Download The metabolism and functions of inositol pentakisphosphate and

627th MEETING, NOTTINGHAM
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We are grateful to the M.R.C., the Leukaemia Research Fund and
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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
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