Acetylcholine stimulation on phosphatidylinositol-inositol phosphohydrolase of rat brain cortex

ACETTLCHOLINE IKOSITOL

STIAIUL.~TION

PHOSPHOH~DI~OL_~SE

ON PHOSPHATIDYI,INOSITOL-01; K,%T BR;\IN

CORTEX

I. Tile aim of the present paper was to stud). if the known effect of acctvlcholine on yl~osI~l~atid~IinositoI turnox,er in\wlved the stimulation of the enz\mic hydrolysis of ~~lrosI)I~atid~Iinositol. 2. rls a first step the distribution of ~~liospliatid_linositol-inositol pliospl~ohydrolase in subcellular fractions from tile rat cerebral cortex was investigated. It was found that the enzyme FVW essentially particle bound lvitll the higher COIKCIIfollowing the distribution of tration in the cholinergic nerve-ending membranes, acet~lcholinesterase. .s, I\ marked effect of acetylclloline on ~~l~ospI~atid~~linositoI~i~~ositol pl~oslhllydrolase activity was obser\.ed in the crude nerve-ending membrane and soluble fractions. The type of effect was dependent on the concentration : IO- 7-~o- 3 31 awtJ.lcholine enhanced the activit\. while IO-” AI produced considerable inhibition. 4. The results favour the hypothesis tllat acct~dcholine affects tlw turno\.er of plrospllatid~lint,sitol during tllc transmission process.

It is well known that ““Pi is rapidly incorporated into the inositol pl~ospllolipids of brain and that this incorporation can be stimulated by acet!dcholine, however, tile exact site of this effect is still unclear (for references see refs I and 2). Redman and Hokin suggested tlrat the acetylchline stimulation occurs at the lel-cl of digI!.ceride kinase but this hypotllesis seems not to be supported by the recent negative hidings of Lapetina and Hawthorne*. On the other hand, Durrcll and co-\fy)rkers” 7 llarre suggested tliat the primaq. effect of acetylcliolinc is the stimulation of the enz\-mic hydrolysis of the pllosphatid~linositol. Furthermore, in this laborator>- it has i,ceu shown that the acetvlcholine stimulation of the pllosphatid,-linositol turnover \vas confined to a fraction of I)liosI~IiatidyIinositoI bound to a brain proteolipiclH \r.lricll shows some properties of a cholinergic rccept&. These findings suggested the present

PHOSPHhTII)TLI~OSITOLINOSITOL

investigation, inositol-inositol cortex

PHOSPHOHT;DROLASE

in which we have studied the subcellular phosphohydrolase

and the effect of acetylcholine

(phosphatidylinositol

219

distribution

of phosphatidpl-

hydrolase)

in rat cerebral

on this enzyme.

Pwfiarntiou of substvatc fey phos~hatid_vlilzositol hydrolase Phosphatidylinositol was obtained from Saccharom>vrs ccmlisiae according to the method of Lettersl”. The yeast was suspended in water, disintegrated 1,~. ultrasonic treatment and extracted with ethanol and thereafter with chloroform~n~ethanol (z: I, \,jv). The extract was evaporated, dissolved in chloroform and fractionated by the use of colums of silicic acid and DEAE-celluloselo. Thin-layer chromatograph>- of the final eluate gave a single band which was dissolved emulsified in waterll to be used as substrate.

in chloroform

and

Assa_v of fdtos~hatitl$i~~ositol hydrolnsc: This enzyme was determined according to the method of Kemp ct c~1.l~.Sanples of the fractions containing 5-50 mg fresh tissue rehomogenized in 40 mM TrisHCl buffer (pH 6.5) were added to 0.5 ml of the buffer-substrate mixture in final concentration 40 mM Tris-HCl buffer (pH 6.5), I miU CaCl, and

