydrolysed at the 1-phosphate position to inositol 3,4-bisphosphate[Ins(3,4)P2]. DEAE-cellulose chromatography of the soluble fraction separated the phosphatase activities into three peaks. The first hydrolysed both Ins(1,3,4,5)P4 and inositol 1,4,5-trisphosphate, the second inositol 1-phosphate and the third Ins(1,3,4)P3 and inositol 1,4-bisphosphate, [Ins(1,4)P2]. Further purification of the third peak on either Sephacryl S-200 or Blue Sepharose could not dissociate these two activities [i.e. with Ins(1,4)P2 and Ins(1,3,4)P3 as substrates]. The dephosphorylation of Ins(1,3,4)P3 could be inhibited by the addition of Li+.     

Accumulation of inositol polyphosphate isomers in agonist-stimulated cerebral-cortex slices. Comparison with metabolic profiles in cell-free preparations.

1. Basal and carbachol-stimulated accumulations of isomeric [3H]inositol mono-, bis-, tris- and tetrakis-phosphates were examined in rat cerebral-cortex slices labelled with myo-[2-3H]inositol. 2. In control samples the major [3H]inositol phosphates detected were co-eluted on h.p.l.c. with Ins(1)P, Ins(4)P (inositol 1- and 4-monophosphate respectively), Ins(1,4)P2 (inositol 1,4-bisphosphate), Ins(1,4,5)P3 (inositol 1,4,5-tris-phosphate) and Ins(1,3,4,5)P4 (inositol 1,3,4,5-tetrakisphosphate). 3. After stimulation to steady state with carbachol, accumulation of each of these products was markedly increased. 4. Agonist stimulation, however, also evoked much more dramatic increased accumulations of a second [3H]inositol trisphosphate, which was co-eluted on h.p.l.c. with authentic Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate) and of three further [3H]inositol bisphosphates ([3H]InsP2(s]. 5. Examination of the latter by chemical degradation by periodate oxidation and/or h.p.l.c. allowed identification of these as [3H]Ins(1,3)P2, [3H]Ins(3,4)P2 and [3H]Ins(4,5)P2 (inositol 1,3-, 3,4- and 4,5-bisphosphates respectively), which respectively accounted for about 22%, 8% and 3% of total [3H]InsP2 in extracts from stimulated tissue slices. 6. By using a h.p.l.c. method which clearly resolves Ins(1,3,4,5)P4 and Ins(1,3,4,6)P4 (inositol 1,3,4,6-tetrakisphosphate), only the former isomer could be detected in extracts from either control or stimulated tissue slices. Similarly, [3H]inositol pentakis- and hexakis-phosphates were not detectable either in the presence or absence of carbachol under the radiolabelling conditions described. 7. The catabolism of [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4)P3 by cell-free preparations from cerebral cortex was also studied. 8. In the presence of Mg2+, [3H]Ins(1,4,5)P3 was specifically dephosphorylated via [3H]Ins(1,4)P2 and [3H]Ins(4)P to free [3H]inositol, whereas [3H]Ins(1,3,4)P3 was degraded via [3H]Ins(3,4)P2 and, to a lesser extent, via [3H]Ins(1,3)P2 to D- and/or L-[3H]Ins(1)P and [3H]inositol. 9. In the presence of EDTA, hydrolysis of [3H]Ins(1,4,5)P3 was greater than or equal to 95% inhibited, whereas [3H]Ins(1,3,4)P3 was still degraded, but yielded only a single [3H]InsP2 identified as [3H]Ins(1,3)P2. 10. The significance of these observations with cell-free preparations is discussed in relation to the proportions of the separate isomeric [3H]inositol phosphates measured in stimulated tissue slices.     

Separation of multiple isomers of inositol phosphates formed in GH3 cells.

A high-performance-liquid-chromatography (h.p.l.c.) separation was developed, which resolves isomers of inositol monophosphate (IP), inositol bisphosphate (IP2), and inositol trisphosphate (IP3) in a single run. In GH3 cells labelled with [3H]inositol, treated with Li+ and thyrotropin-releasing hormone (TRH), radiolabelled components identified as inositol 1-phosphate (I1P), inositol 2-phosphate (I2P), inositol 4-phosphate (I4P), inositol 1,4-bisphosphate [I(1,4)P2], inositol 1,3,4-trisphosphate [I(1,3,4)P3] and inositol 1,4,5-trisphosphate [I(1,4,5)P3] are present, as are multiple unidentified IP2 peaks. After TRH stimulation, both I1P and I4P increase, the increase in I4P preceding that of I1P; I(1,4)P2 and an unknown IP2 increase; and both I(1,3,4)P3 and I(1,4,5)P3 increase, the increase in I(1,4,5)P3 being rapid and transient, whereas the increase in I(1,3,4)P3 is slower and more sustained. The most rapidly appearing inositol phosphates produced after TRH stimulation are I(1,4)P2 and I(1,4,5)P3.     

