Polyphosphoinositide hydrolysis in response to light stimulation of rat and chick retina and retinal rod outer segments

P.iochimica et Biophysica Acta, 970 (1988)205-211

205

Elsevier

BBA12273

Polyphospholnosifide .hydrolysis i n r e s p o n s e to light s t i m u l a t i o n of rat and chick r e t i n a a n d retinal rod o u t e r s e g m e n t s F i o n a A. Millar, Sylvia C. Fisher, C a r o l A. M u i r , E l a i n e E d w a r d s a n d J o h n N. H a w t h o r n e Department of Biochemistry, Universityof Nottingham Medical Schoo~ Queen's Medical Centre, Nottinghmn (U.K.)

(Received15 December1987)

Key words: Pol~phosphoinositide;Lightstimulation;(Rodoutersegment) Phosphoinositides of ehiek and rat retina were labelled with [3Hlinositol. Expesta'e of retinal preparations to light for 30 s caused loss of labelled plmsphatidylinositol 4,5-hisphosplmte and to a smaller extent of the other plmsphoinosifldes. Similar lisht-indueed changes were seen when rod outer segment Weparatin~ were used and, when these were i l l ~ a a t e d in calcium-free media, phe~pha~dylinositol 4 , S - ~ was the only lipid affected. No inositol 1 , 4 , 5 - ~ t e was seen after either 30 s or 5 s of ilmnimttinnof retina or 30 s illumination of red outer segmeats. It is concluded that this ~ plays no direct Part in ve~ebrate photoreceptor light transdu~on, though phospholnositide metabolism misht r e ~ to adaptation mechanisms.

Introduction The sequence of events which occurs in the process of vertebrate visual excitation has not yet been completely defined. Any proposal describing this mechanism has to account for signal transmission from the rhodopsin-co~taining disc to the ROS plasmalemma, between which there is no observable continuity, for the large scale signal Abbreviations:KOS, rod outer segments;Ptdlns,phosphatidylinositol; Ptdlns4P, phosphatidylinositol 4-phosphate; Ptdlns(4,5)P2, phosphatidylinositol4,5-bisphosphate;Ins(l,4, 5)P3, ino~tol1,4,5-trisphmphate;Ins(l,3,4,)P3,inositol1,3,4trisphosphate; RBP, Ringer's bicarbonate pyruvate buffer; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulpbonicacid; HPLC, highpressurefiquidchromatography. Correspondence: J.N. Hawthorne, Departmentof Biochemistry, Unlver~tyof Nottingham Medical School, Queen's MedicalCentre,Nottingham,NG7 2UH.U.K.

amplification thought to occur in the RO8 [1] and for light adaptation, the abil/ty of the system to be equally sensitive to fight both at high and at low background intensities. Two major theories have been postulated to explain the transduction mechanism, one involving Ca 2+ as the primary transducer [2], the other implicating cyclic GMP in this role [3,4]. Recent studies support the cycfic GMP theory for vertebrate retina [5-7]. An enhanced incorporation of labelled precursors into Ptdlns has been shown to occur in rat and frog retinas incubated in the light over those incubated in the dark [8,9]. Phosphorylation of Ptdlns to Ptdlns(4,5)P2 has also been shown to be enhanced in the light [8]. These results are consistent with a classic phosphoinositide effect occurring in the retina. Berridge [10] suggested that receptor-linked hydrolysis of PtdIns(4,5)P2 releases Ins(1,4,5)P3 as a second messenger mobilizing Ca 2+ from intracelluiar stores.

0167-4889/88/$03.50© 1988ElsevierS c i ~ PublishersB.V.(BiomedicalDivision)

206 Polyphosphoinositide b.ydrolysis occurs in response to light in all photoreceptor systems studied so far, but in retina, where nerve cells are also present, the hydrolysis is not necessarily a direct response to light. The light-sensitive area of the photocell, the ROS, is conn.:ted to the remainder of the cell by a thin cilium arid this can be broken by vigorous shaking. ROS can then be purified from the remainder of the retina by Percoll density centrifugation. This investigation examines fight-induced polyphosphoinositide breakdown in whole retina and in isolated ROS. The ROS results show that polyphosphoinositide hydrolysis is a direct response to light in vertebrate photoreceptcx cells, but we have been unable to detect any Ins(1,4,5)P3. Some of the results presented here have been published as a preliminary communication [11]. Materials and Methods