I

containing nd1 phos-

phatid>-linositol. Incubation was carried out for I h at 37 “C with constant shaking. The unit of activity was defined as the amount of enzyme which produced I iu,mole water-soluble organic phosphate per 11. An parallel series of tubes containing the fractions but without substrate was run simultaneously to provide blanks. The reaction was stopped by cooling the tubes in an ice bath and by the addition of 500 ,~l of 6 o/, HClO,. The tubes were then centrifuged at 15 ooo rev./min for 15 min. Samples from the clear supernatant were taken for the assay of water-soluble organic phosphate and Pi. In control experiments it was observed that the incubation of the substrate in the absence of enzyme did not liberate water-soluble organic phosphate or Pi. In the experiments to test the effect of acetylcholine on phosphatidylinositol hydrolase the buffer substrate mixture contained IO-” &I eserine sulphate and different amounts of acetylcholine to achieve the final concentrations indicated in Fig. 2. Controls fey $hosflhatidylinositol lzydrolase The differential assay of total phosphorous and Pi after incubation indicated that in our experimental conditions there is no liberation of Pi. Furthermore, to determine the possible participation of phospholipases B, the amount of carboxylic esters was assayed before and after incubation, this was carried out as follows: the reaction was stopped with chloroform and ethanol, the two phases were separated, washed12 and assayed for carboxylic acid esters as hydroxamates13. It was found that there was no decrease of carboxylic acid esters during the incubation. On replacement of the phosphatidylinositol by synthetic lecithin (Sigma Chemical Co.) as substrate there was liberation of water-soluble phosphate only in the absence of Ca”+, while the addition of I m&I CaC12 (as in all experiments reported here) completely abolished this activity.

0. Chh-ESSA DE SCARNATI.

220

G. KODRIGVEZ DE LORES ARNAIZ

Acetylcholincsterasc Acetylcholinesterase (acetylcholine acetyl-hydrolase, EC 3.1.1.7) was determined by the method Of Ellman ct al.‘” with acetylthiocholine as substrate. The unit of activity was defined as the amount Of enzyme which produced the lrydrolysis of 1 pmole Of acetylthiocholine

per 11.

Total ~hos~hovus ad Pi These were determined

as reported

by Chen et al.‘“.

Protein was determined as described by Lowry d al.l” with bovine plasma albumin as standard. All chemical and enzymatic determinations were run in duplicate. Subcellular fvactionatioga of the cc7rc4val co&-r Groups of 4-6 M?star rats (approx. 100 g body wt) were decapitated and the brains were removed in the cold room (o-4 “C) ; the cerebral cortices were pooled and homogenized in 0.32 PI1sucrose, adjusted to pH 7.0 with Tris base, to give a 20% (w/v) homogenate. This was diluted to 10% with the same suspending medium, and submitted to subcellular fractionation, according to the methods developed in this laboratory, to separate the primary fractions by differential centrifugation17, and the nerve-ending membranes by the use Of a sucrose gradientI”. Essentially the primary fractions were Obtained as follows : nuclear fraction (Fraction NUC) was separated at 000 Xg for IO min (two washes) ; the crude mitochondrial fraction (Fraction MIT) at 11500 ,Xf for 20 min (One wash); the heavy microsomal fraction at 20000 > g for 30 min (Fraction Mic,,) and the light microsomal fraction (Fraction Mic,,,) from the final supernatant fraction (Fraction SUP) at 100000 X, (7 for 60 min. To separate the nerve-ending membranes, the crude mitochondrial fraction (Fraction MIT) was subjected to osmotic shock by dilution with (1 vol. of water followed by liomogenizatioI1. This material was ceutrifuged at 20000 x g for 30 min (l;ractiOn M,), and the supernatant was centrifuged at xooooo i: g for 60 min to separate the synaptic vesicle fraction (Fraction &I,) from the soluble axoplasm fraction ( ITraction RI:,). The bulk fraction M1 (containing nerve-ending membranes) was resuspended in the original homogenizing medium and was layered on a discontinuous densit\- gradient Of sucrose of concentrations 0.8, 0.0, 1.0 and 1.2 $1. After centrifugatjon at 50000 xg details guez

for 2 11 the layered

and for morphology

subfractions

of the fractions,

Arnaiz ct al.1”. The isolated subcellular

and tllc pellet see Kataoka

were

separated.

and De Robertis”

For other and Rodri-

de I-ores

at 4 “C to remove fractions

were

sucrose,

then lyopllilized

fractions I’i

and

were dialyzed during 24 11against

water-soluble

and stored

at -20

organic

phosphate.

o.q(!h NaCl

‘l‘lle

dialyzed

“C.

Preliminary experiments were made using the total particulate and the total soluble fraction of sucrOse homogenates of rat or guinea-pig cerebral cortex Obtained b\- centrifugation at 100ooo ~6 for 60 min. Both fractions showed tile property of

I’HOSPIIATIU~-LI~OSITOL-ISOSITOL

PHOSPHOHSDROLASE

hydrolyzing

with liberation

phosphatidylinositol

but 8oo/:, of the activity

was found associated

221

of water-soluble

with the particulate

organic phosphate, fraction.