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.     

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.     

Purification and properties of inositol-1,4-bisphosphatase from bovine brain.

Inositol-1,4-bisphosphatase has been purified 13,000-fold from bovine brain supernatant. The enzyme is monomeric, with an apparent subunit Mr of 40,000. Maximal hydrolytic rates were observed in Tris buffer, pH 7.8, in the presence of 9 mM-Mg2+. The enzyme acted as a 1-phosphatase, hydrolysing both inositol 1,4-bisphosphate [Ins(1,4)P2] (Km 0.04 mM) and inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] (Km 0.5 mM) to inositol 4-phosphate and inositol 3,4-bisphosphate respectively. Li+ inhibited the hydrolysis of both substrates in an uncompetitive manner, with apparent Ki values of 9.63 mM and 0.46 mM for Ins(1,4)P2 and Ins(1,3,4)P3 respectively.     

Partial purification of inositol polyphosphate 1-phosphomonoesterase with characterization of its substrates and products by nuclear magnetic resonance spectroscopy

A study of the enzyme activities that degrade Ins(1,3,4)P3 in rat brain showed that it was dephosphorylated primarily by a Mg2+-dependent inositol polyphosphate 1-phosphomonoesterase to Ins(3,4)P2 and then to Ins(3)P by a 4-phosphomonoesterase. A less active enzyme activity with the properties of a 4-phosphomonoesterase that converted Ins(1,3,4)P3 to Ins(1,3)P2 was also detected. The inositol polyphosphate 1-phosphomonoesterase was separated from the 4-phosphomonoesterase and the inositol monophosphate phosphomonoesterase by chromatography on phosphocellulose, DE-52 anion exchange and hydroxylapatite columns. Kinetic characterization of the partially purified inositol polyphosphate 1-phosphomonoesterase indicated that both Ins(1,3,4)P3 and Ins(1,4)P2 were substrates with apparent Km values of 0.9 microM and 0.7 microM, respectively. Either substrate was a competitive inhibitor of the other substrate and dephosphorylation of both substrates was directly inhibited by Li+ in an uncompetitive manner. These data strongly suggest that a single enzyme dephosphorylates both Ins(1,3,4)P3 and Ins(1,4)P2. The 4-phosphomonoesterase that dephosphorylated Ins(3,4)P2 to Ins(3)P was insensitive to Mg2+ and Li+ and was probably the same enzyme that degraded Ins(1,3,4)P3 to Ins(1,3)P2. The isomeric configurations of the major inositol polyphosphates formed from the degradation of Ins(1,3,4,5)P4 were determined using 1H- and 31P-NMR spectroscopy, and confirmation of the structures assigned to Ins(1,3,4,5)P4, Ins(1,3,4)P3 and Ins(3,4)P2 was obtained.     

The pathway of myo-inositol 1,3,4-trisphosphate phosphorylation in liver. Identification of myo-inositol 1,3,4-trisphosphate 6-kinase, myo-inositol 1,3,4-trisphosphate 5-kinase, and myo-inositol 1,3,4,6-tetrakisphosphate 5-kinase

Inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) metabolism has been studied in liver homogenates and in 100,000 x g supernatant and particulate fractions. When liver homogenates were incubated in an "intracellular" medium containing 5 mM MgATP, equal proportions of Ins(1,3,4)P3 were dephosphorylated and phosphorylated. Two inositol tetrakisphosphate (InsP4) products and an inositol pentakisphosphate (InsP5) were detected. The InsP4 isomers were unequivocally identified as inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) and inositol 1,3,4,6-tetrakisphosphate (Ins(1,3,4,6)P4) by high performance liquid chromatography separation of inositol phosphates, periodate oxidation, alkaline hydrolysis, and stereo-specific polyol dehydrogenase. Ins(1,3,4)P3 5-kinase is a novel enzyme activity and accounted for 16% of the total Ins(1,3,4)P3 phosphorylation. Ins(1,3,4,6)P4 was also shown to be further phosphorylated to inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P5) by a kinase not previously known to occur in liver. About 75% of Ins(1,3,4)P3 kinase activities were soluble and were partly purified by anion-exchange fast protein liquid chromatography. The two Ins(1,3,4)P3 kinase activities eluted as a single peak that was well resolved from Ins(1,3,4)P3 phosphatase, Ins(1,3,4,6)P4 5-kinase, and Ins(1,3,4,5)P4 5-phosphatase activities. A further novel observation was that 10 microM Ins(1,3,4,5)P4 inhibited Ins(1,3,4)P3 kinase activities by 60%.     

