Rapid formation of inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4-trisphosphate in rat parotid glands may both result indirectly from receptor-stimulated release of inositol 1,4,5-trisphosphate from phosphatidylinositol 4,5-bisphosphate.

Addition of 1 mM-carbachol to [3H]inositol-labelled rat parotid slices stimulated rapid formation of [3H]inositol 1,3,4,5-tetrakisphosphate, the accumulation of which reached a peak 20 s after stimulation, and then declined rapidly towards a new steady state. The initial rate of formation of inositol 1,3,4,5-tetrakisphosphate was slower than that for inositol 1,4,5-trisphosphate. The radioactivity in [3H]inositol 1,3,4,5-tetrakisphosphate fell quickly in carbachol-stimulated and then atropine-blocked parotid slices, suggesting that it is rapidly metabolized during stimulation. Parotid homogenates rapidly dephosphorylated inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate and, less rapidly, inositol 1,3,4-trisphosphate. Inositol 1,3,4,5-tetrakisphosphate was specifically hydrolysed to a compound with the chromatographic properties of inositol 1,3,4-trisphosphate. The only 3H-labelled phospholipids that we could detect in parotid slices labelled with [3H]inositol for 90 min were phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Parotid homogenates synthesized inositol tetrakisphosphate from inositol 1,4,5-trisphosphate. This activity was dependent on the presence of ATP. We suggest that, during carbachol stimulation of parotid slices, the key event in inositol lipid metabolism is the activation of phosphatidylinositol 4,5-bisphosphate-specific phospholipase C. The inositol 1,4,5-trisphosphate thus liberated is metabolized in two distinct ways; by direct hydrolysis of the 5-phosphate to form inositol 1,4-bisphosphate and by phosphorylation to form inositol 1,3,4,5-tetrakisphosphate and hence, by hydrolysis of this tetrakisphosphate, to form inositol 1,3,4-trisphosphate.     

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.     

Quantitative changes in inositol 1,4,5-trisphosphate in chemoattractant-stimulated neutrophils

myo-Inositol 1,4,5-trisphosphate is an intracellular second messenger generated from the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C. In the present study, we have used the abilities of inositol 1,4,5-trisphosphate to inhibit inositol 1,4,5-tris[32P]phosphate binding and to stimulate release of sequestered stores of 45Ca2+ to assay the mass of inositol 1,4,5-trisphosphate in extracts derived from [3H]inositol-prelabeled chemoattractant-stimulated neutrophils. These assays are specific for inositol 1,4,5-trisphosphate since the relative capacity of the extracts to compete with inositol 1,4,5-tris[32P]phosphate binding and to release 45Ca2+ correlated well with the [3H]inositol 1,4,5-trisphosphate content of the extract as determined by high pressure liquid chromatography. No correlation of these activities was observed with the content in the extract of either [3H]inositol 1,3,4-trisphosphate or [3H]inositol 1,3,4,5-tetrakisphosphate, whose formation exhibited kinetics distinct from [3H]inositol 1,4,5-trisphosphate. Thus, within 10 s of stimulation with 10 nM formyl-methionyl-leucyl-phenylalanine, the inositol 1,4,5-trisphosphate content of the extract increased from 0.05 to 0.55 pmol/10(6) cells, equivalent to a change in intracellular concentration from 100 nM to 1.1 microM. These studies demonstrate that neutrophils produce sufficient quantities of inositol 1,4,5-trisphosphate to mobilize Ca2+ from intracellular stores.     

Inositol 1,3,4,5-tetrakisphosphate and not phosphatidylinositol 3,4-bisphosphate is the probable precursor of inositol 1,3,4-trisphosphate in agonist-stimulated parotid gland.

When [3H]inositol-prelabelled rat parotid-gland slices were stimulated with carbachol, noradrenaline or Substance P, the major inositol trisphosphate produced with prolonged exposure to agonists was, in each case, inositol 1,3,4-trisphosphate. Much lower amounts of radioactivity were present in the inositol 1,4,5-trisphosphate fraction separated by anion-exchange h.p.l.c. Analysis of the inositol trisphosphate head group of phosphatidylinositol bisphosphate in [32P]Pi-labelled parotid glands showed the presence of phosphatidylinositol 4,5-bisphosphate, but no detectable phosphatidylinositol 3,4-bisphosphate. Carbachol-stimulated [3H]inositol-labelled parotid glands contained an inositol polyphosphate with the chromatographic properties and electrophoretic mobility of an inositol tetrakisphosphate, the probable structure of which was determined to be inositol 1,3,4,5-tetrakisphosphate. Since an enzyme in erythrocyte membranes is capable of degrading this tetrakisphosphate to inositol 1,3,4-trisphosphate, it is suggested to be the precursor of inositol 1,3,4-trisphosphate in parotid glands.     