The subcellular localization of this phospllatid~~linositol hydrolase activity was studied in the isolated primary fractions and in the subfractions obtained after the osmotic shock. Fig. I shows the subcellular distribution of phosphatid~~linositol hydrolase and acetylcholinesterase. It may be observed that on the basis of the distribution of protein the phospl~atidylinositol hydrolase is not concentrated in any of these fractions. Since the bulk of the activity was found associated with the crude mitochondrial pellet (Fraction MIT) the study was extended to the main fractions obtained after the osmotic shock (Fractions III,, PI, and I&). Here again the phospl~atidylinositol hydrolase preferentially followed the distribution of protein. Acetylcholinesterase sl~owed some concentration in both microsomal fractions (Fractions Xc,, and Micloo) and in Fraction 111,which contains the nerve-ending membranes and mitochondria; furthermore cholinesterase, confirming on a sucrose gradient, the buted in all the fractions

in the soluble Fraction M, there was practically no acetylprevious findingslR. IAfter subfractionation of Fraction 11, pllosyhatid~linositol hvdrolase was almost evenly distri(Table I). Expressing the results by the relative specific

activity, that is on the basis of the protein served that phosphatidvlinositol hvdrolase

content of each fraction, and acetylcholinesterasc

it may be obwere concen-

% 80

NUC

MI

T

PRiMARY

M’Czo

MC,,

FRACTIONS

SUP

b

SUBFRACTION5

M3

@F I!4 IT

Fig. I. Histogram of the percentage of protein, phosphatidy-linositol hydrolasc and acctylcholinesterase in the subcellular fractions of the rat cerebral cortex, taking the sum of the fractious as IOO”,(,. .-lbsolute values per g cerebral cortex for the total homogenate: S7.9 3 7..+ mg for protein, I&+ & x.6 units for ~hosphatidylinositol hydrolase and 229.0 & 4.0 wits for acetylchohnestcrasc. The results are the mean of 3 espcriments i S.E. Perccutage recovery for the prtmary fractious: So, 87 and 90; for the subfractions of Fraction MI: TOO,95 and 78 for protein, phosphatidylinositol hydrolase and acetylcholinesterase, respectively.

3.7

20.7

1.4

7,s

I.3 3.0

7 ..1

10,s

S.,j

474

,jI.S

LO.0

C).X S.S

s.j

trated

in I~ractions

nI, 0.0and $1,x.0,wliicb

contain

cbolinergic

ucrvc>-ending

il!eIlI-

branes’“. Tlw effect of the acetylcboline on phospllatidvllnositol bydrolasc was studied in ITraction JI, aud in the fiual soluble Fraction SlTP. Acet!Iclloline at concentrations of IO-“-IO-~ 11 markedly enhanced the activity of the pllosI’llatid~linosito1 l~ydrolasc. On the other band, an inhibitory effect was observed wit11 IO-” A1 acetylcboline, while wit11 IO+ II1 acetylclioline tliere was practically no cliange on the activity of pl~ospbatidylinositol hydrolase in I’ractiou RI, and some inhibition in l’raction SVI’ (Fig. 2). Preliminary esperimeuts with tile nerve-ending membrane fraction (Fraction RI, 1.0) have also sl~~wn that IO-” 11 acet~4cl~oline produced an eulrancenlent iu pbospllatid!~linositol b~~drolasc activit>-.

Fried4 ct ~d.~“-~” and Tlron~pson21 reported that in guinea-pig and rat brain, respectiveI!-, the products of the activity- of pllospllatid\~linositol lrvdrolase were inositol pbospllate and diglyceride. At variance wit11 the results reported llere, the aboxymentioned investigators rnainlv cousidercd tlw soluble euzvmc. ITriedel ct al.?” found some plwspbatidylinositol hydrolase associated wit11 the isolated nerve endings but it was solubilized by osmotic shock”“. Tlleir use of wllolc brain and the experimental conditions emplqed iu the I~oruogenization ma\- account for this difference to our findings. We found that the plwspl~atidylinositol hyclrolase was found mostly particle bound aud mainly associated vvitll tile crude mitocboudrial fraction (Fraction 11 IT) and tire nerve-ending membranes. 11 similar subcellular distribution was reported for

PHOSPHATIDYLINOSITOLINOSITOL

NONE

10-T 10-G 10-s 10-q 10m3 M [Ace+ylcholl ne 1

223

PHOSPHOHYDROLASE

WNE

10-6

10-5

10-q

10-3

M [Acetylchol~nel

Fig. L. Effect of the presence of different concentrations

of acetylcholinc on phosphatitlylinositol hydrolasc in the crude nerve-ending membrane Fraction 31, and soluble Fraction SUP. The results came from two separate experiments (:--~- and O--O) and are expressed as perccntagc considering the activity in the absence of acctylcholinc as IOO$;~.