Different patterns of agonist-stimulated increases of 3H-inositol phosphate isomers and cytosolic Ca2+ in bovine adrenal chromaffin cells: comparison of the effects of histamine and angiotensin II

Bovine adrenal chromaffin cells (BCC) were used to compare histamine- and angiotensin II-induced changes of inositol mono-, bis-, and trisphosphate (InsP1, InsP2, and InsP3, respectively) isomers, intracellular free Ca2+ ([Ca2+]i), and the pathways of inositol phosphate metabolism. Both agonists elevated [Ca2+]i by 200 nM 3-4 s after addition, but afterwards the histamine response was much more prolonged. Histamine and angiotensin II also produced similar four- to fivefold increases of Ins(1,4,5)P3 that peaked within 5 s. Over the first minute of stimulation, however, Ins(1,4,5)P3 formation was monophasic after angiotensin II, but biphasic after histamine, evidence supporting differential regulation of angiotensin II- and histamine-stimulated signal transduction. The metabolism of Ins(1,4,5)P3 by BCC homogenates was found to proceed via (a) sequential dephosphorylation to Ins(1,4)P2 and Ins(4)P, and (b) phosphorylation to inositol 1,3,4,5-tetrakisphosphate, followed by dephosphorylation to Ins(1,3,4)P3, Ins(1,3)P2, and Ins(3,4)P2, and finally to Ins(1 or 3)P. In whole cells, Ins(1 or 3)P only increased after histamine treatment. Additionally, Ins(1,3)P2 was the only other InsP2 besides Ins(1,4)P2 to accumulate within 1 min of agonist treatment [Ins(3,4)P2 did not increase]. These results support a correlation between the time course of Ins(1,4,5)P3 formation and the time course of [Ca2+]i transients and illustrate that Ca2(+)-mobilizing agonists can produce distinguishable patterns of inositol phosphate formation and [Ca2+]i changes in BCC. Different patterns of second-messenger formation are likely to be important in signal recognition and may encode agonist-specific information.     

Metabolism of inositol phosphates in alpha 1-adrenoceptor-stimulated and homogenized cardiac myocytes of adult rats.

Previous studies demonstrated the accumulation of inositol mono- and poly-phosphates in carbamoylcholine-stimulated cultured cardiac ventricular myocytes of adult rats [Berg, Guse & Gercken (1989) Biochim. Biophys. Acta 1010, 100-107]. Stimulation with noradrenaline (50 microM) in the presence of propranolol (10 microM) led to a time-dependent pattern of inositol mono- and poly-phosphates in cultured cardiac-ventricular myocytes. Ins(1,4,5)P3 and Ins(1,3,4,5)P4 increased in a rapid initial phase. The degradation products of Ins(1,4,5)P3, namely Ins(1,4)P2 and Ins(4)P, accumulated between 1 and 15 min. Ins(1,3,4,5)P4 was rapidly dephosphorylated to Ins(1,3,4)P3, which was then metabolized to Ins(1,3)P2 and Ins(3,4)P2. The last two InsP2 isomers and their degradation products, Ins(1)P and Ins(3)P (determined as an enantiomeric mixture), increased at extended stimulation periods. To confirm the pathway of Ins(1,3,4,5)P4 degradation, homogenates of isolated ventricular myocytes were incubated with [3H]INs(1,3,4,5)P4. The subsequent products were Ins(1,3,4)P3, Ins(3,4)P2, Ins(1,3)P2 and InsP. The effect of noradrenaline was antagonized by prazosin (0.1 microM), but not by yohimbine (0.1 microM), indicating phosphoinositidase activation via the alpha 1-adrenoceptor.     

The quantitative spectrum of inositol phosphate metabolites in avian erythrocytes, analysed by proton n.m.r. and h.p.l.c. with direct isomer detection.