Formation and metabolism of inositol 1,3,4,5-tetrakisphosphate in liver

The inositol lipid pools of isolated rat hepatocytes were labeled with [3H]myo-inositol, stimulated maximally with vasopressin and the relative contents of [3H]inositol phosphates were measured by high performance liquid chromatography. Inositol 1,4,5-trisphosphate accumulated rapidly (peak 20 s), while inositol 1,3,4-trisphosphate and a novel inositol phosphate (ascribed to inositol 1,3,4,5-tetrakisphosphate) accumulated at a slower rate over 2 min. Incubation of hepatocytes with 10 mM Li+ prior to vasopressin addition selectively augmented the levels of inositol monophosphate, inositol 1,4-bisphosphate, and inositol 1,3,4-trisphosphate. A kinase was partially purified from liver and brain cortex which catalyzed an ATP-dependent phosphorylation of [3H]inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate. Incubation of purified [3H]inositol 1,3,4,5-tetrakisphosphate with diluted liver homogenate produced initially inositol 1,3,4-trisphosphate and subsequently inositol 1,3-bisphosphate, the formation of which could be inhibited by Li+. The data demonstrate that the most probable pathway for the formation of inositol 1,3,4,5-tetrakisphosphate is by 3-phosphorylation of inositol 1,4,5-trisphosphate by a soluble mammalian kinase. Degradation of both compounds occurs first by a Li+-insensitive 5-phosphatase and subsequently by a Li+-sensitive 4-phosphatase. The prolonged accumulation of both inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in vasopressin-stimulated hepatocytes suggest that they have separate second messenger roles, perhaps both relating to Ca2+-signalling events.     

Metabolism of inositol 1,3,4-trisphosphate to a new tetrakisphosphate isomer in angiotensin-stimulated adrenal glomerulosa cells

Angiotensin stimulates rapid and prominent increases in inositol polyphosphates and their metabolites in bovine glomerulosa cells labeled with [3H]inositol. In addition to the early formation of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and inositol 1,3,4-trisphosphate (Ins-1,3,4-P3), as well as their intermediate product, inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4), delayed increases in two new InsP4 isomers were consistently observed by high resolution high performance liquid chromatography. Studies on the metabolism of purified Ins-1,3,4,5-P4 preparations, labeled with [3H]inositol and 32P to monitor sites of dephosphorylation, were performed in permeabilized glomerulosa cells. In addition to rapid degradation of Ins-1,3,4,5-P3 to Ins-1,3,4-P3 and then to Ins-3,4-P2, there was delayed formation of one of the putative InsP4 isomers observed during AII stimulation in intact cells. The kinetics of formation of the new InsP4 isomer, and the lack of phosphate in its 5 position based on isotope ratios, were consistent with its origin from Ins-1,3,4-P3. This was confirmed by the conversion of [3H]Ins-1,3,4-P3 to the new InsP4 isomer in permeabilized cells by a kinase distinct from that which phosphorylates Ins-1,4,5-P3. These results have demonstrated that the dephosphorylation sequence of Ins-1,4,5-P3 metabolism is accompanied by a complex cycle of higher phosphorylations with formation of new intermediates of potential significance in cellular regulation.     

L-myo-inositol 1,4,5,6-tetrakisphosphate (3-hydroxy)kinase.

