Methods for the analysis of inositol phosphates

Interest in the inositol phospholipids was stimulated by the simultaneous discoveries that the products of hydrolysis of these lipids could serve as messengers to activate to synergistic signaling pathways in hormonally responsive cells, namely, inositol 1,4,5-trisphosphate which causes the release of Ca2+ from intracellular stores and diacylglycerol which promotes the activation of protein kinase C. At the same time, Berridge and co-workers introduced relatively simple approaches to study the inositol phospholipid cycle. These included the use of [3H]inositol to label the inositol metabolites, all of which are confined to this cycle, and of Li+ to decrease the rate of degradation of the inositol phosphates. Water-soluble inositol phosphates and chloroform-soluble inositol phospholipids could then be separated by solvent partition and the inositol phosphates further separated by use of an anion-exchange resin. However, the subsequent application of high-performance liquid chromatography as a separation technique indicated the existence of many isomers of the inositol phosphates formed by different pathways of dephosphorylation and phosphorylation. Mapping of these metabolic pathways may be substantially complete, but novel pathways may still be discovered. We review both old and new methods of analysis of the inositol phosphates for the measurement of mass and radioactivity. Although the complexity of the cycle sometimes demands the use of sophisticated methods of separation and rigorous identification, older and inexpensive methods may still be useful for some purposes.     

Rapid dephosphorylation of protein kinase C substrates by protein kinase A activators results from inhibition of diacylglycerol release

The biochemical events encompassing the dephosphorylation of protein kinase C substrates by protein kinase A activators have been investigated in a neurotumor cell line, NCB-20. Treatment of [32P]orthophosphate-labeled cells with protein kinase A activators (e.g. forskolin, dibutyryl cAMP, prostaglandin E1) resulted in an inhibition of protein kinase C activity due to a failure of the protein kinase C complex to translocate into the membrane. Phospholipase C activity, as measured by the synchronous release of diacylglycerol and inositol phosphates (inositol 1,4,5-trisphosphate, inositol 1,4-bisphosphate, and inositol 1-phosphate) in response to bradykinin, was inhibited up to 50% following exposure to protein kinase A activators. At the same time, phospholipase C-specific inositol phospholipid substrates (phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate) were found to accumulate in NCB-20 cells following treatment with protein kinase A activators. This suggests that phospholipase C may be altered through protein kinase A-mediated protein phosphorylation. Second messenger generation (inositol phosphates, diacylglycerol, and Ca2+) is therefore inhibited through cyclic AMP-mediated shutdown of the inositol lipid cycle at the level of phospholipase C.     

Inositol-Limited Growth, Repair, and Translocation in an Inositol-Requiring Mutant of Neurospora crassa

The biochemical consequences of inositol limitation in an inositol auxotroph of Neurospora crassa have been examined as a means of disclosing the cellular role of inositol. The cellular levels of inositol in the inl mutant were proportional to the concentration of inositol in the growth medium whereas inositol phosphate levels remained relatively constant at about 0.1 mumol/g (dry weight). After 72 h of growth, about 57-fold more protein per milligram (dry weight) was released by the mutant grown on limiting inositol than by the inositol-supplemented control. When the inositol-limited growth medium was osmotically buffered with 1% NaCl, 3% NaCl, or 6% sorbitol, there was about 33, 74, or 54%, respectively, less protein released by the mutant. These results are consistent with cell lysis occurring in the mutant grown on limiting inositol because of a structurally weakened cell wall and membrane deterioration. When sufficient inositol for normal mycelial growth was supplied to an inositol-deficient mycelium, there was within 2 h a rapid incorporation of inositol to 85% of control levels. This incorporation occurred without significant growth by any area of the mycelium. About 10 to 15% of the total cell inositol was translocated forward from the older mycelial areas to the growing tips; only 2 to 5% of the total cell inositol was translocated backward toward the older mycelial areas. Possible mechanisms of translocation are discussed.     

Effect of protein kinase A on inositide metabolism and rap 1 G-protein in human erythroleukemia cells.

Human erythroleukemia (HEL) cells phosphorylate [3H]inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate; they also contain all the enzymes to sequentially dephosphorylate [3H]inositol 1,4,5-trisphosphate and [3H]inositol 1,3,4,5-tetrakisphosphate to inositol. alpha-Thrombin, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, and sodium fluoride caused the formation of [3H]inositol phosphates in HEL cells that were previously labeled with [3H]inositol. This indicates agonist-induced activation of phospholipase C and hydrolysis of the inositol phospholipids. Pretreatment of the HEL cells with iloprost, a prostacyclin analog that increases cellular cyclic AMP levels, dramatically reduced the formation of inositol phosphates and the increase of [3H]phosphatidylinositol 4,5-bisphosphate. The inhibitory effects of iloprost were associated with the phosphorylation of a 24-kDa protein, which was detected with an antiserum obtained against the rap 1 protein. The catalytic subunit of protein kinase A inhibited formation of polyphosphoinositides during phosphorylation of the rap 1 protein in membranes. This rap 1 protein might have functional relevance in the inhibition of agonist-induced inositide metabolism.     