Materials. Male Wis~.ar rats (250-350 g) were obtained from the University of Nottingham Joint Animal Breeding Unit, Sutton Bonington, Leics. 1- or 2-day-old chicks were obtained from Herm a n ' s Poultry, Bilsthorpe, Notts. myo-[2-3H]Inositol (15 Ci/mmol) was obtained from Amersham International Amersham, Bucks. Silica gel 60H was obtained from Merck, Darmstadt, F.R.G., scintillation fluid and methanol were obtained from Fisons, Loughborough, chloroform was obtained from B.D.H., Poole, Dorset and all other reagents were obtained from Sigma Chemical Co., Poole, Dorset. Isolation, labelling and illumination of whole retinas. Dissection, incubation with [3H]inositol and ROS preparation were all performed under dim red light. 20 rats were dark-adapted for 24 h and killed 2-3 h after their normal light cycle would have begun, i.e., about 10 a.m. The eyes were removed and placed in 50 ml of buffer A: 130 mM NaCI, 10 mM KCI, 5 mM MgCI 2 and 5 mM Hepes (pH 7.5), which was kept ice-cold. As the retinas were dissected out, they were collated in a glass vial cooled with ice and containing the same buffer. For labelling, they were transferred into 2 ml Ringer's bicarbonate pyruvate buffer (RBP): 120 mM NaCI, 2 mM KCI, 2 mM MgSO4, 2 mM

C~C12, 25 mM NaHCO 3 and 10 mM sodium pyruvate, containing 20 pCi/ml [3H]inositol. The retLaas were shaken in a water bath at 37°C and gen.tly gassed with O2/CO z (95 : 5). After 2 h, the retinas were separated into two roughly equal portions. To one, 1 ml 20~ trichloroaeetic acid was added. The other was exposed to bright light (7.2 lumens per m2) for 30 s before addition of trichloroacetic acid. For the removal of chick retinas the animals were killed after 24 h dark adaptation and the head was held in position while the eye was sliced open with a razc~ blade, leaving the eye cup exposed in ~ita. Vitreous humour was removed and the retina detached with forceps. The remaining incubation and fight stimulation procedures were as described above. Preparation of rat and chick ROS. The method is based on that of Shuster and Farber [12]. After labelling with [aH]inositol the retinas were vortex-mixed for 30 s then centrifuged for 1 rain at 2000 rev/min in a conical tube to remove retinal debris. Of the supernatant, 1.5 ml was transferred to a 10 ml polycarbonate centrifuge tube ,and replaced by 1.5 ml buffer A. After vortex mixing for a further 5 s, centrifugation was repeated and the supernatant was transferred as before. The process was continued until 3.75 ml of supernatant had been collected. To this, 4.25 ml of Percoll-buffer A solution was added with gentle mixing. This solution was made by mixing 9 ml Percoll with 1 ml buffer A, (pH 6.0), the latter being at 10-fold concentration. The mixture was then centrifuged at 20000 rev/min for 1 h (M.S.E. Europa 24, using 8 × 50 ml rotor with 10 ml adaptors). The ROS band near the centre of the gradient was collected using a plastic pipette tip. The volume was made up to 8 ml with buffer A (pH 7.5) and centrifuged at 5000 rev/min for 20 rain. The pellet was resuspended in 2 ml of buffer A which contained 0.5 mM EGTA in some experiments. As with retinal preparations, 1 ml was then quenched direct!y in the dark with trichloroacetic acid and 1 ml was illuminated for 30 s before quenching. In ROS preparations from chick retina, a layer of pigment epithelium material was seen just below the ROS layer in the gradient. This was avoided when the ROS material was collected.