Homogenates of primary-cultured murine bone macrophages contain an enzyme capable of synthesizing myo-[3H]inositol pentakisphosphate from myo-[3H]inositol tetrakisphosphate fractions derived from myo-[3H]inositol-labelled mouse macrophages and chick erythrocytes. D-myo-inositol 1,3,4,5-tetrakis[32P]-phosphate present in the same incubations was not phosphorylated. Since the myo-[3H]inositol-labelled tetrakisphosphate fractions used as substrates consist of a mixture of L-myo-inositol 1,4,5,6-tetrakisphosphate (60-85%) and a periodate-resistant tetrakisphosphate(s) whose characteristics are consistent with those of D-myo-inositol 1,3,4,5-tetrakisphosphate (the preceding paper [Stephens, Hawkins, Carter, Chahwala, Morris, Whetton & Downes (1988) Biochem. J. 249, 271-282] ), these data suggest the existence of a kinase that phosphorylates L-myo-inositol 1,4,5,6-tetrakisphosphate to give a myo-inositol pentakisphosphate. A similar activity was identified in homogenates of rat cerebrum, liver, heart and parotid gland. D-myo-Inositol 1,3,4,5-tetrakis[32P]phosphate in the same incubations was not a substrate. The activity was almost entirely soluble in all the tissues investigated and was found at its greatest specific activity in brain cytosol. The activity was purified 120-fold from a rat brain homogenate by (NH4)2SO4 fractionation and anion-exchange chromatography. The activity was clearly distinct from D-myo-inositol 1,4,5-trisphosphate (3-hydroxy)kinase. Incubation of this partially purified preparation with L-myo-[3H]inositol 1,4,5,6-tetrakisphosphate from chick erythrocytes and [gamma-32P]ATP resulted in the formation of L-myo-[3H]-inositol [1-32P]1,3,4,5,6-pentakisphosphate. The enzyme is therefore identified as an L-myo-inositol 1,4,5,6-tetrakisphosphate (3-hydroxy)kinase.     

Chemoattractant and guanosine 5''-[gamma-thio]triphosphate induce the accumulation of inositol 1,4,5-trisphosphate in Dictyostelium cells that are labelled with [3H]inositol by electroporation.

The analysis of the inositol cycle in Dictyostelium discoideum cells is complicated by the limited uptake of [3H]inositol (0.2% of the applied radioactivity in 6 h), and by the conversion of [3H]inositol into water-soluble inositol metabolites that are eluted near the position of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] on anion-exchange h.p.l.c. columns. The uptake was improved to 2.5% by electroporation of cells in the presence of [3H]inositol; electroporation was optimal at two 210 microseconds pulses of 7 kV. Cells remained viable and responsive to chemotactic signals after electroporation. The intracellular [3H]inositol was rapidly metabolized to phosphatidylinositol and more slowly to phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. More than 85% of the radioactivity in the water-soluble extract that was eluted on Dowex columns as Ins(1,4,5)P3 did not co-elute with authentic [32P]Ins(1,4,5)P3 on h.p.l.c. columns. Chromatography of the extract by ion-pair reversed-phase h.p.l.c. provided a good separation of the polar inositol polyphosphates. Cellular [3H]Ins(1,4,5)P3 was identified by (a) co-elution with authentic [32P]Ins(1,4,5)P3 and (b) degradation by a partially purified Ins(1,4,5)P3 5-phosphatase from rat brain. The chemoattractant cyclic AMP and the non-hydrolysable analogue guanosine 5'-[gamma-thio]triphosphate induced a transient accumulation of radioactivity in Ins(1,4,5)P3; we did not detect radioactivity in inositol 1,3,4-trisphosphate or inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. In vitro, Ins(1,4,5)P3 was metabolized to inositol 1,4- and 4,5-bisphosphate, but not to Ins(1,3,4,5)P4 or another tetrakisphosphate isomer. We conclude that Dictyostelium has a receptor- and G-protein-stimulated inositol cycle which is basically identical with that in mammalian cells, but the metabolism of Ins(1,4,5)P3 is probably different.     

Formation of inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate from inositol 1,3,4,5-tetrakisphosphate and their pathways of degradation in RBL-2H3 cells

Previous studies with antigen-stimulated rat basophilic leukemia (RBL-2H3) cells indicated the formation of multiple isomers of each of the various categories of inositol phosphates. The identities of the different isomers have been elucidated by selective labeling of [3H]inositol 1,3,4,5-tetrakisphosphate with [32P]phosphate in the 3'-or 4',5'-positions and by following the metabolism of different radiolabeled inositol phosphates in extracts of RBL-2H3 cells. We report here that inositol 1,3,4,5-tetrakisphosphate, when incubated with the membrane fraction of extracts of RBL-2H3 cells, was converted to inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate. Further dephosphorylation of the inositol polyphosphates proceeded rapidly in whole extracts of cells, although the process was significantly retarded when ATP (2 mM) levels were maintained by an ATP-regenerating system. The degradation of inositol 1,4,5-trisphosphate proceeded with the sequential formation of inositol 1,4-bisphosphate, the inositol 4-monophosphate (with smaller amounts of the 1-monophosphate), and finally inositol. Inositol 1,3,4-trisphosphate, on the other hand, was converted to inositol 1,3-bisphosphate and inositol 3,4-bisphosphate and subsequently to inositol 4-monophosphate and inositol 1-monophosphate (stereoisomeric forms were undetermined). The possible implications of the apparent interconversion between inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in regulating histamine secretion in the RBL-2H3 cells are discussed.     