The identification of a novel inositol lipid, phosphatidylinositol trisphosphate (PIP3), in rat cerebrum using in vivo techniques

Rats received intraventricular injections of 20 uCi of [3H]-myo-inositol, and were sacrificed 24 hrs later by high-power head-focused microwave fixation. Two inositol lipid extraction methods were compared: The Hauser and Eichberg method yielded higher recovery of inositol lipids, but a lower inositol phosphate content. The Schacht method yielded reduced radiolabel in the lipid fractions, but increased water soluble phosphates. Both methods extracted a novel inositol lipid (PIP3) which contained inositol tetrakisphosphate (IP4) as its polar head group. This was determined by alkaline hydrolysis and analyzed by high performance liquid chromatography with authentic IP4 standard. Furthermore, preliminary studies of the fatty acid composition indicated a similarity with other inositol lipids. The radiolabel ratio of PIP2:PIP3 was 5:1. In summary, we have isolated a novel inositol phospholipid in rat brain, PIP3, the parent compound for inositol tetrakisphosphate (IP4).     

Protein kinase C phosphorylates human platelet inositol trisphosphate 5'-phosphomonoesterase, increasing the phosphatase activity

Phosphoinositide breakdown in response to thrombin stimulation of human platelets results in the formation of the calcium-mobilizing messenger molecules inositol 1,4,5-trisphosphate and inositol 1,2-cyclic-4,5-trisphosphate and of diglyceride, which activates protein kinase C. We find that protein kinase C phosphorylates and thereby increases the activity of inositol 1,4,5-trisphosphate 5'-phosphomonoesterase, a phosphatase that hydrolyzes these molecules to inert compounds. The 5'-phosphomonoesterase phosphorylated using [gamma-32P]ATP comigrates on SDS-polyacrylamide gels with a protein (40 kd) phosphorylated rapidly in response to thrombin stimulation of 32PO4-labeled platelets. Peptide maps of proteolytic digests of these two phosphorylated proteins indicate that they are the same. We propose that platelet Ca2+ mobilization is regulated by protein kinase C phosphorylation of the inositol 1,4,5-trisphosphate 5'-phosphomonoesterase. These results explain the observation that phorbol ester treatment of intact human platelets results in decreased levels of inositol trisphosphate and decreased Ca2+ mobilization upon subsequent thrombin addition.     

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.     

Basic fibroblast growth factor does not initiate mitogenic signalling via phosphoinositide hydrolysis in PC12 cells

Basic fibroblast growth factor (b FGF) was found to be equally potent mitogen as compared to alpha-thrombin to reinitiate DNA synthesis in quiescent PC12 cells. Whereas thrombin was found to be an activator of phospholipase C as judged by a rapid increase in the formation of inositol triphosphate, inositol biphosphate and a massive accumulation of inositol phosphate when 20 mM LiCl was present as an inhibitor of inositol mono phosphatases, basic FGF failed to induce the breakdown of the polyphosphoinositides in quiescent PC12 cells to any appreciable levels, however, a simultaneous increase in the level of diacylglycerol was observed. b FGF also failed to stimulate protein kinase C which is believed to be activated by diacylglycerol. It is therefore concluded that bFGF receptor mediated 'signalling is not via phospholipase C activation and bFGF's early mitogenic responses and DNA synthesis are initiated independent of the inositol lipids and protein kinase C activation. Thus bFGF must have its own unique signal transducing mechanism independent of inositol pathways.     

Regulation of casein kinase-2 (CK2) activity by inositol phosphates

Casein kinase 2 (CK2) was one of the first protein kinases to be discovered and has been suggested to be responsible for as much as one-fifth of the eukaryotic phosphoproteome. Despite being responsible for the phosphorylation of a vast array of proteins central to numerous dynamic cellular processes, the activity of CK2 appears to be unregulated. In the current study, we identified a protein kinase activity in rat liver supernatant that is up-regulated by inositol 1,3,4,5-tetrakisphosphate (IP4) and inositol hexakisphosphate (IP6). The substrate for the inositol phosphate-regulated protein kinase was identified as a phosphatidylcholine transfer protein-like protein. Using the phosphorylation of this substrate in an assay, we purified the inositol phosphate-regulated protein kinase and determined it to be CK2. Bacterially expressed recombinant CK2, however, showed very high basal activity and was only modestly activated by IP6 and not regulated by IP. We found that an endogenous component present in rat liver supernatant was able to inhibit both recombinant and liver-purified CK2 basal activity. Under these conditions, recombinant CK2 catalytic activity could be increased substantially by IP4, inositol 1,3,4,5,6-pentakisphosphate (IP5), and IP6. We concluded that, contrary to the previously held view, CK2 can exist in a state of low constitutive activity allowing for its regulation by inositol phosphates. The ability of the higher inositol phosphates to directly stimulate CK2 catalytic activity provides the first evidence that these signaling molecules can operate via a direct control of protein phosphorylation.     

