Exp. Eye Res. (1990) 50, 397405

Stimulator-y and Inhibitory Actions of Excitatory Amino Acids on lnositol Phospholipid Metabolism in Rabbit Retina. Evidence for a Specific Quisqualate Receptor Subtype Associated with Neurones NEVILLE Nuffield Laboratory of Ophthalmology,

N.OSBORNE

University of Oxford, Walton Street, Oxford OX2 6A W, U.K.

(Received 13 June 1989 and accepted in revised form 28 September 1989) The effectsof excitatory aminoacidagonistson [3H]inositolphosphates(InsPs)levelshave beenexamined in rabbit retinal tissuesunderbasalconditionsand after agoniststimulation.Quisqualate(QA) isthe most effective excitatory aminoacid agonistat stimulatingInsPsaccumulationwith an EC,, value of 0.1 PM. The responses for maximally effective concentrationsof QA with either ibotenate or kainate were not additive, which suggested that all the excitatory aminoacid agonistswhich stimulateInsPsaccumulation (quisqualate,kainate, NMDA, glutamate,ibotenate,aspartate)have a commonsiteof action. Noneof the following antagonists: m-2-amino-5-phosphonovalerate(APV), nL-2-amino-4-phosphonobutyrate (APB) and glutamate dimethyl ester(GDEE),prazosin,ketanserinor atropine influencedthe excitatory amino agoniststimulation of InsPs.Thesedata suggestthe presenceof a specificQA-receptor subtypein the retina. QA. and to a lesserextent other excitatory amino acidagonists,were alsoeffectivein stimulatingInsPs accumulationand the mobilizationof internal calcium levelsin 3-S-day-old retinal cultures but not in the older cultures (25-30 days old), which lack neuronesbut contain Miiller cells.The QA receptor subtypeslinked to InsPsaccumulation in the retina are therefore presenton neurones. Kainate and NMDA had a weak inhibitory action on the effect of the carbachol-inducedstimulation of InsPsat 50 ,UM.The NMDA action was abolishedby APV, whereasthis antagonisthad no effect on the action of kainate. Experimentswith tetrodotoxin, cadmiumand verapamilindicate that the kainate and NMDA action on the carbachol-inducedstimulation of InsPsdoesnot take placethrough an indirect releaseof substances from neighbouringneurones.The modeof action of NMDA and kainate in reducing agonist-mediatedInsPsformation in the retina still requireselucidation. Key words : excitatory amino acids; inositol phospholipidmetabolism: retina; quisqualate. 1. Introduction Excitatory amino acids (glutamate, aspartate) are major excitatory transmitter candidates in the mammalian retina (Ehinger and Dowling, 198 7 ; Ehinger et al., 1988). Three distinct ionotropic excitatory amino acid receptor subtypes have been defined electrophysiologically (Watkins and Evans, 1981). They have been named N-methyl-D-aspartate (NMDA), quisqualate (QA) and kainate (KA) (Watkins and Evans, 1981) or A,, AA, and AA,, respectively (Fagg, Foster and Ganong, 1986). The KA and QA subtypes mediate fast excitatory ionic responses carried by Na+ ions, while the NMDA receptors induce ionic changes that are longer in duration and involve movement of Ca2+ ions in addition to Nat (Cotman and Iverson, 198 7). The activation of NMDA receptors can be selectively inhibited by the competitive antagonist DL2-amino-5-phosphonovalerate (APV) as well as other substances (Foster and Fagg, 1984). Apart from being rapid synaptic transmitters, excitatory amino acids have been shown to enhance inositol phospholipid turnover via receptor-mediated processes, which generally have a pharmacological profile distinct from the subtypes defined above. Generally, QA in particular and ibotenate (an NMDAselective agonist) are potent agonists, while glutamate is less effective and KA and NMDA are weak or 00144835/90/040397+09