Inhibition of protein kinase C by staurosporine promotes elevated accumulations of inositol trisphosphates and tetrakisphosphate in human platelets exposed to thrombin

We have examined regulation by protein kinase C (Ca2+/phospholipid-dependent enzyme) of thrombin-induced inositol polyphosphate accumulation in human platelets. When platelets are exposed to thrombin for 10 s, the protein kinase C inhibitor staurosporine causes inositol phosphate elevations over control values of 2.7-fold (inositol 1,4,5-trisphosphate (Ins(1,4,5)P3], 1.9-fold (inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4], and 1.2-fold (inositol 1,3,4-trisphosphate). In the same period, phosphatidic acid and diacylglycerol are unaffected. The myosin light chain kinase inhibitor ML-7 has no effect on inositol phosphate accumulations. Staurosporine does not inhibit Ins(1,4,5)P3 3-kinase and 5-phosphomonoesterase activities in saponin-permeabilized platelets incubated with exogenous Ins(1,4,5)P3 unless the platelets have been exposed to thrombin and protein kinase C is consequently activated. The protein kinase C agonist beta-phorbol 12,13-dibutyrate increases the Vmax of the 3-kinase 1.8-fold, with little effect on Km. Our results provide strong evidence for a role for protein kinase C in regulating inositol phosphate levels in thrombin-activated platelets. We propose that endogenously activated protein kinase C removes Ins(1,4,5)P3 by stimulating both 5-phosphomonoesterase and Ins(1,4,5)P3 3-kinase. Initial activation of phospholipase C does not appear to be affected by such protein kinase C. Inhibition of protein kinase C by staurosporine decreases 5-phosphomonoesterase activity. The resulting elevated Ins(1,4,5)P3, as substrate for Ins(1,4,5)P3 3-kinase, promotes production of Ins(1,3,4,5)P4, which also may accumulate through decreased 5-phosphomonoesterase activity and elevated Ca2+ levels. These factors apparently counteract the inhibitory effect on 3-kinase, yielding a net increase in Ins(1,3,4,5)P4.     

Degradation of inositol 1,3,4,5-tetrakisphosphates by porcine brain cytosol yields inositol 1,3,4-trisphosphate and inositol 1,4,5-trisphosphate

Inositol 1,3,4,5-tetrakisphosphates (Ins(1,3,4,5)P4), 32P-labelled in positions 4 and 5 were prepared enzymatically, using [4-32P]-phosphatidylinositol 4-phosphate (PtdInsP) and [5-32P]phosphatidylinositol 4,5-bisphosphate (PtdInsP2) as substrates, respectively. Degradation studies of Ins(1,3,4,5)P4, using an enriched phosphatase preparation from porcine brain cytosol, led to the formation of two inositol trisphosphate isomers which were identified as inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). This novel degradation pathway of Ins(1,3,4,5)P4 to Ins(1,4,5)P3 provides an additional source for the generation of Ins(1,4,5)P3, involving a 3-phosphatase.     