207 Better separations were obtained on the gradient if the tube contained material from no more than six retinas. Lipid extraction. The trichloroacetic acid-treated samples were centrifuged at 650 x g for 5 rain and the supcrnatants removed. The pellets were washed in 1.5 ml 5% trichloroacetic acid containing 1 mM EDTA followed by a wash in 1.5 ml distilled water. The pellets were then r~,spended in 1.5 ml chloroform/methanol/concentrated HC1 (100: 100:1 v/v) and allowed to stand for ~0 min before centrifogation at 3000 × g for 10 rain. The pellets were further extracted with 1.5 ml of the same solvent followed by 1.5 ml chloroform/ methanol/concentrated HC1 (200 : 200 : 1). The three supernatants from the lipid extractions were combined for each sample and made biphasic by the addition of 1.5 ml chloroform and 1.5 ml 0.1 M HCI. The upper phase was then removed and the lower phase containing the lipids was washed twice with synthetic upper phase (chloroform/ methanol/0.1 M HCI containing 10 mM inositol, 80:40:30). Total lipid P was determined by the Bartlett [13] method. The samples were then dried under a stream of N 2, resuspended in 100 ~1 chloroform/4-methanol (1:1) and spotted onto thin-layer plates of silica gel 60H spread in 1% potassium oxalat¢. For visualisation of the polyphosphoinositides a small quantity (approx. 5 pg P) of a Ptdlns4P/Ptdlns(4,5)P2 mixture prepared from brain was added to each spot. Preparation details are given in a previous paper [14]. The thin-layer plates were run in one dimension in a chloroform/acetone/methanol/acetic acid/ water (40:15:13:12:7) solvent system. After iodine staining, spots were scraped off and radioactivity was determined by scintillation counting in a Packard Tficarb 4640 scintillation counter. HPLC of inositol phosphates. Portions (0.75 ml) of the trichloroacetic acid supemat~nt from retinal or ROS experiments were extracted five times with 2.5 ml diethyl ether and then neutralized with 0.1 M Tris base. To increase recovery of inositol phosphates after f=ceze-drying. 0.1 ml of 50 mM D-mannitol was added [15]. Samples were then freeze-dried overnight. The dry residue was dissolved in 2 ml water and stored at - 2 0 °C until required for HPLC. In some experiments, a simpler acetone precipitation was used. This avoided acid conditions and

gave cleaner samples for HPLC. The retinal or ROS samples were quenched by the addition of an equal volume of acetone. After centrifugation at 3000 × g for 5 rain, acetone was removed in a stream of nitrogen, mannitol was added and the sample was freeze-dried. The residue was then taken up in 2 ml water and any insoluble material was centrifuged down before applying the solution to the HPLC column. Where lipids were to be studied, the acetone precipitate was treated exactly as the trichloroacetic acid precipitate, described in the section on fipid extraction above. The HPLC method was essentially that of Batty et al. [16]. The apparatus used was a Beckman 332 Gradient Liquid Chromatogram (Beckman-RIIC Ltd., High Wycombe, Bucks), a modular dual pump system comprising a 420 Microprocessor System Controller, two Model ll0B single piston reciprocating pumps, a gradient mixing chamber and a Model 210 sample injection valve, connected to a Model 160 fixed-wavelength ultraviolet detector at 254 nm. Nucleotide markers were used to register approximate positions of [3H]inositol phosphates [15]. In addition, 32p-labelled Ins(1,4,5)P3 was prepared from erythrocytes by the method of Downes et al. [17]. As noted by lrvine et al..[15] the Ins(1,4,5)P3 preparation contained another 32p-labelled compound which was ehited from the HPLC column in the Ins(1,3,4)P3 position. This was probably inositol 1,2-cyclic 4,5-trisphosphate [18,19]. ?~.mpies were !oaded on to a Partisphere 5SAX column, 4.7 mm internal diameter, length 11 cm, particle size 5 ttm with AX guard cart_ridge(Whatman, Maidstone, Kent). The column was connected to a Whatman Solvecon precolumn and the flow rate was 1.25 ml/min. Labelling of chick ROS. In some experiments, the chick ROS were prepared from unlabelled retinas and then incubated with [3H]inositoL ROS from 12 retinas were incubated in 2 ml RBP buffer with added 1.6 mM cytidine and 40/tCi/ml [3H]inositol for 1 h at 37°C as described for the retinal labelling. Statistical treatment. Since the phosphoinositide labelling varied from one retinal or ROS preparation to the next, in each experiment the percentage loss of radioactivity caused by fight was calculated. The Student's t-test was then applied to these percentage changes.