$03.00/O

ineffective (Sladeczek et al., 1985 ; Nicoletti et al., 1986a, b; Sugiyama, Ito and Hirono, 1987; Godfrey et al., 1988; Doble and Perrier, 1989). However, in some reports QA has been shown to be less effective than ibotenate (Schoepp and Johnson, 1988) and even glutamate (Nicoletti et al., 1988) in stimulating inositol phospholipid metabolism. Excitatory amino acids have also been shown to have a modulatory effect on cholinergic or adrenergic receptors coupled to phosphoinositide hydrolysis (Baudry, Evans and Lynch, 1986; Nicoletti et al., 1986a, b; Schmidt et al., 1987: Jope andLi, 1989). In some instances excitatory amino acids have been shown to inhibit only phosphoinositide hydrolysis stimulated by carbachol and noradrenaline (Baudry et al., 1986 ; Schmidt et al., 198 7). In other studies excitatory amino acids were found to inhibit phosphoinositide hydrolysis stimulated by noradrenaline (Jope and Li, 1989) and not by carbachol (Nicoletti et al., 1986a). The contrasting effects reported in these studies may be due to differences of method and tissues used in each laboratory. The aim of the present study was threefold: first, to examine the pharmacological profile of various excitatory amino acid receptor subtypes involved in the stimulation of inositol phosphates (InsPs) turnover in rabbit retinal slices: secondly, to address the questions of whether these receptors are associated with retinal 0 1990 AcademicPressLimited

398

N N. OSBORNE

neurones and/or glial cells and whether they mobilize an internal store of Ca*+ ; and thirdly, to see whether excitatory amino acids inhibit phosphoinositide hydrolysis stimulated by carbachol and/or noradrenaline.

2. Materials and Methods

Subsequently, @5 ml of distilled water and 0.5 ml chloroform were added to each tube. The coverslips and retinal cells were crushed with a glass rod and the tubes were vortexed before allowing a complete separation of the aqueous phase from the lower lipid phase. The water soluble [3HJInsPs were then extracted using Dowex as described by Berridge, Downes and Hanley ( 1982).

Retinal Cell Culture Retinas from l-S-day-old postnatal Dutch or New Zealand rabbit pups were dissociated by a modified trypsinization procedure, as previously described (Dutton, Currie and Tear, 1981; Beale et al., 1982). Briefly, freshly dissociated retinas were incubated for 15 min in solution 1 of Dutton et al. (198 1) containing 0.2 5 mg ml-’ trypsin, centrifuged, and the supernatant decanted. A mixture of solution 1 containing 0.13 mg ml-’ DNAse and 0.67 mg ml-l soya bean trypsin inhibitor was added and repetitive pipetting carried out. Cellular debris and undissociated tissue were removed until a fairly homogeneous suspension was obtained. The suspension was then centrifuged at 180 g for 5 min and the cells were suspended in a culture medium which consisted of 85 % Eagle’s Minimal Essential Medium with Earle’s salt (Gibco) (MEM) to achieve isotonicity, and 0.01% gentamicin, 10% horse serum (Gibco), plus additional glucose, to give a final concentration of 33 mM. The suspended cells were placed in 0.5 ml of culture medium on 13 mm diameter glass coverslips in Multidish (Falcon 3008) wells at a density of 4000-5000 cells per mm2. The cultures were grown at 35.5”C in a humidified atmosphere containing 5% carbon dioxide. The culture medium was changed after 3 days and thereafter at 2-3-day intervals. Excitatory Cultures

Amino Acid Stimulation of InsPs in Retinal

Cultures at either 3-5 days or 25-30 days after plating were incubated for 6 h at 3 5*5”C (in a 5 % CO, humidified atmosphere) in MEM plus glucose (final concentration 33 mM) containing 3 x lo-’ M 3H myoinositol. An incubation time of 6 h was routinely used, since trial experiments showed that at this time isotopic equilibrium was reached (see Ghazi and Osborne, 1988a). The cultures were then rinsed with MBM containing 5 mM LiCl (Li, MEM) and each culture was transferred to another Falcon muItiwei1 which contained 0.50 ml Li, MEM plus a further 0.25 ml of this solution either alone or containing an antagonist. The cultures were then incubated for 10 min at 35.5% after which 0.25 ml Li, MEM solution containing agonists was added. Control cultures were simply supplied with the vehicle solution. After 45 min at 35.5% the reaction was stopped by transferring four coverslips into 1 ml of chloroform :methanol (1.2, v/v) in a glass tube.