The spectrum of inositol phosphate isomers present in avian erythrocytes was investigated in qualitative and quantitative terms. Inositol phosphates were isolated in micromolar quantities from turkey blood by anion-exchange chromatography on Q-Sepharose and subjected to proton n.m.r. and h.p.l.c. analysis. We employed a h.p.l.c. technique with a novel, recently described complexometric post-column detection system, called 'metal-dye detection' [Mayr (1988) Biochem. J. 254, 585-591], which enabled us to identify non-radioactively labelled inositol phosphate isomers and to determine their masses. The results indicate that avian erythrocytes contain the same inositol phosphate isomers as mammalian cells. Denoted by the 'lowest-locant rule' [NC-IUB Recommendations (1988) Biochem. J. 258, 1-2] irrespective of true enantiomerism, these are Ins(1,4)P2, Ins(1,6)P2, Ins(1,3,4)P3, Ins(1,4,5)P3, Ins(1,3,4,5)P4, Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, Ins(1,3,4,5,6)P5, and InsP6. Furthermore, we identified two inositol trisphosphate isomers hitherto not described for mammalian cells, namely Ins(1,5,6)P3 and Ins(2,4,5)P3. The possible position of these two isomers in inositol phosphate metabolism and implications resulting from absolute abundances of inositol phosphates are discussed.     

Inositol 1,4,5-trisphosphate and inositol 1,2-cyclic 4,5-trisphosphate are minor components of total mass of inositol trisphosphate in thrombin-stimulated platelets. Rapid formation of inositol 1,3,4-trisphosphate

Stimulation of human platelets by thrombin leads to rises of both inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) within 10 s. The mass of Ins(1,4,5)P3 was measured in platelet extracts after conversion to [3-32P]Ins(1,3,4,5)P4 with Ins(1,4,5)P3 3-kinase and [gamma-32P]ATP. Basal levels were equivalent to 0.2 microM and rose to 1 microM within 10 s of stimulation by thrombin. The mass of Ins(1,3,4)P3 was more than 10-fold greater than that of Ins(1,4,5)P3 between 10 and 60 s of thrombin stimulation. These results indicate that the majority of InsP3 liberated by phospholipase C in stimulated platelets must be the non-cyclic Ins(1,4,5)P3 in order to allow rapid phosphorylation by Ins(1,4,5)P3 3-kinase to Ins(1,3,4,5)P4 and then dephosphorylation to Ins(1,3,4)P3 by 5-phosphomonoesterase. A significant proportion of the InsP3 extracted from thrombin-stimulated platelets under neutral conditions is resistant to Ins(1,4,5)P3 3-kinase but susceptible after acid treatment, implying the presence of inositol 1,2-cyclic 4,5-trisphosphate (Ins(1,2cyc4,5)P3. The relative proportion of Ins(1,2cyc4,5)P3 increases with time. We suggest that such gradual accumulation is attributable to the relative insensitivity of this compound to hydrolytic and phosphorylating enzymes. Therefore, early Ca2+ mobilization in platelets is more likely to be effected by Ins(1,4,5)P3 than by Ins(1,2cyc4,5)P3.     

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.     

Hydrolysis of inositol phosphates by plant cell extracts.

A gel-filtered soluble fraction prepared from suspension-cultured Nicotiana tabacum cells hydrolysed inositol mono-, bis- and tris-phosphates. At a concentration of 7.5 microM the rates of hydrolysis followed the sequence Ins(1,4,5)P3 greater than Ins(1,4)P2 greater than Ins(4)P congruent to Ins(1)P. The major products of Ins(1,4,5)P3 hydrolysis identified by h.p.l.c. were Ins(1,4)P2 and Ins(4,5)P2. Ins(1,4)P2 was hydrolysed exclusively to Ins(4)P. The inclusion of Ca2+ in the incubation buffer markedly stimulated the hydrolysis of all the inositol phosphate substrates. Under identical conditions, Ca2+ inhibited the hydrolysis of inositol phosphates by soluble extracts prepared from rat brain. Half-maximal stimulation of Ins(1,4)P2 hydrolysis was obtained at free [Ca2+] of 0.6 and 1.2 microM when the Mg2+ concentration in the incubations was 0.3 and 1.0 mM respectively. This effect of Ca2+ was exerted solely by increasing the Vmax. of hydrolysis without affecting the Km for Ins(1,4)P2. Again, in contrast with brain, the hydrolysis of inositol bis- or mono-phosphates was insensitive to high concentrations of Li+. We conclude that plants contain specific Li+-insensitive inositol phosphate phosphatases that are regulated by low concentrations of Ca2+ in a manner which is different from that observed in mammalian tissues.     