208 Results

Phosphoinositide changes Illumination o f rat retina for 30 s caused a marked loss o f labelled PtdIns(4,5)P 2 but appreciable losses o f P t d I n s 4 P and PtdIns also occurred (Table I). Since retina contains nerve cells as well as the photoreceptor cells, the p h o s phoinositide changes could b e taking place in either or b o t h cell types. To localise the changes m o r e precisely, retinal rod outer segments were prepared and illuminated in the same way. Table II shows that all three phosphoinositides o f R O S responded by loss o f label, that in PtdIns(4,5)P~. being most marked. This phosphoinositide response is therefore associated with photoreceptor cells, though secondary phosphoinositide breakd o w n may occur in other parts o f the retina. W h e n R O S were illuminated in buffer containing E G T A there was a m o r e m a r k e d loss o f

PtdIns(4,5)P 2 and n o significant changes in the other phosphoinositides. In whole retina o f chick (Table III) the only significant fight response was again seen in PtdIns(4,5)P 2. In chick R O S (Table IV) the major change was in this lipid, b u t s o m e loss o f label f r o m P t d I n s 4 P was also seen. W h e n the R O S were incubated in calcium-free buffer, as with the rat preparations, P t d I n s ( 4 , 5 ) P 2 was the only lipid responding to light. Similar p h o s p h o i n o s i t i d e recoveries a n d changes in response to light were seen w h e n trichloroacetic acid quenching was replaced b y acetone treatment.

Changes in inositolphosphates F o r the detection b y H P L C o f inositol phosphates the water-soluble p h o s p h a t e fraction f r o m about 40 chick retinas was used. T o reduce inositol p h o s p h a t a s e activity, the incubation buffer

TABLE I EFFECT OF LIGHT ON [3H]INOSITOL-LABELLED PHOSPHOINOSITIDES OF RAT RETINA The second column for each lipid represents dpm per Fg ,'.orallipid P, Each figure is a mean from three experiments. The P values (paired t-test) give significance of the fight effect; S.D. are provided. Ptdlns(4,5)P 2 Dark Light P

dpm 1818 904

dpm/Fg P 170 97

Ptdlns4P $ change 42.9+2.6 < 0.01

dpm 5088 3283

Ptdlns

dpm/Fg P 476 354

~ change 25.6=1:2.9 < 0.01

dpm 39652 27133

dpm/Fg P 3709 2927

$ change 21.1~-6.5 < 0.05

TABLE 1I EFFECT OF LIGHT ON [3H]INOSITOL-LABELLED PHOSPHOINOSITIDES OF RAT ROS Means from six (buffer A) or five (buffer A with EGTA) experiments are given. Other details are as described in Table I. n.s. no significant change. Ptdlns(5,4) P2 dpm dpm/Fg P Buffer A Dark Light P

583 529

64 55

Buffer A with 0.5 mM EGTA Dark 496 62 Light 321 43 P

% change

Ptdlns4P dpm dpm/Fg P

% change

Ptdlns dpm

dpm/pg P

~ change

14.1:i: 14.3 < 0.05

2056 1970

225 205

8.9+4.0 < 0.01

26165 2869 2 3 4 0 0 2437

15.1+9.4 < 0.05

30.64-7.9 < 0.001

1636 1483

203 199

2.0:[:14.0 n.s.

15 219 14156

-0.7+18.6 n.s.

1890 1903

209 TABLE III EFFECT OF LIGHT ON [3H]INOSITOL-LABELLED PHOSPHOINOSITIDES OF CHICK RETINA Means from six experiments are given. Other details are as given in Table I. ~tdIns(4,5)P2 Dark Light P

dpm 7167 5808

dp:a//tg P 23~ 200

Ptdlns4P 9~change 14.5:i: 12.2 < 0.01

dpm 25087 22027

Ptdlns dpm//~g P 820 758

.%change 7.6+24.4 n.s.

dpm 151096 148328

~pm//~g P 4939 5106

~ change -3.4~-20.1 n.s.