Excitatory Slices

Amino Acid Stimulation of 1nsPs in Retinal

Dutch and New Zealand adult rabbits were injected with a lethal dose of pentobarbitone and the retinas (mostly free of pigment epithelium) were rapidly removed and cross-chopped (0.350 mm x 0.350 mm) with a McIlwain tissue chopper. The slices were washed in oxygenated HEPES-Ringer buffer (NaCl 150 rnM, KC1 5-O mM, MgSO,. 7H,O 1.2 mM, CaCl, .2H,O 1.2 mM, glucose 15 mM, HEPES 20 mM), resuspended in fresh HEPES-Ringer buffer and incubated for 30 min at 37°C. The medium was once again renewed with the addition of 3 x lo-’ M [3H]inositol (Amersham International, specific activity 19.6 Ci mmol) and incubated for 45 min. The slices were next washed with modified oxygenated HEPES-Ringer buffer, where 5 mM LiCl was substituted for the same amount of NaCl and then suspended in approximately 1.5-2 ml (for tissue slices from a pair of retinas) of the same buffer. Aliquots of the suspended slices (0.10 ml) were added to 0.19 ml HEPES-Ringer/LiCl buffer and incubated for 10 min in a shaking water bath (3 7’C). During this period antagonists (0.01 ml) were added when required. Agonists (0.01 ml) were then added to the tubes and the incubation was continued for a further 45 min. The incubations were terminated by adding 0.940 ml chloroform/methanol (1: 2 by vol), 0.3 10 ml chloroform and 0.3 10 ml water. The samples were centrifuged (1000 g) for 10 min and the upper phases removed for assay of E3H]InsPs. Total [3H]InsPs were absorbed on to Dowex and extracted with 1 M ammonium formate/O*l M formic acid using the ‘batch ’ method of Berridge et al. (1982). In some experiments, individual [3H]inositol phosphates were separated through a Dowex column by sequential elution using formate solutions of increasing strength (see Cutcliffe and Osborne, 1987; Osborne, 1988), as originally described by Berridge et al. (1982). Measurement of Mobilized Intracellular

Caz+

Retinal cultures, at 3-5 days or 25-30 days after seeding, were incubated in Eagle’s Minimum Essential Media containing 5 x 10m5M Quin2/AM for 60 min at 35.5”C. The final concentration of the Quin2/AM solvent dimethylsulphoxide (DMSO) was about 0.4 %. Control cultures were incubated in MEM containing only 0.4% DMSO without Quin2/AM. The cultures

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were then removed and washed twice in a HEPESbuffered saline solution (20 mM HEPES, 145 mM NaCl, 5 mM KCl, 1 mM MgSO,. 33 mM glucose, 1 mu CaCl,, pH 7.4) containing 0.05 % bovine serum albumin (BSA), and were left to incubate in this solution for 10 min prior to fluorescence measurements. Preliminary trials established that at that time the hydrolysis of Quin2/AM to Quin2 was complete, as the QuinZ/AM emission spectrum (430 nm) had shifted to that of Quin2 (492 nm). The coverslips containing the cultured cells were then placed diagonally at an angle of about 45” in a quartz cuvette containing 0.98 ml of the HEPES-buffered solution without BSA (buffer). Fluorescence was recorded in a Perkin-Elmer Spectrophotometer at excitation and emission wavelengths of 339 nm (4-nm slits) and 492 nm (lo-nm slits) respectively, directly after the addition of an agonist (in 0.01 ml of buffer). Antagonists (in 0.01 ml of buffer) or buffer alone were added 75 set prior to the addition of the agonists. At the end of each experiment the cells were lysed with 0.1 Y0 Triton X100, and the Quin2 fluorescence was measured in the presence of 1 mM CaCl, (maximal) and 2 mM EGTA (minimal). An analysis of unloaded cells showed that the measured autofluorescence did not change significantly when 0.4% DMSO, agonists or antagonists were added. There was, however, a slight (10%) increase in autofluorescence when the unloaded cells were lysed by Triton X-100. This autofluorescence was taken into consideration when determining the absolute intracellular calcium concentration [Caz+],. [Ca2+li was calculated from the observed relative fluorescence values according to the following equation (Tsien, Pozzan and Rink, 1982) :

