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627th MEETING, NOTTINGHAM commitment to differentiation or are reflections of the new differentiated states. We are grateful to the M.R.C., the Leukaemia Research Fund and the Royal Society for financial support. Barker, C. J., Morns, A. J., Kirk, C. J. & Michell, R. H. (1988) Bioctiem. Soc. Trctns. 16, 984-985 Berridge, M. J. ( 1 987) Annic. Rev. Biochem. 56, 159- 193 Berridge, M. J. & Irvine, R. F. ( 1 984) Nature (London) 312, 3 15-32 1 Berridge, M. J. & Michell, R. H. (cds.) ( I 988) Philos. Truns. R. Soc. London. Ser. B. 320, 237-246 Downes, R. H. & Michell, R. H. ( 1985) in Molecitlur Mechanisms of Transmembrclne Signalling (Cohen, P. & Houslay, M. D., eds.), pp. 3-56, Elsevier, Amsterdam French, P. J., Bunce, C. M., Brown, G., Creba, J. A. & Michell, R. H. ( 1988)Biochem. Soc. Truns. 16,985-986 Hawthorne, J. N. ( 1 960) J. Lipid Rex. I , 255-280 Hokin, M. R. & Hokin, L. E. (1953) J. Biol. Cliem. 203,967-977 Homans, S. W., Ferguson, M. A. J., Dwek, R. A,, Rademacher, T. W., Anand, R. & Williams, A. F. (1988) Nurure (London) 333, 369-272 Irvine, R. F., Moor, R. M., Pollock, W. K. & Smith, P. M. (1988) I’hilos. Trrrns. R. Soc. London Ser. B.320, 28 1-298 King, C. E., Stephens, L. R., Hawkins, P. T., Guy, G . R. & Michell, R. H. ( 1987) Biochem. J. 244,209-2 I7 Kypta, R. M., Ulug, E. T., Goldberg, Y . & Courtneidge ( 1988) Cold Spring Harbor Symp. @itant. Bid. 53, in the press 3 Low, M. G. & Saltiel, A. R. ( 1 988) Science 239,268-275 Maccallum, S. H., Hunt, P. A., Michell, R. H. & Kirk, C. J. (1989) Riochem. Soc. Trans. 11,88-89 Michell, R. H. ( 1975) Biochim. Biophys. Actu 4 IS, 8 I - 147 Michell, R. H., Kirk, C. J., Jones, L. M., Downes, C. P. & Creba, J. A. (1 981) Philos. Trans. R. Soc. London Ser. B. 296, 123- 137 Michell. R. H., Kirk, C. J., Maccallum, S. H. & Hunt, P. A. (19880) Philos. Trans. R. Soc. London, Ser. B. 320, 239-246 Michell, R. H., King, C. E., Piper, C. J., Stephens, L. R., Bunce, C. M., Guy, G. R. & Brown, G. ( 1 9 8 8 b ) J. Gen. Physiol. in the press Morrison, D., Kaplan, D., Piwnica-Worms. H., Keller, T., Mamon, H., Cohen, B., Rapp, U., Schafthausen, B., Cantley, L. & Roberts, T. ( 1 988) Cold Spring Harbor Symp. @urn/. Biol. 53, in the press Muller. E., Hegewald, H., Jaroszewicz. K., Cumme, G. A,, Hoppe, H. & Frunder, H. (1986) Biochem. J. 235, 775-783 Nishizuka, Y . (1984) Nature (London) 308,693-693 Nishizuka, Y .(1986) Science 233,305-3 12 Stephens, L. R.. Hawkins, P. T., Carter, N., Chahwala, S., Morris, A . J., Whetton, A. D. & Downes, C. P. (1988) Biochem. J. 249, 271-282 Whitman, M.. Downes, C. P., Keeler, M., Kellcr, T. & Cantley. L. ( 1988) Nature (London) 332,644-646 Wong, N. S, Barker, C. J., Shears, S. B., Kirk, C. J . & Michell, R. H. ( 1988) Biochem. J. 252, 1-5 Received 1 August 1988 The metabolism and functions of inositol pentakisphosphate and inositol hexakisphosphate DAVID CAWENTER,* MICHAEL R. HANLEY,? PHILLIP T. HAWKINS,? TREVOR R. JACKSON,? LEONARD R. STEPHENS* and MARIO VALLEJOT 1-M.R.C. Molecular Neurobiology Unit, University of Cambridge Medical School, Hills Road, Cambridge C B 2 ZQH, U.K. and *Smith Kline and French Research Ltd, The Frythe, Welwyn, Herts. A L 6 9 A R , U.K. Over the last few years, the investigation of inositol lipid metabolism has elucidated a fundamental mechanism by which cells respond to a variety of external stimuli (Berridge & Irvine, 1984; Downes & Michell, 1985). Activation of appropriate cell-surface receptors stimulates the hydrolysis of a membrane lipid, phosphatidylinositol 4,5-bisphosphate, to form two informational products, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P,]. DAG and Ins( 1,4,5)P, are now generally recognized as authentic ‘second-messengers’ in intracellular communication, controlling the activity of protein kinase C and the elevation of cytosolic C a 2 + , respectively (Nishizuka, 1984; Streb et al., 1983). These findings have provided the impetus for more detailed investigations of inositol metabolism in cells, and these have recently revealed that cells contain a hitherto unsuspected and quite startling diversity of inositol phosphates (see for example; Batty et al., 1985; Heslop et ul., 1985, Hawkins et ul., 1986; Hansen er al., 1986; Balla et al., 1987; Shears et al., 1987; lnhorn er al., 1987; Stephens et al., 1 9 8 8 ~Dean ; & Moyer, 1988).Some of these compounds are produced via the receptor-stimulated formation and subsequent metabolism of the second-messcnger Ins( 1,4,5)P3,but it is becoming increasingly clear that others are normal constituents of quiescent cells with no clear connection with Abbreviations used: DAG, diacylglycerol; Ins/’, Ins/’?, Ins/’,, Ins/’,,, Ins/’,, Ins/’(,, inositol mono-, bis-, tris-, tetrakis-. pentakisand hexakis-phosphate with locants designated where appropriate; DMEM, Dulbecco‘s modified Eagle’s medium; FCS, fetal calf serum. VOl. 17 hormone-stimulated events. Two compounds which appear to fit into this latter category are inositol pentakisphosphate (InsP,) and inositol hexakisphosphate (Ins/’(,). Preliminary evidence first suggested that the levels of InsP, and Insl’, went up (Morgan et al., 1987),down (Tilly et al., 1987)or even oscillated (Heslop et al., 1985) in response to cellular stimulation by agonists coupled to inositol lipid hydrolysis. However, we have measured the levels of [‘HIlnsP, and/or [‘HIlnsP, in a variety of cell.typ+ labelled with [‘H]inositol and stimulated with the appropriate agonists, and can find no evidence for rapid changes in the levels of these two compounds. The systems we have studied are: bradykinin-stimulated NG 1 15-40 1L neuroblastomaglioma hybrid cells, carbachol-stimulated 1321N 1 astrocytoma cells, ADP-stimulated turkey erythrocytes and vasopressin-stimulated L.6 myoblasts (see Fig. 1 and data not shown). If InsP, and InsP6 are not directly involved in the inositollipid signal-transduction pathway, what do they do? These compounds have only recently kp observed in mammalian cells, but they have been known‘ as major constituents of plant seeds (as phytic acid) and avian erythrocytes .for a number of years (Posternak, 1903; Jackson et ul., 1982; Dyer, 1940; Johnson & Tate, 1969; Bartlett, 1980). Are there any clues in this older literature as to their possible functions in mammalian cells? Unfortunately, the answer appears to be no. It seems that Ins/’5 has a rather specialided function as a modulator of haemoglobin oxygen affinity in avian erythrocytes (Bartlett, 1980) and Ins/:, is generally assigned the role of a phosphorus and/or energy store in seeds, though convincing evidence for this is still lacking. Preliminary evidence suggests that Ins/-’, and Ins/’, can accumulate in mammalian tissues to levels of the order 10-100 p~ (Szwergold et al., 1987 and our own unpublished work). This poses a problem, in that one might expect these concentrations to be insoluble in the presence of an intracellular concentration of free MgZf of the order of 1 mM BIOCHEMICAL SOCIETY TRANSACTIONS 4 v1 c 01 30 5 cd 4 -.-.- Ins P 0 z g 20000~ 3: Ins P2 L 15 000 . 20. 0 I 10 000 . 5000 I / 0 10 20 30 40 T i m e (min) Fig. 2. Extracellular hydrolysis of inositol phosphates by NIH 3T3 cells 0 01 5 30 Time (min) Fig. 1. Vasopressin-stimulated formation of itiositol phosphutes in L6 cells L6 cells were grown to 75% confluence in tissue-culturetreated 6-well plates in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal calf serum (FCS) at 37°C and 8% (v/v) CO, in air. For the final 72 h in culture, cells were grown in DMEM with 5 pM-myo-inositol, 2 pCi of [3H]myo-inositol/ml (New England Nuclear, 14 Ci/ mmol) and 10% (v/v) dialysed FCS. Labelled cells were washed with a balanced salts solution (130 mM-NaC1; 5 mMKCI, 0.5 m~-MgC1,,0.