TABLE IV EFFECT OF LIGHT ON [3H]INOSITOL-LABELLED PHOSPHOINOSITIDES OF CHICK ROS Means from seven (buffer A) or five (buffer A with EGTA) experiments are given. Other details are as given in Table I. Ptdlns(4,5) P2 Buffer A Dark Light p

Ptdln.~P

Ptdlns

dpm

dpm//~g P

% change

dpm

dpm//~g P

% change

dpm

dpm//tg P

~ cl:ange

403 305

47 35

25.5+11.9 < 0.001

3312 3116

383 355

7.3:1:6.9 < 0.02

17718 17823

2051 2030

1.0+13.8 n.s.

16.0+3.6 < 0.001

2468 2390

388 397

-2.3+12.4 n.s.

30906 29428

4859 4888

-0.6+10.7 n.s.

AMP

AOP GI~

Buffer A with 0.5 mM EGTA Dark 606 95 Light 480 80 P

c o n t a i n e d 10 m M LiCI. N o inositol 1,4,5-trisp h o s p h a t e w a s detected in either fight o r d a r k i n c u b a t i o n s for 30 s, b u t i l l u m i n a t i o n increased t h e radioactivity in b o t h inositol m o n o p h o s p h a t e a n d inositol b i s p h o s p h a t e p e a k s (Figs. I a n d 2). A f t e r correction for t h e radioactivity applied to t h e c o l u m n , there w a s a 329~ increase in m o n o p h o s p h a t e a n d a 009~ increase in b i s p h o s p h a t e . T h e first s m a l l peak, u n a f f e c t e d b y light, is p r o b a b l y d u e to glycerophosphoinositol. A n o t h e r exp e r i m e n t o f this t y p e gave similar results, b u t in a third e x p e r i m e n t n o effect o f light o n t h e inositol monophosphate and bisphosphate peaks could be detected. Since Ins(1,4,5)P3 m a y h a v e b e e n h y d r o l y s e d to t h e lower p h o s p h a t e s d u r i n g t h e 30 s illumination, a 5 s i l l u m i n a t i o n period w a s u s e d in f u r t h e r experiments. A g a i n , n o t r i s p h o s p h a t e w a s detected a n d light d i d n o t significantly increase the radioactivity o f t h e m o n o - or b i s p h o s p h a t e peaks. A large-scale e x p e r i m e n t in w h i c h R O S were prepared f r o m 132 chick retinas gave sufficient

25

2(

LLL

ATP

GTP

L

L

II~mm lira (mini

Fig. 1. HPLC pattern of [3H]inosilol phosphates from chick retina. Incubation was in the dark for 30 s, medium contained 10 mM LiCI. Arrows indicate elution of marker substances detected by ultraviolet absorption. Discontinuous lines show the 32p-labelled Ius(1,4,5)P 3 from erythrocytes (second peak) and the 1,2-cycfic compound obtained with it (see Materials and Methods).

210

2~-

AMP A~P T

iP

G[

20-

[3H]Ins(1,3,4)P3 from parotid gland being added for comparison. Details of the chemical procedure are given by Irvine et al. [15]. The only 32p-labelled InsP3 ran in the position of the 1,4,5-trisphosphate and was clearly separated from [3H]Ins(1,3,4)P3. Thus chick retina contains only the usual PtdIns(4;5)P2. Discussion

k 18

t~j~ 20

21.

78

32

38

Retentionllme(min) Fi& 2. HPLC pattern of [3H]mosito] phosphatesfrom chick retina, Illumination was for 30 s in the same medium as for Fig. 1. Other details are given in the legend to Fig. 1.