Agonist-Stimulated Inositol Polyphosphate Formation in Cerebellum

The accumulation of inositol polyphosphates in the cerebellum in response to agonists has not been demonstrated. Guinea pig cerebellar slices prelabeled with [3H]inositol showed the following increases in response to 1 mM serotonin: At 15 s, there was a peak in 3H label in the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], decreasing to a lower level in about 1 min. The level of 3H label in the putative second-messenger inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] increased rapidly up to 60 s and increased slowly thereafter. The accumulation of 3H label in various inositol phosphate isomers at 10 min, when steady state was obtained, showed the following increases due to serotonin: inositol 1,3,4-trisphosphate [Ins(1,3,4)P3], eight-fold; Ins(1,3,4,5)P4, 6.4-fold; Ins(1,4,5)P3, 75%; inositol 1,4-bisphosphate [Ins(1,4)P2], 0%; inositol 3,4-bisphosphate, 100%; inositol 1-phosphate/inositol 3-phosphate, 30%; and inositol 4-phosphate, 40%. [3H]Inositol 1,3-bisphosphate was not detected in controls, but it accounted for 7.2% of the total inositol bisphosphates formed in the serotonin-stimulated samples. The fact that serotonin did not increase the formation of Ins(1,4)P2 could be due to the fact that Ins(1,4)P2 is rapidly degraded or that Ins(1,4,5)P3 is metabolized primarily by Ins(1,4,5)P3-3'kinase to form Ins(1,3,4,5)P4. In the presence of pargyline (10 microM), [3H]Ins(1,3,4,5)P4 and [3H]Ins(1,3,4)P3 levels were increased, even at 1 microM serotonin. Ketanserin (7 microM) completely inhibited the serotonin effect, indicating stimulation of serotonin2 receptors. Quisqualic acid (100 microM) also increased the levels of [3H]Ins(1,4,5)P3, [3H]Ins(1,3,4,5)P4, and [3H]Ins(1,3,4)P3, but the profile of these increases was different.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Thrombin and phorbol ester stimulate inositol 1,3,4,5-tetrakisphosphate 3-phosphomonoesterase in human platelets

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), which mobilizes intracellular Ca2+, is metabolized either by dephosphorylation to inositol 1,4-bisphosphate(Ins-(1,4)P2) or by phosphorylation to inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4). It has been shown in vitro that Ins(1,3,4,5)P4 is also dephosphorylated by a 5-phosphomonoesterase to inositol 1,3,4-trisphosphate. However, we have found that exogenous Ins(1,3,4,5)P4 is dephosphorylated to predominantly Ins(1,4,5)P3 in saponin-permeabilized platelets in the presence of KCl (40-160 mM). This inositol polyphosphate 3-phosphomonoesterase activity is independent of Ca2+ (0.1-100 microM), and it was also observed when the ionic strength of the incubation medium was increased with Na+. The action of KCl appears to be due to activation of a 3-phosphomonoesterase as well as an inhibition of the 5-phosphomonoesterase, because the dephosphorylation of Ins(1,4,5)P3 to Ins(1,4)P2 was completely inhibited by KCl. The 3-phosphomonoesterase may be regulated by a protein kinase C, since both thrombin and phorbol dibutyrate increase 3-phosphomonoesterase activity and this is inhibited by staurosporine. The formation of Ins(1,4,5)P3 from Ins(1,3,4,5)P4 reported here provides an additional pathway for the formation of the Ca2+-mobilizing second messenger in stimulated cells.     

Interconversion of inositol (1,4,5)-trisphosphate to inositol (1,3,4,5)-tetrakisphosphate and (1,3,4)-trisphosphate in permeabilized adrenal glomerulosa cells is calcium-sensitive and ATP-dependent

The metabolism of [3H]inositol (1,4,5)-trisphosphate was followed in permeabilized bovine adrenal glomerulosa cells. At low Ca++ concentration (pCa = 7.2), more than 90% of [3H]inositol (1,4,5)-trisphosphate had disappeared within 2 min, while two other metabolites, [3H]inositol (1,3,4)-trisphosphate and [3H]inositol (1,3,4,5)-tetrakisphosphate appeared progressively. At higher Ca++ concentrations (pCa = 5.7 and 4.8), the formation of these two metabolites was markedly increased, but completely abolished if the medium was ATP-depleted. The peak levels for the generation of [3H]inositol (1,3,4,5)-tetrakisphosphate (1 min) preceded those of [3H]inositol (1,3,4)-trisphosphate and were closely correlated. These results suggest that, in adrenal glomerulosa cells, the isomer inositol (1,3,4)-trisphosphate is generated from inositol (1,4,5)-trisphosphate via a calcium-sensitive and ATP-dependent phosphorylation/dephosphorylation pathway involving the formation of inositol (1,3,4,5)-tetrakisphosphate.     

Detection of inositol 1,4,5-trisphosphate kinase in retina. A direct demonstration of phosphorylation of inositol 1,4,5-trisphosphate by ATP.