triphosphoinositide

diesterase’” and favours the view that both enzvmic activities may be due to the same enzyme2”. In the sucrose gradient to separate the nerve-ending membranes the phosphatidylinositol hydrolase followed the distribution of acetylcholinesterase (Table I), indicating that both enzymes are concentrated in the cholinergic nerve-ending membranes18. Furthermore, it was demonstrated that the effect of acetylcholine on phosphatidylinositol hydrolase depends on the concentration, because between IO-’ and IO+ RI it enhances the enzyme activity, while at IO+M acetylcholine produces considerable inhibition (Fig. 2). Durell and Soddz3 found that acetylcholinesterase and the stimulating effect of acetylclioline on the incorporation of ‘“Pi to phosphatidic acid were associated with the isolated nerve endings. They also reported that the intact structure was needed for the acetylcholine stimulation, since this effect was abolished by freezing. However, we found that the integrity of the nerve ending is not needed for the effect of acetvlcholine on phosphatidylinositol hydrolase since this may be observed even after the osmotic disruption of these particles. \Ve think that although the integrity of the nerve ending might be necessary for the overall action of acetylcholine on phosphatidylinositol turnover--which involves a series of steps with the corresponding enzymes and cofactorsPthe primary effect of the transmitter on the cleavage of phosphatidylinositol may occur directly on the membrane-bound phosphatidylinositol hydrolase (Fig. 2). Durell and co-workers observed a stimulation of phosphatidylinositol hvdrolysis by acetylcholine using the crude brain mitochondrial fraction of rats previously injected with labelled inositol. However, they could not find any acetylcholine effect

224

0.

CAMSSSA

DE

SC_-ZRNATI,

G.

ROI)RIGC-EZ

1111 LORES

ARNAIZ

on the partially purified pllosphatid~li~~ositol hydrolasefi,‘. It is now evident that to observe this effect the concentration of acetylcholine used is extremely important, since at IO-” Al there is no effect and at l+$er concentrations the enzvme ma\- ex’cn be inhibited. Our work brings direct evidence that tlw acetylclwlinc effect on the cleavage of phosphatidylinositol is due to an activation of the phosphatidylinositol hydrolasc. However, the possibilit\r of an effect on other steps of the phospllatid~linositol metabolism are not discarded. Although l’llosphatidylinositol hydrolase is widely spread in brain membranes, it is preferentially localized in acetylcholinesterase-ricll nerve-ending membranes (Table I) where the proteolipid having cholinergic receptor properties is also localized”. This observation supports tlw findings of Lunt et al.“, in which the acetylcl~oline effect on the metabolism of l’l~osphatid~liIlosito1 was associated with a fraction of I~llospllatidq’linositol bound to a proteolipid showing receptor properties. It was then suggested that this tightl?, bound pool of pliospl~atid~linositol could play a role at the postsynaptic membrane in relation with the function of such a proteolipid’“. The present demonstration that the phosphatidylinositol hydrolase is associated with the cholinergic nerve-ending membranes and is stimulated by acetylcholine is in line with the above hypothesis and may throw new light on some aspects of the role of acetvlclioline in Ventral svnaptic transmission. ACKh-0WLEI)GEMENTS

The authors are greatly indebted to Prof. E. De Kohertis, Director of Institute, for his interest and encouragement during this work. This work was supported by Grants of the Instituto de Farmacologia (Consejo National de Investigaciones Cientificas y T&nicas, Xrgentina) Leg. 4qqi70 and National Institutes of Health (5 ROI NS 06953-05 NEUA) U.S.A. 0. C. de S. is a Fellow of the Consejo National de Investigaciones Cientificas y T&micas, Xrgentina.

PHOSPHATIDYLINOSITOL-IXOSITOL

G. Rodriguez de Lores _k-naiz, M. Alberici and E. De Robertis, f. Nezwochem., 14 I<. 0. Friedel. J. D. Brown and J. Durell, B&him. Biophys. Acta, 144 (1967) 684. Ii. 0. Friedel, J. D. Brown and J. Durell, J. Neuvochem., 16 (1969) 371. W. Thompson, Can. J. Biochem., 45 (1967) 553. 2.2 Ii. RI. W. Keough and W. Thompson, J. ~~~~z~~ec~e~~~., 17 (rg70) I. 23 J. Durell and XI. A. Sodd, J. Neurochem., 1.3 (1966) 487. 24 E. Do Robertis, Science, 171 (1971) 963. 18 rg 20 21

225

PHOSPHOHYDROLASE (1957)

215.