Formation of inositol 1,3,4,6-tetrakisphosphate during angiotensin II action in bovine adrenal glomerulosa cells

Vol. 148, No. 1, 1987 October 14, 1987

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 199-205

FORWWION OF INC6I!l0Ll,3,4,6-TEI'RAKISPHOSPHAI'EDURING AC!IIONINBCVINEADRJW&GLC&lERDLL)ljACELLS Gaetan

Tamas Balla,

Guillemette,

Albert

Ehdocrinolcgy and Reproduction National Institute of Child Health National Institutes of Health,

J.

Baukal,

ANGICYIENS~ II

and Kevin

J.

Qtt

Research Branch, and Human Development, Bethesda, MD 20892

Received August 10, 1967 Angiotensin II stimulates the formation of several inositol polyphosphates in cultured bovine adrenal glomerulosa cells prelabelled with [3H]inositol. Analysis by high performance anion exchange chromatography of the inositol-phosphate compounds revealed the existence of two additional inositol tetrakisphosphate (InsP4) isomers in proximity to Ins-1,3,4,5-P4, the known phosphorylation product of Ins-1,4,Ertrisphosphate and precursor of Ins-1,3,4-trisphosphate. Both of these new compounds showed a slow increase after stimulation with angiotensin II. The structure of one of these new InsP4 isomers, which is a phosphorylation product of Ins-1,3,4-P3, was deduced by its resistance to periodate oxidation to be Ins-1,3,4,6-P4. Tne existence of multiple cycles of phosphorylation-dephosphorylation reactions for the processing of Ins-1,4,5-P4 may represent a new aspect of the inositol-lipid related signalling mechanism in agonist-activated target cells. SW2

o

1987

Academic

Press,

Inc.

Angiotensin zone through of angiotensin (4,5)

to

yield

messenger role

by a mechanism

me

Q2+-n-obilizing

its

steroidogenic

receptors

II to such receptors Ins-1,4,5-P3 in

various

involving

Elimination

occurs mainly another

exerts

(6)

(8,9).

of

abbreviations

trisphosphate; diacylglycerol; sat:

both

Ihe

signal

Binding

breakdown of PtdIns-4,5-P2 of

active

which

serve

a second

water-soluble

species,

pool

to an intracellular

Ins-1,4,5-P3

dephosphorylating

pathway by which Ins-1,4,5-P3

glomerulosa

Ca2+ frcm a non-mitochondrial

of the Ins-1,4,5-P3

by an active

on the adrenal

in rapid

and DPG (7),

tissues

binding

effect

in the plasma membrane (l-3).

results

has been shown to release

Ins-1,4,5-P3,

(11-13) .

II

receptor

seems to be extremely mechanism

is converted

(14).

rapid

and

Tne discovery

of

to Ins-1,3,4-P3

used are: Ins: inositol; Ins-1,4,5-~3: inositol PtdIns-4,5-P2: p hosphatidylinositol 4,5-bisphosphate; B3TA: ethylene-bis(oxyethylenenitrilo)-tetraacetic

(10)

through

Ins-

1,4,5DPG: acid. 0006-291X/87 $1.50

199

Copyright 0 I987 by Academic Press, Inc. All rights of reproduction in any form reserved.

BIOCHEMICAL

Vol. 148, No. 1, 1987

1,3,4,5-P4

(15,16)

mobilizing

InsP3

regulatory

components of cellular

We have glomerulosa

raised isomer

recently cells

P4 isolner

(18).

structural

analysis

the Ins-1,3,4,6-P4

the to

possibility

InsP4

described

is

of this

that

relevant,

RESEARCH COMMUNICATIONS

this

conversion

in that

Ca2+ homeostasis a new metabolic

by which Ins-1,3,4-P3 In the present

AND BIOPHYSICAL

new InsP4 isomer,

provides

the

pathway

in

adrenal

to a novel

additional

which indicates

D-E,-

data on the that

it

is

isomer of IP4.