The metabolism of myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] consists of two pathways: dephosphorylation by 5-phosphomonoesterase(s) produces inositol 1,4-bisphosphate, and phosphorylation by Ins(1,4,5)P3 3-kinase yields inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. The requirements for Ins(1,4,5)P3 kinase activity in retina were characterized. Apparent Km values for ATP and Ins(1,4,5)P3 are 1.4 mM and 1.3 microM respectively. A direct demonstration of phosphorylation of Ins(1,4,5)P3 by [gamma-32P]ATP was achieved. Characterization of the 32P-labelled product revealed that it had the expected chromatographic and electrophoretic properties of Ins(1,3,4,5)P4.     

Formation of [3H]Inositol Metabolites in Rat Hippocampal Formation Slices Prelabelled with [3H]Inositol and Stimulated with Carbachol

Rat hippocampal formation slices were prelabelled with [3H]inositol and stimulated with carbachol for times between 7 s and 3 min. The [3H]inositol metabolites in an acid extract of the slices were resolved with anion-exchange HPLC. Carbachol dramatically increased the concentration of [3H]inositol monophosphate, [3H]inositol bisphosphate (two isomers), [3H]inositol 1,3,4-trisphosphate, [3H]inositol 1,4,5-trisphosphate, and [3H]inositol 1,3,4,5-tetrakisphosphate. The levels of [3H]inositol 1,4,5-trisphosphate rose most rapidly; they were maximally elevated after only 7 s and declined toward control levels in 1 min followed by a more sustained elevation in levels for up to 3 min. When [3H]inositol 1,4,5-trisphosphate was incubated with hippocampal formation homogenates in an ATP-containing buffer it was very rapidly metabolised. After 5 min [3H]inositol 1,4-bisphosphate, [3H]inositol 1,3,4-trisphosphate, and [3H]inositol 1,3,4,5-tetrakisphosphate could be detected in the homogenates. Under similar experimental conditions [3H]inositol 1,3,4,5-tetrakisphosphate is metabolised to [3H]inositol 1,3,4-trisphosphate and an inositol bisphosphate isomer that is not [3H]inositol 1,4-bisphosphate. We conclude that like other tissues the primary event in the hippocampus following carbachol stimulation is the activation of phosphatidylinositol 4,5-bisphosphate selective phospholipase C.     

Formation and metabolism of inositol 1,4,5-trisphosphate in human platelets.

1. myo-[3H]Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], when added to lysed platelets, was rapidly converted into [3H]inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], which was in turn converted into [3H]inositol 1,3,4-trisphosphate [Ins(1,3,4)P3]. This result demonstrates that platelets have the same metabolic pathways for interconversion of inositol polyphosphates that are found in other cells. 2. Labelling of platelets with [32P]Pi, followed by h.p.l.c., was used to measure thrombin-induced changes in the three inositol polyphosphates. Interfering compounds were removed by a combination of enzymic and non-enzymic techniques. 3. Ins(1,4,5)P3 was formed rapidly, and reached a maximum at about 4 s. It was also rapidly degraded, and was no longer detectable after 30-60 s. 4. Formation of Ins(1,3,4,5)P4 was almost as rapid as that of Ins(1,4,5)P3, and it remained detectable for a longer time. 5. Ins(1,3,4)P3 was formed after an initial lag, and this isomer reached its maximum, which was 10-fold higher than that of Ins(1,4,5)P3, at 30 s. 6. Comparison of the intracellular Ca2+ concentration as measured with fura-2 indicates that agents other than Ins(1,4,5)P3 are responsible for the sustained maintenance of a high concentration of intracellular Ca2+. It is proposed that either Ins(1,3,4)P3 or Ins(1,3,4,5)P4 may also be Ca2+-mobilizing agents.     

The role of haem in the regulation of rat liver tryptophan metabolism.

Anion-exchange h.p.l.c. analysis of [3H]inositol phosphates derived from glucose-stimulated isolated pancreatic islets that had been prelabelled with myo-[3H]inositol revealed that the predominant inositol trisphosphate was the 1,3,4-isomer [Ins(1,3,4)P3]. The 1,4,5-isomer [Ins(1,4,5)P3] was also detectable, as was a more polar inositol phosphate with the chromatographic properties of inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. Glucose-induced accumulation of Ins(1,3,4)P3 was augmented by Li+ and occurred after maximal accumulation of Ins(1,4,5)P3. These findings suggest a possible role for Ins(1,3,4)P3 or its probable precursor Ins(1,3,4,5)P4 in stimulus-secretion coupling in pancreatic islets.     

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.