W+li = & (F- Fmin)/(Fmax - F) where F is the observed fluorescence, Fmin is the

calcium-free Quin2 (excess EGTA) fluorescence, and Fmax is the fluorescence at a high calcium concentration. An apparent Kd value of 120 nM was used for the Quin2/Ca2+ complex at 22°C (Tsien, 1980; Tsien et al., 1982). The viability of the Quin2-unloaded cells as assessed by Trypan blue (0.2 %) exclusion was found to be greater than 95 %, and essentially the same as in non-loaded (control) cells. Materials

[3H]Myoinositol was obtained from Amersham International (Amersham, U.K.). Most of the drugs and fine chemicals were from Sigma (Poole, Dorset). All excitatory amino acids were dissolved in buffer and the pH of the resultant solution was checked. Glutamate solution around 1 mM reduced the pH below 7.2, and this was corrected before use.

3. Results Studies in Retinal Slices

Quisqualate (QA) is the most effective of the excitatory amino acids tested for their stimulation of InsPs production in rabbit retinal slices (Table I). At a concentration of 10 ,UM none of the excitatory amino acid agonists tested significantly stimulated InsPs production, with the exception of QA (Fig. 1). At concentrations of 100 ,UM or 500 ,UM QA is still the most effective of all the excitatory amino acid agonists at stimulating InsPs production, although all the agonists tested potentiated the production of InsPs, in particular kainate and ibotenate (Table I). The responses of 100 puM (maximally effective concentration) of QA and 500 ,LAM of either ibotenate or kainate were not additive (Table I), suggestingthat the

TABLE I

Effect of various excitatory amino acid agonistson the stimulation of inositol phosphate(InsPs) in rabbit retinal slices

Stimulation of ~-

InsPsaccumulation above control (%)

Substance(s)

Quisqualate

100 /LM 5ooph4

Ibutenate

100 ,uM

Kainate

100 /LM

500yM soopi

Glutamate

100 JAM 500/~M 100 /AM 500/~M 100 /LM 500,lLM

NMDA Aspartate

Results

are mean

Quisqualate

100 PM + ibotenate 500 FM

Quisqualate

100 ,&f fibotenate 500 PM

values+_s~.~.

where

n = 3-6.

228+14 217+16 84+7 110+12 83+8

lOlfl1 71+9 93f6 64+7 88If;8 56+9 72+_9 260+20 249+21

--.~---..

400

N. N OSBORNE

,’

A

/

5:- $L-d -10

-9

The elution profile of [“H]InsPs from retinal slices subjected to Dowex chromatography showed that the vast majority of water-soluble InsPs in the basal unstimulated state are inositol- 1-phosphate (InsPI. Stimulation of the slices with QA resulted in a dramatic increase in the labelled InsP fraction (Fig. 2). Peaks corresponding to inositol-1,4-bisphosphate (InsP,) and inositol-1,4,5-triphosphate (InsP,) also manifested a slight increase after QA stimulation, but this was minor compared with InsP. The QA, kainate, aspartate, NMDA and glutamate actions on InsPs production were not significantly diminished by 1 mM of APV (d-amino-S-phosphonovalerate). APB (2-amino-4-phosphobutyrate), PDA (cis 2,3-piperidinedecarboxylic ester) or GDEE (glutamate-diethylester) (see Table II). Furthermore. prazosin, a&opine and ketanserin at 1 ,UM did not have an antagonistic effect (Table II). Thus the QA receptor subtype in the retina which induces a stimulation of InsPs is not influenced by the antagonists APV, APB, PDA and GDEE and is therefore similar to the QA receptors described in the brain (Recasens et al., 19 8 8 : Godfrey et al., 1988). The excitatory amino acids kainate and NMDA at 50 PM significantly inhibited the carbachol but not the noradrenaline-induced stimulation of InsPs (Table III). In contrast, QA at the same concentration potentiated both the carbachol and noradrenaline responses. Importantly, only the inhibitory action of NMDA on