Material3: Myo-~3H]inositol, [3Hlinositol-1,4,5-P3, [3H]inositol-l,3,~~ P4 and [ H]mnositol-1,4-P2 were obtained from New England Nuclear (b collagenase, CNA-se (type I) were from Sigma Chemical (lo.; and [Ile il angiotensin II fran Peninsula Labs. Anberlite MS 3A was from Aldrich Chemical (31. OJture media were prepared by the NM Media Unit. Preparation and incubation of cells: Bovine adrenal glomerulosa cells --were prepared and cultured as described previously (18). Briefly, the outer 0.5 nm slices of bovine adrenal glands containing the glomerulosa tissue were minced and treated with collagenase followed by mechanical dispersion. ells were plated in a density of 5 x 105/ml and kept in culture for 3 days. Q7 the second day of the culture the madium was changed to one containing [3H]inositol (20 pCi/ml) but no carrier inosit01 (see 18 for more details). After labelling for 24 hrs, cells were washed and preincubated at 37OC for 20 min before the addition of angiotensin II (50 nM). The reaction was terminated and inositol phosphates extracted as described previously (18). Samples were then applied to an HPLC column (Absorbosphere SAX, 5 fl, 250 mn, Alltech Applied Sci., Deerfield, IL) and eluted with a linear gradient of ammonium phosphate as detailed elsewhere (6). Preparation of radiolabelled inositol phosphates: EiiitTZEE~~on the properties of an InsP3-kiriase preparation prepared from the adrenal cortex (19) by the method of Hansen (20), we observed that Ins-1,4,5-P3 was rapidly converted to Ins-1,3,4,5-P4, which was then dephosphorylated to Ins-1,3,4-P3. 'Ihe latter product was again phosphorylated to produce one of the new InsP4 isomers that we cbserved in the stimulated glcmerulosa cells. This reaction sequence produced almost exclusively the new InsP4 isomer in prolonged incubat' ns. Taking advantage of this property of our preparation, 2 pci Of u3 [ HlIns-1,4,5-P3 was incubated with 1.5 mg enzyme protein in 2.0 ml medium (50 mM Tris/HCl pH 5 mM ATP, 1 mM dithiothreitol, 8.05 5 mu M9"2 , 5 mM Na-pyrophosphate, 10 M CXl,) which contained 0.2 UM Ins-1,4,5-P3. Incubation was carried out for 4 hrs at 37 OC, and the reaction was stopped with 160 ~1 HC104 (60 %). After centrifugation, HClO4 was extracted fran the Sample (21), which contained about 60% of its radioactivity as the new InsP4. After purification on HPLC and desalting by sequential chromatography on Bio Had Iy; lx8 and Lowex W-50 (H+), lyophylization gave 0.5-0.7 &i of the new InsP4 isomer, which was checked for purity by HPK before periodate oxidation. Ins-1,3,4-P3 was prepared fran [ HIIns-1,3,4,5-P4 (NEN, 1 Ci/mnol) by incubation with erythrocyte membrane 5-phosphatase (22)

*

200

Q2+-

additional

(17).

is phosphorylated

study we provide

it

of

Vol. 148, No. 1, 1987

BIOCHEMICAL

AND BIOPHYSKAL

RESEARCH COMMUNICATIONS

Pericdate oxidation: Periodate oxidat$on was performed acyrding to (23) and (24) with slight qifications. [ HIIns-1,3,4-P3 and [ HIIns-1,3,4,5P4, as well as the [ Hllabelled new InsP4 isomer (0.1 ~01 of each), were each separately incubated with 0.5 ml of 0.1 M NaI04 for 7-11 days in the dark at room temperature. Sodium-borohydrate (50 mg) was then added from an aqueous solution dropwise and the reaction was continued for 5-8 hours. The excess borohydrate was reacted with 1 N HCl until Hz evolution ceased. Samples were then applied to 5 ml Sephadex 615 columns (preswollen in 0.1 M phosphate buffer) and eluted with 0.1 M phosphate buffer. Fractions containing 3H activity (2-4 ml) were pooled and diluted with distilled water to 30 ml. MgCl2 (1 mM final) and alkaline intestinal phosphatase (1000 IU, Sigma , Type VII-L) was added after the pH was adjusted to 10 with NaOH. The reaction was left to proceed for 6 hr at 37 "C. A mixture of unlabeled myo-inositol and altritol (prepared from altrose according to (23)) was added to each of the samples before lyophylization. !the residue was distilled with 1% methanolic HCl and finally deionized with a mixed-bed ion-exchange resin (Amberlite, MB-3A, Aldrich Chemical Co., Milwaukee, Wis). me resulting polyols were separated by paper chromatography (23) as described by Irvine et al. (23). The strips were then bisected and one half was developed by the silver-nitrate method (25) with the modification of Frahn and Mills (26). The other half was cut into 1 cm pieces and analyzed for radioactivity in a f+spectrometer. RESULTS AND DISaJSSICN ____.~ Bovine adrenal 24 hr contained

mainly

under basal

Ins-&P,

Fig.

glomerulosa

1.