radioactive inositol phosphates for HPLC. Illumination was for 30 s followed by quenching with trichloroacetic acid. In neither dark nor light samples could inositol trisphosphates be detected, though lipid analysis showed 18% loss of [3H]inositol label from PtdIns(4,5)P2. Inositol monoand bisphosphates were detected, radioactivity in both peaks being greater after illumination. Confirmation of Ptdlns(4,5)Pz structure Since Ins(1,3,4)P3 had been detected after illumination of Limulus photoreceptors [20] and some of our early experiments suggested the formation of this compound, we investigated the possibility that vertebrate retina contained PtdIns(3,4)P2. Briefly, retinas from 12 chicks were incubated with 30 /~Ci/ml [32p]orthophosphate for 1 h. After precipitation with trichloroacetic acid, phosphollpids were extracted and the polyphosphoinositides separated by thin-layer chromatography (TLC) as described already. The PtdInsP2 band was scraped from the plates and the lipid was extracted into chloroform/ methanol/concentrated HCI (200:100:1, v/v). After washing, the PtdInsP2 was deacylated in methanolic NaOH, the glycerol moiety was removed by periodate and dimethylhydrazine and the resulting [32p]InsP3 was separated by HPLC

Retinal phosphoinositide responses to light may be associated specifically with photoreceptor cells and/or with neurons which make synapses with these cells. Studies with whole retina do not distinguish between these possible sites and there is evidence for a major phosphoinositide response in the hor+.zontal nenrons of toad retina [9]. Our results indicate that breakdown of PtdIns(4,5)P2 is a specific response to light in the rod outer segments of rat and chick retina. The ROS preparations were virtually homogenous under phase-contrast microscopy. Inner segments of the photoreceptor cells had been removed and there was little contamination by retinal fragments. Such contamination, for instance by neuronal material, would not affect the results, since the neurons would not be able to respond to light unless there was a synaptic connection with a photoreceptor cell. Such connections are destroyed during ROS preparation. A specific loss of Ptdlns(4,5)P2 was seen by Ghalayini and Anderson [21] when frog retinas were illuminated. In these experiments, ROS membranes were isolat,:A after trichloroacetic acid precipitation of the stimulated retina. The phosphoinositide change was seen in these membranes but attempts to prepare ROS before light exposure resulted in loss of polyphosphoinosifides. We have overcome this problem and shown lightinduced loss of phosphoinositides in ROS prepared from [3H]inositol-labeUed retinas or labelled with [3H]inositol after preparation. Hayashi and Amawaka [22] labelled ROS from frog retina with [y-32p]ATP in the presence of added PtdIns4P. A 5 s light fl~sh caused 20% loss of label from only PtdIns(4,5)P2. Giusto and Ilincheta de Boscbero [23] have also studied the biosynthesis of polyphosphoinositides from labelled ATP in ROS from bovine retina, but not the effect of light.

The fight stimulus iv the present, work causes loss of label from all Uiree phosphoinositides in R O S unless E G T A is present, w h e n only PtdIns(4,5)P2 is affected. Current thinking suggests that this is the i m p o r t a n t response, receptor activation causing hydrolysis o f PtdIns(4,5)P 2 to release Ins(1,4,5)P 3 as a calcium-mobilizing seco n d messenger. I n m a n y systems, however, loss of b o t h polyphosphoinositides and sometimes of PtdIns as well is observed [24]. O n e explanation is t h a t the p h o s p h o l i p a s e C is specific for PtdIns(4,5)P 2, though this has n o t b e e n shown in vitro, and that the loss o f label from P t d I n s 4 P a n d P t d I n s is due to their conversion to PtdIns(4,5)P 2 rather t h a n their hydrolysis. T h e present results show a specific light-induced loss o f Ptdlns(4,5)P2 from R O S in the absence of external Ca 2+ (Tables II and IV), E G T A being added. Even though buffer A contains no a d d e d C a 2+, loss o f P t d I n s 4 P a n d P t d I n s is seen unless E G T A is present. This may indicate calcium-dep e n d e n t hydrolysis o f these phosphoinositides. Our lipid results suggest that Ins(1,4,5)P 3 may b e produced in response to light in the chick retina, b u t that it is hydrolysed in less than 5 s. Brown et al. [25] obtained similar results with t o a d retina. Ins(1,4,5)P 3 increased with 250 m s illumination but declined after 1 s. It seems unlikely that this Ins(1,4,5)P 3 acts to mobilize calcium, since in the vertebrate retina light reduces the cytoplasmic Ca 2+ concentration in the p h o t o receptor cells [26-28]. Acknowledgements This work was s u p p o r t e d b y the Wellcome Trust. Re[erences

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