-8

-7

-6

-5

I

-4

I

-3

I

-2

Log [Caxentration]

FIG. 1. Dose-dependent curves for the stimulation of inositol phosphates (InsPs) formation by carbachol and quisqualate in adult rabbit retinal slices. EC,, values of 11.5, and 0.1 ,UM were obtained for carbachol and quisqualate, respectively. Values are means + S.E.M. of percentage stimulation (above control) from three to four separate experiments. (Individual experiments were always carried out in triplicate.)

agonists are acting at the same recognition sites. The effect of QA on InsPs production is dose-dependent (Fig. l), with an EC,, vaIue of 01 ,UM. The extent of stimulation produced by QA is greater than that produced by the cholinergic receptor agonist carbachol (Fig. 1) or by noradrenaline (see Osborne and Ghazi, 1988).

3000

-

A-----A

l -

r: d d

2500

-

2000

-

0

Control 0

QA

1500 -

1000

-

500

-

0 Fraction -__-

---I

number

---2

-_-3 Solution

---4

5

number

FIG. 2. Rabbit retinal sliceswere prelabelledwith [3H]inositoland then incubatedin the presence of quisqualate(100 PM) or vehicle solution for 30 min, containing 5 mu Li+ asdescribedelsewhere(Cutcliffe and Osborne,1987). Aliquots of 750 ,~l of the aqueousphasewere appliedto Dowex-1 anion-exchangecolumns.Five 1 ml fractionswerecollectedfor eachsolutionused, and the radioactivity in each fraction was determined.Stepwiseelution was carried out with: distilledwater (solution 1) for fractions l-5; 5 mM sodium tetraborate/60 mix sodium formate (solution 2) for fractions 6-10: 0.2 M ammonium formate/O.l M formic acid (solution 3) for fractions 11-15; 04 M formic acid (solution 4) for fractions 16-20: and 1.0 M ammonium formate/O-1M formic acid (solution 5) for fractions 21-25. The resultsshown are the meansfrom triplicate determinations.

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401

TABLE II Efect of excitatory amino acid antagonists and other substances on the InsPs accumulation induced by excitatory

amino acid agonists Agonist

APV

APB

GDEE

(1 rnM)

(1rnM)

(1 rnM)

170 75 109 70 111

164 94

183

111

101 115

69 115

66 116

Quisqualate (10 ,UM) NMDA (1 mM) Kainate (1 mM) Aspartate (1 mM) Glutamate (1 mM)

Prazosin (1 PM)

Atropine (1 PM)

Ketanserin (1 PM)

169

188

181

188

96 107

98 121

94 114

100 116

71 109

59 121

66 120

80 117

PDA (1 rnM)

Results are expressed as a percentage of the control value ([W]InsPs accumulation in the absence of any agonist or antagonist) and are the mean value of three to four independent experiments each carried out in triplicate.

TABLEIII

~--

Agonist ~Buffer Carbachol (500

Control

94+5

100

262f22

PM)

+ kainate (50 ,UM) + NMDA (50 ,/AM)

248+21

171*17*

200f 340f

18*

19 140f18

+QA (50 FM) NoradrenaLine (5 50 ,UM)

APB (1 mM)

+ kainate (50 PM)

118+17

+ NMDA (50 ,uM) + QA (50 ,%M)

129+14 210+ 19

191f15 200f17 345+19 162+20 140 + 14 131+15

230+19

APV (1 mM) 108f7 270f6 200+11 266+ ll** 369 + 14 131f14 120+

16

119+19 240&21

GDEE (1 mM) 110+7 249+20 210+18 212+8 340+ 10 139flO 118+14 12Ok16 215+30

Results are shown as percentage increase in InsPs above basal control and represent the means f S.E.M. from three independent experiments. The excitatory amino acid agonists were added 5 min prior to the addition of carbachol. *P -C 0.05, compared with carbachol. ** P < 0.05. compared with carbachol + NMDA.