Basal

glomerulosa

of

with

[3H]inositol

as shown on the HPLC elution

Ins-1,4,5-P3

contained

and Ins-1,4-P2

a larger

based upon its

HpLC elution

large

in the levels

increases

prelabelled

the two isomers of Ins-monophosphate, conditions

levels

cells

cells

peak which

characteristics.

with even larger

changes in Ins-1,4-P2.

tion

of Ins-4-P

was followed

In addition

to these

and

profiles

in

but the

is assumed to be InsP5 II

stimulated

and Ins-1,3,4-P3,

!lhe selective

by a delayed

Ins-l-P

were low,

Angiotensin

of Ins-1,4,5-P3

for

increase

together

and early

stimula-

in the amount of Ins-

1-P.

were eluted

in the

The central

peak increased

and was identified other

tissues

region

changes

of extracts

rapidly

(20,27-29).

upon stimulation

levels

peak of this

The other increased

two peaks

continuously

group was recently

identified 201

(18),

fram AII-stimulated

as the 1,3,4,5-tetrakisphosphate

responses and their The first

InsP4

well-characterized

three

peaks cells.

with angiotensin

II

isomer described

in

showed much slower

during

AI1 stimulation.

by us as a new In@4

BIOCHEMICAL

Vol. 148, No. 1, 1987

0

10

20

30

AND BIOPHYSICAL

50

40 Time

RESEARCH COMMUNICATIONS

60

70

80

(minutes)

profiles obtained frcxn control (upper Panel) or Fig. 1. HEW elution angiotensin II-stimulated (lower panel) glomerulosa cells. Cultured bovine adrenal glamerulosa cells were prelabelled with [3H]inositol for 24 hrs, and after several washesthey were stimulated with angiotensin II (50 134) for 15 minutes. After extraction, inositol phosphates were resolved on a SPX HPLCcolumn eluted with a linear gradient of ammonium phosphate (---) . The results shown are representative of at least 15 similar observations. isomer produced frcm Ins-1,3,4-P3 which phosphorylates that this

Ins-1,4,5-P3

isaner was neither

phate migration

by a kinase distinct

(18).

(18).

a cyclic

Double label

‘IB distinguish periodate

oxidation

Ins-1,2,3,4-P4

Since it was produced frc4n Ins-1,3,4-P3, Ins-1,2,3,4-P4

between these two possibilities,

there are two vicinal

inositol

ring

al.

in the case of Ins-1,3,4-~3.

indicated

-OH

groups

only two

or Ins-1,3,4,6-P4. we used the

method of Grado and Ballou (23).

although these are in the trans position,

(24)

studies

form nor a product of sin-pie phos-

remained for its structure:

possibility

from the 3-kinase

If the structure

(on positions

periodate

5 and 6);

is able to open the

between them during long exposure as showed by Irvine

is not susceptible

to periodate

is

et

CX the other hand, Ins-1,3,4,6-P4

oxidation 202

because of the lack of vicinal

Vol. 148, No. 1, 1987

-OH groups

BIOCHEMICAL

(similar

reduction

to

Ins-1,3,4,5-P4).

of the aldehyde

and the resulting

tosraphy

!Lhese experiments

Ins-1,3,4-P3

was converted

InsP4 isomer gave inositol InsP4 isomer

to periodate

Ins-1,3,4,6-P4.

periodate

as the final

product.

Ins-1,3,4-P3

Our results

did

not

innmediately

behind

reveal

the

The resistance that

its

its

Ins-1,3,4,5-P4,

isomer

of

structure

is

finding

of

to be resistant is

the

which is very

of this

to a new InsP4

structure

structure

where

and the new

is phosphorylated

that

groups

conditions

with the recent

They found this

SUggeSting

under

indicates

and

by paper chrome-

Ins-1,3,4,5-P4

are consistent

homogenate.

oxidation,

that

both

oxidation

the phosphate

were analyzed

revealed

oxidation

(30) that

isomer in liver

polyols

RESEARCH COMMUNICATIONS

periodate

to altritol,

!Ihese results

Shears et al.

After

groups by Na-borohydrate,

were hydrolyzed (24) .

AND BIOPHYSICAL

to

Ins-1,3,4,6-P4.

third

peak

likely

eluting

to be another

InsP4 isaner. The physiological by which Ins-1,4,5-P3

importance is transformed

lations-dephosphorylations

is

elimination,

lic

produces

route

tions

(17).

ther

Ins-P4

lated

cells,

that

can

tion

to

InsP5

messenger

erytrocytes

(31)

ligands

functo ano-

in angiotensin-stimunetwork for

by adrenocortical

and nmnnnalian

expend considerable

203

cytosol

tissues

shown to (29,321.

energy to modify

produced during

remains to be determined.

that

In this way, Ins-1,3,4,6-

between InsP3-s and InsP5, a compound already

avian

the

metabo-

We have also observed

for publication).

of the inositol-polyphosphates

by agonist

this

is also phosphorylated

polyphosphates.