Involvement of a specific guanine nucleotide binding protein in receptor immunoglobulin stimulated inositol phospholipid hydrolysis

The role of a specific guanine nucleotide binding (G protein) protein in coupling murine B lymphocyte receptor immunoglobulin to inositol phospholipid hydrolysis was investigated. Using an in vitro system with isolated membranes, we have observed specific enhancement of GTP binding subsequent to ligand-induced receptor crosslinking. Induced increases were inhibited by pretreatment with pertussis toxin which catalyzed ADP-ribosylation of a 43 kDa substrate. Involvement of this G protein with receptor immunoglobulin-induced inositol phospholipid hydrolysis was evidenced by the ability of pertussis toxin to block this response. This report, then, indicates that the B lymphocyte antigen receptor belongs to a family of receptors which are linked to inositol phospholipid hydrolysis through a G protein.     

Role for protein kinase C in the modulation of glomerular PGE2 production by angiotensin II

Angiotensin II increased PGE2 release from superfused glomeruli, and stimulated labeled inositol phosphate production. 12-O-Tetradecanoyl phorbol -13-acetate (TPA, 10(-7) M), which stimulates protein kinase C activity in soluble fractions of glomerular homogenates, suppressed angiotensin II actions on inositol phosphate production and PGE2. By contrast, 4a phorbol 12,13 di-decanoate and phorbol had no effect on protein kinase C activity or angiotensin II induced increases in inositol phosphate or PGE2. 1-(5-Isoquinolinyl)-2-methylpiperazine (H-7), which inhibits protein kinase C activity in soluble fractions of glomerular homogenates, prevented TPA induced suppression of angiotensin II actions on inositol phosphate production and PGE2. Moreover H-7 prolonged the time course of angiotensin II induced inositol phosphate production and enhanced angiotensin II actions on glomerular PGE2 production. The results support a role for inositol phospholipid hydrolysis through the phospholipase C pathway in the mediation of angiotensin II actions on PGE2 in glomeruli and are consistent with negative modulation of these actions by protein kinase C.     

Pertussis toxin blocks activin A-induced production of inositol phosphates in rat hepatocytes.

The present study was conducted to examine an involvement of G protein in the action of activin A in rat parenchymal liver cells. Activin A induced a dose-dependent increase in inositol phosphates in cells prelabelled with [3H]inositol. The effect of activin A was completely blocked by pretreatment of the cells with pertussis toxin. In contrast, pertussis toxin had little effect on angiotensin II-induced production of inositol phosphates. Both activin A and angiotensin II inhibited glucagon-mediated production of cAMP. Pretreatment of the cells with pertussis toxin blocked the inhibition induced by both activin A and angiotensin II. In permeabilized cells, activin A augmented production of inositol phosphates. Activin-mediated production of inositol trisphosphate was enhanced by GTP-gamma S and was attenuated by GDP-beta S. These results suggest that a pertussis toxin-sensitive G protein(s) may be involved in the action of activin A in hepatocytes.     

Differentiated human NT2-N neurons possess a high intracellular content of myo-inositol

myo-Inositol plays a key role in signal transduction and osmotic regulation events in the CNS. Despite the known high concentrations of inositol in the human CNS, relatively little is known about its distribution within the different cell types. In this report, inositol homeostasis was studied in NT2-N cells, a unique cell culture model of human CNS neurons. Differentiation of precursor NT2 teratocarcinoma cells into NT2-N neurons by means of retinoic acid treatment resulted in an increase in inositol concentration from 24 to 195 nmol/mg of protein. After measurement of intracellular water spaces, inositol concentrations of 1.6 and 17.4 mM were calculated for NT2 and NT2-N cells, respectively. The high concentrations of inositol in NT2-N neurons could be explained by (1) an increased uptake of inositol (3.7 vs. 1.6 nmol/mg of protein/h, for NT2-N and NT2 cells, respectively) and (2) a decreased efflux of inositol (1.7%/h for NT2-N neurons vs. 9.0%/h for NT2 cells). Activity of inositol synthase, which mediates de novo synthesis of inositol, was not detected in either cell type. The observation that CNS neurons maintain a high intracellular concentration of inositol may be relevant to the regulation of both phosphoinositide signaling and osmotic stress events in the CNS.     