z i .-E %

203

c

150

E t 4 0 &

100

a”

50

transmitters or the activation of phospholipase C by Ca2+, the action of Ca2+, verapamil and tetrodotoxin on the formation of InsPs was investigated. Figure 3 shows that neither Cd2+, verapamil or tetrodotoxin had any significant effect on the production of InsPs induced by QA. Studieson Retinal Cultures

-t 0 C

VP

Cd

TTX

FIG. 3. The effect of ion channel blockerson quisqualatemediatedproduction of InsPs.Retinal slicesprelabelledwith [3H]inositol were stimulated with quisqualate (100

,UM)

for

30 min in the absence(C) or presenceof verapamil (VP, 10 PM). cadmium ions (Cd, 300 pM) or tetrodotoxin (lTx, 300 nM). The data are means+~.~.~. for four separate experiments.

the carbachol stimulation of InsPs production is significantly reduced by APV (see Table III) while APB and GDEE are without effect. In order to rule out the possibility that the increase in InsPs produced by QA is not an indirect effect mediated, for example, by the release of neuro-

In a previous study (Ghazi and Osborne, 1988a) we have shown that carbachol, noradrenaline and serotonin produce a stimulation of InsPs in primary rabbit retinal cultures of 3 days which contain neurones and glia, while in older cultures (2 5 days or more) which are non-neuronal in character (essentially putative Miiller cells), only carbachol and noradrenaline are effective in stimulating InsPs. These studies were interpreted as showing that S-I-IT, receptors (i.e. serotonin receptors linked to the stimulation of InsPs and antagonized by ketanserin) are associated solely with neurones in the retina and are absent from Miiller cells. In the present study it is shown that QA, like serotonin, is only effective in stimulating InsPs production in the 3-S-day-old retinal cultures and not in the older 25-30-day-old retinal cultures (Table IV).

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TABLE IV Effect of QA, ibotenate, kainate and carbachol on stimulation of InsPs in 3-s-day-old and 25-30-day-old retinal cultures

Stimulation of InsPs (%) Substance

3-S-days-old

25-30-days-old

205 f 36 92$-10 90+_14 1665 19

17*9 6&3 lo+5

QA (100 PM)

Ibotenate (500 pM) Kainate (500 PM) Carbachol (500 ,!LM) Resultsare expressedas

meansk?..~.~.

78k9

when

n = 5.

TABLE V Ejfect of QA, ibotenate,kainate andcarbacholin mobilizing intracellular Ca2+stores (increasein [Caz+]J in 3-5-dayold cultures and 2%30-day-old retinal cultures

Cultures Substance

3-S-days old

Carbachol (500 ,uM) QA (100 PM) Ibotenate (500 pM) Kainate (500 pM)

328f20n~

Resultsare the

means

300+21 190*9

221+14nM nra

nM

190flonM + S.E.M.

when

-_~ 25-30-days old lo+_8

nM

9*3nM 7+5nM

n = 9.

The present results also confirm previous findings (Ghazi and Osborne, 1988a) that carbachol, in contrast, stimulates InsPs production in both young and old retina cultures (Table IV). As for the retinal slices, excitatory amino acid agonists other than QA stimulate InsPs production to about the same extent in the 3-S-day-old retinal cultures (see Table I). The effect of QA and carbachol on the mobilization of intracellular Ca2+ in the two ages of cultures is shown in Table V. Both QA and carbachol mobilize Ca”+ stores in 3-S-day-old cultures, but QA is not effective in the older 25-30-day-old cultures. As expected, ibotenate, kainate and NMDA were less effective in mobilizing Ca2+ stores, and GDEE did not nullify the caicium response by QA in the young cultures. As described previously, the agonist-induced increase in intracellular Ca2+ was unchanged when EGTA, cadmium, lanthanum (two inorganic calcium channel blockers) or nifedipine (an inhibitor of voltagedependent calcium channels) were present in the extracellular media. This clearly demonstrates that Ca2+ is released from intracellular stores (Ghazi and Osborne, 1988b). 4. Discussion Excitatory amino acid receptors in the CNS, including the retina, have been classified into subtypes