of

mechanism

is a more complex metabolic

be converted

However, the reason why cells structure

that

potential

as Ins-1,3,4,6-P4

there

et al. submitted

in

with

Ins-1,3,4-~3

now identified

P4 might be the link be present

that

through phosphory-

an alternative

the possibility

molecules

of inositol

Ins-1,3,4,6-P4 (Guillemette

additional

of reactions

The discovery

clear.

but also raised

indicates

the disposition

yet

series

products

pathway provided

r&r finding isomer,

to other

not

inositol-tris-tetrakisphosphate of Ins-P3

of the complex

homnal

the

activa-

Vol. 148, No. 1, 1987

We are grateful advice during

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

to Drs. John S. Fawcett and John Morel1 the structural

analysis

for their

of Ins-1,3,4,6-P4.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

Fakunding, J. L., Chow, R. and Catt, K. J. (1979) Endocrinology 105, 327-333. Fakunding, J. L. and Qtt, K. J. (1980) Endocrinology 107, 13451353. Cdpponi, A. M., LOW, P. D., Jornot, L. and Valloton, M. B. (1984) J. Biol. Chem. 259, 8863-8869. Farese, R. V., Larson, R. E. and Davis, J. S. (1984) hdocrinology 114, 302-304. Enyedi, P., Buki, B., Mucsi, I. and Spat, A. (1985) ml. Cell. Endocrinol. 41, 105-112. Balla, T, Guillemette, G., Morgan, R. 0. and Qtt, BaLkal, A. J., K. J. (1986) Proc. Natn. &ad. sci. U.S.A. 83, 9323-9327. Kojima, I., Kojima, K., Kreutter, D. and Rasmussen, H. (1984) J. Biol. Own. 259, 14448-14457. Berridge, M. J. and Irvine, R. F. (1984) Nature (London) 312, 315321. Nishizuka, Y. (1984) Nature 308, 6931698. Streb, H., Irvine, R. F., Berridge, M. J. and Schulz, I. (1983) Nature (London) 306, 67-69. wt, A., Bradford, P. G.r McKinney, J. S., Rubin, R. P. and Putney, J. W. Jr (1986) Nature 319, 514-516. BadCal, A. J., Guillemette, G., Rubin, R., Spat, A. and Catt, K. J. (1985) Biochem. Biophys. Res. @annun. 133, 532-538. Spat, A., Fabiato, A. and Rubin, R. P. (1986) Biochem. J. 233, 929-932. Storey, D. J.r Shears, S. B., Kirk, C. J. and Michell, R. H. (1984) Nature (London) 312, 374-376. Batty, I. R., Nahorski, S. R. and Irvine, R. F. (1985) Biochem. J. 232, 211-215. Irvine, R. F., Letcher, A. J., ~eslop, J. P. and Berridge, M. J. (1986) Nature (London) 320, 631-634. Irvine, R. F. and Moor, R. M. (1986) Biochem. J. 240, 917-920. G., Baukal, A. J. and Qtt, Balla, T., Guillemette, K. J. (1987) J. Biol. Chem. 262, 9952-9955. Guillemette, G., Baukal, A. J., Balla, T. and Catt, K. J. (1987) Biochem. Biophys. Res. Gmnnun. 142, 15-22. HanSen, C. A., Mah, S. and Williamson, J. R. (1986) J. Biol. Chem. 261, 8100-8103. Dzwnes, C. P., Hawkins, P. T. and Irvine, R. F. (1986) Biochem. J. 238, 501-506 R. H. (1982) Biochem. J. Ewnes, C. P., Mussat, M. C. and Michell, 203, 169-177. Grado, C. and Ballou, C. E. (1961) J. Biol. &em. 236, 54-60. Irvine, R. F., Ietcher, A.J., Lander, D.J. and Dowries, C. P. (1984) Biochem. J. 223, 237-243. Travelyan, W. E., Procter, D. P. and Harrison, J. S. (1950) Nature (London) 166, 444-445. Frahn, J. L. and Mills, J. A. (1959) mst. J. Chem. 12, 65-89. Hawkins, P. T., Stephesn, L. and Dowries, C. P. (1986) Bzhem. J. 238, 507-516. Biden, T. J. and Wollheim, C. B. (1986) J. Biol. Chem. 261, 1193111934. 204

Vol. 148, No. 1, 1987

29. 30. 31. 32.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Morgan, R. O., chang, J. P. and Catt, K. J. (1987) J. Biol. Chem. 262, 1166-1171. Shears, S. B., Parry, J. B., Tang, E. K. Y., Irvine, R. F., Michell, R. H. and Kirk, C. J. (1987) Biochem. J. (in press). Johnson, L. F. and Tate, M. E. (1969) a. J. Chem. 47, 63-73. Heslop, J. P ., Iwine, R. F., Tashjian, A. H. and Berrirge, M. J. (1985) J. Exp. Biol. 119, 395-401.

205