Altered signal transduction secondary to surface IgM cross-linking on B-chronic lymphocytic leukemia cells. Differential activation of the phosphatidylinositol-specific phospholipase C

To further study the mechanisms by which surface Ig triggering activates the inositol phospholipid signaling pathway, we have used B cells from chronic lymphocytic leukemia patients which, as previously described, display two patterns of response upon sIg cross-linking: in one group this cross-linking induces an inositol phosphate release, an intracellular free Ca2+ concentration elevation and a subsequent cell proliferation; in a second group none of these events occur although there is an increased class II Ag expression following anti-mu stimulation as in the first group. We have been able to demonstrate that the phosphatidyl inositol specific phospholipase C (PI-PLC) can be activated in permeabilized B cells from the first group by direct stimulation, with GPT gamma S, of a guanine nucleotide binding (G) protein. In addition, since anti-mu + GTP gamma S stimulate an increased inositol phosphate production in these cells, this suggests that surface Ig cross-linking activates PI-PLC via a G protein. However, in cells from the second group no inositol phosphate is released after GTP gamma S stimulation although PI-PLC can be directly activated by high Ca2+ concentrations. This reflects in these cells, an interruption of the signaling cascade sIg/G protein/PI-PLC at the level of the G protein or at the G protein/PI-PLC coupling. In cells from both groups PMA treatment, which is known to alter phosphatidyl inositol metabolism in B cells, completely inhibits PI-PLC activation even by high Ca2+ concentrations. These studies show that the phosphatidyl inositol-dependent signaling cascade after surface Ig triggering can be altered at different levels in B cells.     

High-performance liquid chromatographic analysis of radiolabeled inositol phosphates

Separation of inositol phosphates by low-pressure anion-exchange chromatography yields unsatisfactory results, while previously described anion-exchange HPLC methods require such extensive processing times that they preclude efficient sample analysis. Using a low-capacity Vydac nucleotide anion-exchange column, we have developed a method which allows complete separation of myo-inositol, inositol 1-phosphate, inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate in approximately 10 min followed by a 5-min column regeneration time. This method provided exceptional reproducibility and quantitative recovery of each inositol phosphate. One column was used for over 300 separations with no loss in performance or alteration in elution pattern. A modified procedure with a 14-min gradient was developed to separate the 1,3,4- and 1,4,5-isomers of inositol trisphosphate. These separation procedures were used to characterize the kinetics of degradation of inositol phosphates by lysates of erythrocytes and neutrophils. We conclude that these procedures are applicable for rapid and quantitative analysis of radiolabeled inositol phosphates in cellular extracts.     

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

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

Post-translational regulation of phosphatidylglycerolphosphate synthase in response to inositol

Phosphatidylglycerolphosphate synthase (Pgs1p) catalyses the committed step in the synthesis of cardiolipin (CL). This is the only step of CL synthesis that is regulated by inositol. We have shown previously that Pgs1p enzyme activity is decreased within minutes after supplementation with inositol, but PGS1 expression is unaltered. We utilized an epitope-tagged Pgs1p to determine if the rapid decrease in activity following inositol was because of degradation or inactivation of the protein. In this report, we show that, in response to inositol, the decrease in CL content and Pgs1p enzyme activity are associated with increased phosphorylation of Pgs1p, but not with degradation or mislocalization of the protein. This is the first evidence of phosphorylation of a phospholipid biosynthetic enzyme in response to inositol and identifies a new mechanism of inositol-mediated regulation.     

Purification and characterization of inositol-1,3,4-trisphosphate 5/6-kinase from rat liver using an inositol hexakisphosphate affinity column.

The metabolism of inositol 1,3,4-trisphosphate is a pivotal branch point of inositol phosphate turnover; its dephosphorylation replenishes cellular inositol pools, its phosphorylation at the 6-position supports the synthesis of inositol pentakisphosphate, and its phosphorylation at the 5-position produces inositol 1,3,4,5-tetrakisphosphate (Shears, S.B. (1989) J. Biol. Chem. 264, 19879-19886). In order to increase understanding of the control of inositol-1,3,4-trisphosphate kinase activity, the enzyme was highly purified from rat liver by precipitation with polyethylene glycol, MonoQ ion-exchange chromatography, heparin-agarose affinity chromatography, and a novel affinity chromatography procedure that utilized Affi-Gel resin to which InsP6 was coupled (Marecek, J.F., and Prestwich, G.D. (1991) Tetrahedron Lett. 32, 1863-1866). The final purification was about 26,000-fold, with a 6% yield. This final preparation performed both 5- and 6-kinase activities in the ratio of approximately 1:5. The affinity of the enzyme for inositol 1,3,4-trisphosphate was 0.04 microM, the highest yet determined for an inositol phosphate kinase. Both inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4,6-tetrakisphosphate were competitive inhibitors of the kinase (Ki values of 2-4 microM). The enzyme was determined to have a molecular mass of 36 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinase activity was unaffected by Ca2+/calmodulin, protein kinase A, or protein kinase C.