Cross-linking membrane IgM induces production of inositol trisphosphate and inositol tetrakisphosphate in WEHI-231 B lymphoma cells

The addition of anti-IgM to the immature B lymphoma cell line WEHI-231 resulted in breakdown of phosphatidylinositol 4,5-bisphosphate, generating diacylglycerol and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). These reactions have recently been demonstrated in mature resting B cells stimulated with anti-IgM, as well. In addition to Ins(1,4,5)P3, inositol tetrakisphosphate (InsP4) and inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) were rapidly generated in WEHI-231 cells upon stimulation of the antigen receptor with anti-IgM. These two inositol polyphosphates are probably generated from Ins(1,4,5)P3 by phosphorylation to yield InsP4 and removal of the 5-phosphate from InsP4 to yield Ins(1,3,4)P3. It is possible that these inositol polyphosphates play a second messenger role in mediating the biologic effects of antigen-receptor signaling. It had previously been shown that anti-IgM also causes an increase in cytoplasmic free calcium. Therefore, the relationship between Ca2+ elevation and phosphoinositide breakdown was investigated. Although elevation of cytoplasmic Ca2+ with ionophores can trigger phosphoinositide breakdown, this required levels of Ca2+ well beyond those normally seen in response to anti-IgM. Thus, the Ca2+ elevation seen in response to anti-IgM cannot be the event controlling phosphoinositide breakdown. WEHI-231 cells have been shown to have a calcium storage compartment that releases Ca2+ in the presence of Ins(1,4,5)P3; therefore, it is likely that anti-IgM stimulates phosphoinositide breakdown as a primary event and this leads to the elevation of cytoplasmic Ca2+.     

Epidermal growth factor and angiotensin II stimulate formation of inositol 1,4,5- and inositol 1,3,4-trisphosphate in hepatocytes. Differential inhibition by pertussis toxin and phorbol 12-myristate 13-acetate

The ability of epidermal growth factor (EGF) and angiotensin II to stimulate production of inositol trisphosphate and mobilize intracellular Ca2+ in hepatocytes was compared using quin2 fluorescence to monitor changes in Ca2+ levels and high performance liquid chromatography to resolve the inositol trisphosphate (InsP3) isomers. Both EGF and angiotensin II stimulated an increase in free intracellular Ca2+ concentration ([Ca2+]i) as well as a rapid increase in the production of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Concentrations of angiotensin II which gave a rise in [Ca2+]i equivalent to that seen with maximal doses of EGF produced an equivalent increase in Ins(1,4,5)P3 formation. Both EGF and angiotensin II stimulated the formation of the Ins(1,3,4)P3 and inositol 1,3,4,5-tetrakisphosphate isomers. The formation of the Ins(1,3,4)P3 isomer lagged behind production of Ins(1,4,5)P3 but eventually reached higher levels in the cell. The initial rise in [Ca2+]i and InsP3 levels stimulated by EGF and angiotensin II was not affected by reducing the external Ca2+ concentration below 30 nM with an excess of [ethylenebis(oxyethylenenitrilo)] tetraacetic acid. Treatment of hepatocytes for 30-180 s with 1 micrograms/ml phorbol 12-myristate 13-acetate prior to the addition of EGF blocked the EGF-stimulated production of Ins(1,4,5)P3 and the increase in [Ca2+]i. Phorbol 12-myristate 13-acetate attenuated the production of Ins(1,4,5)P3 generated by angiotensin II over the concentration range of 10(-10) to 10(-8) M; however, the Ca2+ signal was only inhibited at the 10(-10) M dose of angiotensin II. Treatment of rats with pertussis toxin for 72 h prior to isolating hepatocytes blocked the ability of EGF to increase Ins(1,4,5)P3 and Ins(1,3,4)P3 but did not inhibit the ability of any concentration of angiotensin II to stimulate formation of InsP3 or inositol tetrakisphosphate. The observation that pertussis toxin selectively abolishes EGF-stimulated inositol lipid breakdown suggests that EGF and angiotensin II use different mechanisms to activate phospholipase C in hepatocytes.     