N. OSBORNE

on the basis of electrophysiological experiments (see Introduction). The effects of glutamate, aspartate and their analogues in the retina have been reviewed elsewhere (Miller and Slaughter, 198 5, 1986 ; Massey and Redburn, 1987). In the mudpuppy retina, electrophysiological studies with excitatory amino acid agonists and antagonists suggest that six distinct glutamate receptors might exist (Coleman, Massey and Miller, 1986). While variations exist in retinas from different species, it appears generally that glutamate, kainate and QA mimic the effect of the natural transmitter in the outer plexiform layer, hyperpolarizing ON bipolar cells and depolarizing OFF bipolar and horizontal cells (Lasater and Dowling, 1982; Slaughter and Miller, 1983a, b; Ishida and Neyton, 1985), although in some studies the effect of QA and kainate is not consistently antagonized by GDEE and y-n-glutamylglycine, respectively (Rowe and Ruddock, 1982a, b). In the inner plexiform layer. QA and NMDA receptors seem to be associated with amacrine cells while ganglion cells are excited by kainate, NMDA and QA (Massey and Redburn, 1987) although a recent study has shown kainate receptors associated with rabbit retinal amacrine cells (Linn and Massey, 1989). It should be pointed out that a unique APB receptor has been described as occurring in the retina (Slaughter and Miller, 1981). its major pharmacological effect being that of inhibiting the ON pathway. In the present study APB at 1 mu neither stimulates InsPs production nor antagonizes the responses of any excitatory amino acid agonist. In 1986, Nicoletti et al. (1986a, b) showed for the iirst time that activation of excitatory amino acid receptors in the brain enhances inositol phospholipid hydrolysis. The most potent of the excitatory amino acid agonists were ibotenate and quisqualate, an observation which has been generally confirmed in different brain regions and various neuronal cultures (Sladecsek et al., 1985; Schmidt et al., 1987; Godfrey et al., 1988; Recasens et al,, 1988; Schoepp and Johnson, 1988). This study shows that a similar situation exists in the retina. The EC,, value for QA stimulation of InsPs in the retina (0.1 PM) is significantly less than that for the stimulation of InsPs by other agonists, namely carbachol (EC,,, value 11.5 PM) (see Ghazi and Osborne, 1988a; Cutcliffe and Osborne, 1987; Osborne and Ghazi, 1989). However, the QA effect is not nullified by any of the antagonists used (see Table II), in particular GDEE. Since the other excitatory amino acids stimulate InsPs to a lesser extent than QA and these effects are not antagonized by the substances tried, it is suggested that they mediate their effects by stimulating InsPs through the same sites as QA. This QA site or receptor is distinct from previously defined subclasses of excitatory amino acid receptors based on physiological experiments. This new QA subtype of receptor, linked to stimulation of InsPs and shown to occur in the retina, appears to