% mM-MgSO,, 10 mM-glucose, 1.0 mMCaCI,, 20.0 mM-sodium Hepes pH 7.4) and then maintained at 37°C in this buffer supplemented with 10 mM-LiC1 for 15 rnin, before addition of agonist (Arg-vasopressin, AVP) or vehicle (Con). Incubations were terminated by addition of an equal volume of 10% (v/v) perchloric acid and neutralized extracts then loaded on to 1.8 cm Ag-1 x 8 (200-400) formate-form columns (Hawkins et d., 1986). The columns were eluted sequentially, as follows: inositol, 10 ml of H,O; Ins/: 10 ml of 0.2 M-ammonium formate/O.l M-formic acid Ins/’,, 10 ml of 0.4 M-ammonium formate/0.l M-formic acid; InsP,, 10 ml of 0.8 M-ammonium formate/O.l M-formic acid; InsP4, 10 ml of 1.0 M-ammonium formate/O.l M-formic acid; Ins!’, + (,, 6 ml of 2.0 M-ammonium formate/0.1 M-formic acid. Each fraction was solubilized by addition of 10 ml of Quickzint 401 (Zinsser Analytic) and its radioactivity determined by scintillation counting. (Cosgrove, 1980). Perhaps some form of compartmentation or complexing of Ins/’, and Ins/’, occurs inside the cell. In this context, the accumulation of high levels of Ins/>, in lower organisms leads to the formation of dense precipitates or refractile bodies (Lapan, 1975) and the high level of Ins/’, in avian erythrocytes is thought to be maintained by binding to NIH 3T3 cells were grown to confluence in 35 mm dishes (approx. 3 x 10, cells) in DMEM containing 10% (v/v) FCS at 37°C and 8% (v/v) CO, in air. Cells were removed from the incubator, given one wash, and the medium replaced with 1.0 ml of 130 mM-NaCI, 5.0 mM-KCI, 20.0 mM-sodium Hepes, 0.5 mM-MgCI,, 0.2 mM-MgSO,, 10.0 mM-glucose, 1.0 mM-CaCI,, pH 7.3. The cells were then incubated for 15 min at 37°C before additions of the various substrates (25 p l , containing approx. 5000 d.p.m.). Incubations were continued at 37°C and after various times terminated by addition of 100 pl of 50% (v/v) perchloric acid. Neutralized extracts were prepared and loaded on to 1.8 cm Ag-1 X 8, (200-400) formate-form columns (Hawkins et al., 1986). The columns were sequentially eluted as described for Fig. 1. The data shown are the means fSD ( t i = 3) for the radioactivity remaining in the original compound: ( 0 )[14C]lns3P,0.5 p ~ , Amersharn; ( 0 )Ins( 1,4,S)P,, 0.5 nM, New England Nuclear; ( A ) [‘H]lns(1,3,4,S)P4and ( 0 ) [,H]lns( 1,3,4,5,6)/’,, both at approx. 0.6 nM, synthesized via phosphorylation of [3H]Ins(1,4,5)P, using a 25-40% (w/v) ammonium sulphate fraction from turkey erythrocytes. haemoglobin. However, Insf’, appears to be ‘n.m.r.-visible’ and hence soluble in slime moulds (Martin et al., 1987) and in lettuce seeds and potato tubers (Delfini et al., 1985; Kimc et al., 1982) even though present in very high concentrations. The metabolic pathways to and from Ins/’, and Ins/’, are still obscure. Two inositol tetrakisphosphates [Ins(3,4,5,6)P4 and Ins( 1,3,4,6)P4]have been identified as potential biosynthetic precursors of Ins( 1,3,4,5,6)P, in mammalian and avian cells (Stephens, 1988a,b,c). The origin of Ins(3,4,5,6)P4is unknown, but the absence of a phosphate in position - 1 of the inositol ring suggests that it is not immediately derived from a known inositol phospholipid. One possibility is that it is made via direct phosphorylation of inositol, as has been postulated for InsP, synthesis in plants (e.g. English et ul., 1966).Ins( 1,3,4,6)P4can be formed 1989 S 627th MEETING, NOTTINGHAM via phosphorylation of Ins( 1,3,4)/13(Shears ef al., 1987) and hence, in principle, the synthesis of InsP, may be indirectly linked to the hormone-stimulated formation of Ins( 1,4,S)P, via the production and subsequent degradation of Ins( 1,3,4,S)f, (Batty et al., 198.5; lrvine et al., 1986). To the best of our knowledge, the synthesis of Insf, has never been convincingly demonstrated in vitro. It is clear that we are still far from unravelling the complexities of inositol phosphate