The metabolism of tris- and tetraphosphates of inositol by 5-phosphomonoesterase and 3-kinase enzymes

Phospholipase C cleaves phosphatidylinositol 4,5-bisphosphate to form both inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,2-cyclic 4,5-trisphosphate (cInsP3). The further metabolism of these inositol trisphosphates is determined by two enzymes: a 3-kinase and a 5-phosphomonoesterase. The first enzyme converts Ins(1,4,5)P3 to inositol 1,3,4,5-tetrakisphosphate (InsP4), while the latter forms inositol 1,4-bisphosphate and inositol 1,2-cyclic 4-bisphosphate from Ins(1,4,5)P3 and cInsP3, respectively. The current studies show that the 3-kinase is unable to phosphorylate cInsP3. Also, the 5-phosphomonoesterase hydrolyzes InsP4 with an apparent Km of 0.5-1.0 microM to form inositol 1,3,4-trisphosphate at a maximal velocity approximately 1/30 that for Ins(1,4,5)P3. The apparent affinity of the enzyme for the three substrates is InsP4 greater than Ins(1,4,5)P3 greater than cInsP3; however, the rate at which the phosphatase hydrolyzes these substrates is Ins(1,4,5)P3 greater than cInsP3 greater than InsP4. The 5-phosphomonoesterase and 3-kinase enzymes may control the levels of inositol trisphosphates in stimulated cells. The 3-kinase has a low apparent Km for Ins(1,4,5)P3 as does the 5-phosphomonoesterase for InsP4, implying that the formation and breakdown of InsP4 may proceed when both it and its precursor are present at low levels. Ins(1,4,5)P3 is utilized by both the 3-kinase and 5-phosphomonoesterase, while cInsP3 is utilized relatively poorly only by the 5-phosphomonoesterase. These findings imply that inositol cyclic trisphosphate may be metabolized slowly after its formation in stimulated cells.     

The metabolism of inositol 1,3,4-trisphosphate to inositol 1,3-bisphosphate

We previously demonstrated a pathway for the metabolism of inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) to inositol 3,4-bisphosphate (Ins(3,4)P2) in calf brain extracts. Inositol polyphosphate 1-phosphatase, a Mg2+-dependent, lithium ion-inhibited enzyme, specifically hydrolyzes Ins(1,3,4)P3 to Ins(3,4)P2 and Ins(1,4)P2 to Ins 4-P (Inhorn, R. C., Bansal, V. S., and Majerus, P. W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2170-2174). Now we have found an alternative pathway for the metabolism of Ins(1,3,4)P3 in crude calf brain extracts. Along this pathway, Ins(1,3,4)P3 is first converted to Ins(1,3)P2 which is further hydrolyzed to Ins 1-P. This pathway involves a 4-phosphatase and a 3-phosphatase which do not require Mg2+ and are not inhibited by lithium ions. A similar 4-phosphatase also degrades Ins(3,4)P2 to Ins 3-P. Three different inositol bisphosphates formed from calf brain supernatant are each further metabolized by a separate enzyme. The three inositol monophosphates, i.e. Ins 1-P, Ins 3-P, and Ins 4-P, are converted to inositol by inositol monophosphate phosphatase (Ackermann, K. E., Gish, B. G., Honchar, M. P., and Sherman, W. R. (1987) Biochem. J. 242, 517-524).     

The pathway for the production of inositol hexakisphosphate in human cells

The yeast and Drosophila pathways leading to the production of inositol hexakisphosphate (InsP(6)) have been elucidated recently. The in vivo pathway in humans has been assumed to be similar. Here we show that overexpression of Ins(1,3,4)P(3) 5/6-kinase in human cell lines results in an increase of inositol tetrakisphosphate (InsP(4)) isomers, inositol pentakisphosphate (InsP(5)) and InsP(6), whereas its depletion by RNA interference decreases the amounts of these inositol phosphates. Expression of Ins(1,3,4,6)P(4) 5-kinase does not increase the amount of InsP(5) and InsP(6), although its depletion does block InsP(5) and InsP(6) production, showing that it is necessary for production of InsP(5) and InsP(6). Expression of Ins(1,3,4,5,6)P(5) 2-kinase increases the amount of InsP(6) by depleting the InsP(5) in the cell, and depletion of 2-kinase decreases the amount of InsP(6) and causes an increase in InsP(5). These results are consistent with a pathway that produces InsP(6) through the sequential action of Ins(1,3,4)P(3) 5/6-kinase, Ins(1,3,4,6)P(4) 5-kinase, and Ins(1,3,4,5,6)P5 2-kinase to convert Ins(1,3,4)P(3) to InsP(6). Furthermore, the evidence implicates 5/6-kinase as the rate-limiting enzyme in this pathway.