metabolism in cells, but progress in this arena is likely to be crucial to any understanding of the functions of Ins/’, and Ins/-’,. Nature has clearly used inositol phosphates as a means of signalling information in at least one case, namely Ins( 1,4,S)P,. Vallejo et al. ( 1987) considered the intriguing possibility that InsP, and Ins/’, are also used as signals, but in the extracellular domain. They showed that injection of InsP, and InsP,, but not Ins/-’,, into the nucleus tractus solitarius (a discrete brain stem nucleus implicated in cardiovascular regulation) results in dose-dependent, reversible changes in heart rate and blood pressure. Furthermore, they showed that [3H]lnsl’, and [‘HIInsP, are synthesized in intact brain after labelling with [3H]inositol it? vivo, and that these labelled compounds accumulate in a region-specific manner. These results suggest that while InsP, and InsP6 may have more general ‘house-keeping’ roles in cells, at least in the brain, they may also function as extracellular messengers. Such a situation would be directly analogous to that previously described for glycine, glutamate and ATP. While the data thus far presented by no means prove that InsP, and InsP, act extracellularly, this hypothesis has the great virtue of making clear predictions about the properties of these molecules, e.g. they must be released from cells under appropriate circumstances, they must reversibly activate their target cells via a physiologically meaningful mechanism and there must be an effective means of removing them from the extracellular environment. In this latter respect, we have recently identified a phosphatase(s) which is present on the outside of a variety of cell types and is capable of hydrolysing Insf,. The relative rates of hydrolysis of InsP,, hf4, Ins( 1,4,5)P3and inositol 3-phosphate (Ins3P) applied to the outside of intact, washed NIH 3T3 cells are shown in Fig. 2. We have not yet tested the ability of these cells to hydrolyse Insf,. On permeabilization of these cells with digitonin (SO pg/ml), the rate of Ins( 1,4,5)/-’, hydrolysis increased to match that of Ins!’,; the rate of Ins/-’, hydrolysis, however, remained unchanged, suggesting that the majority of this activity resides on the extracellular face of the cell (data not shown). The much higher rate of hydrolysis of Ins/-’, and Insf, relative to Ins/-’ suggests that the activity o n NIH 3T3 cells is unlike previously characterized. nonspecific cell-surface phosphatases (e.g. alkaline phosphatases), since these latter enzymes show a marked preference for the hydrolysis of isolated phosphate groups rather than those present in vicinal pairs (see Grado & Ballou, 1961). Clearly, this activity must be further characterized in terms of substrate specificity, substrate affinity, products, cellular distribution and sensitivity to inhibitors, before its role can be properly evaluated. Enzymes capable of hydrolysing Ins/’, and Ins/’(,, termed phytases (Posternak & Posternak, 1929) have been widely studied in plants where they are responsible for the mobilization of InsP, in the seed (see, e.g. Tomlinson & Ballou, 1962; Lim & Tate, 1973).They have also been described in intestinal epithelial cells (Rao & Ramakrishnan, 1985),where they may function in the digestion of dietary inositol phosphates Vol. 17 beforc inositol readsorption. The data presented hcre, however, raise the possibility that ‘phytases’ may be morc widespread constituents of the cell surface of mammalian cells. Balla, T., Guillcmette, G., Baukal. A. J. & Catt, K. J. ( I 987) Biochem. Biophys. Hes. Commun. 148, 199-205 Bartlett, G. R. ( 1 980) Am. Zoo/. 20, 103- I 14 Batty, I. K., Nahorski, S. K. 62 Irvine, R. F. ( I 985) Niochem. J. 232, 21 1-215 Berridge, M. J. & Irvine, R. F. ( I 984) Ncitiire (Loridori) 312, 3 15-32 I Cosgrovc, D. J. 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