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exist in other CNS tissues (Recasens et al., 1988 ; Godfrey et al., 1988). The possibility exists that the QA effect on InsPs stimulation in the retina is indirect and mediated through the release of other neuro-transmitters. This is unlikely to be the case for several reasons. (1) Tetrodotoxin or Cd2+ had no significant effect on the response. Cd2+ blocks Ca2+ entry at the nerve terminal and thus prevents the secretion of neurotransmitters, while tetrodotoxin inhibits the conduction of Na+-dependent action potentials, thereby preventing the secretion of neurotransmitters from spiking neurones. (2) QA was also very effective in stimulating InsPs in 3-5-day-old retinal cultures, where it is unlikely that any secreted substances have an influence on their own. The culture solution would significantly dilute any substance secreted from the neurone, for example. (3) An indirect effect of QA through an increase in the levels of intracellular Ca2+ does not seem to occur, since verapamil and Ca2+, at concentrations that prevent the entrance of extracellular Ca2+ through voltage-operated channels and therefore inhibit K+induced formation of InsPs (Kendall and Nahorski, 1985). have no influence. Separation of total accumulated InsPs (after 30 min stimulation with QA) showed that the vast majority of tritium label was associated with InsP fraction. It should be pointed out that any interpretation of data from Dowex-1 column separation must be made with caution, as it is clear that the formation and degradation of InsP, (inositol- 1,4,5-triphosphate) have not been fully established ; several inositol polyphosphates (inositol-1,3,5-triphosphate. inositol tetrakisphosphate, inositol-cyclic triphosphate etc.) are produced in stimulated cells (Michell, 1986 ; Osborne, Tobin and Ghazi, 1988b). The Dowex-1 column elution profile of inositol polyphosphates from retinal tissue treated with QA is similar to studies where other agonists, e.g. carbachol and substance P, have been used (see CutcliIIe and Osborne, 1987; Osborne and Ghazi, 1989). In this context it is interesting to note that desimipramine also stimulates phospholipid hydrolysis, but in this case it has been postulated that this is via a membrane effect rather than through specific receptors (Osborne, 198 7a). The elution profile of the polyphosphates of desimipramine-treated tissues shows that the InsP, fraction is elevated in comparison to the InsP fraction (Osborne, 1987a). Previous studies have established that young primary retinal cultures (3-5 days old) contain a mixture of neurones and glial cells, while older cultures (2 5-30 days old) are devoid of neurones and consist of retinal glial cells identified on the basis of their vimentin staining as being primarily Miiller cells (Osborne, 1987b; Ghazi and Osborne. 1988a; Osborne et al., 1988a). The finding that QA stimulates InsPs pro28

403

duction in the younger cultures, but not in the older cultures, strongly suggests that receptors linked to phosphoinositide turnover in the retina are associated purely with neurones. Earlier studies of ours have shown that retinal serotonin and substance P receptors associated with inositol phospholipid hydrolysis are also only present on neurones, whereas some retinal muscarinic and alpha,-adrenergic receptors linked to the same second messenger are additionally located on Miiller cells (Ghazi and Osborne 1988a; Osborne and Ghazi, 1989). It is worth noting in this respect that Miiller cells of non-mammalian retinas (Brew and Attwell, 1987) and astrocytes of the brain (Bowman and Kimelberg, 1984) have been shown to contain glutamate receptors by electrophysiological studies. Furthermore, QA (and other excitatory amino acid agonists to a lesser degree) stimulates InsPs accumulation in astrocytes (Pearce, Morrow and Murphy, 1986). It is a generally accepted fact that, following agonistinduced stimulation of phosphoinositide turnover, the formed InsP, mobilizes an intracellular store of Ca2+ (Berridge and Irvine, 1984; Berridge, 1984a, b). We have recently employed the fluorescent calcium indicator, Quin2, to study agonist-induced stimulation of Ca2+ mobilization in retinal cultures (Ghazi and Osborne, 1988b). Using a similar approach, it is shown here that QA stimulates the free intracellular calcium concentration in young cultures, but not in older cultures (see Table V). Furthermore, this effect is not antagonized by GDEE. It must therefore be concluded that QA acts via receptors which stimulate both inositol phospholipid hydrolysis and Ca”+ mobilization. These receptors occur only on retinal neurones and they are sensitive to ibotenate, kainate and NMDA, but these agonists are less effective than QA. The present study demonstrates that some excitatory amino acid agonists are also able to inhibit agonist-stimulated formation of InsPs in retinal slices. That QA does not possess this characteristic may be explained by the fact that it is a powerful stimulator (even at 50 ,uM) of InsPs production on its own (see Fig. 1). In contrast, kainate and NMDA at 50 ,MM have a negligible effect on their own, and yet weakly diminish the carbachol effect on InsPs production (see Table III). The blockade of carbachol-stimulated InsPs formation in the retina by NMDA and kainate is in good agreement with previous observations on tissues from different brain areas (Baudry et al., 1986 : Godfrey et al., 1988; Jope and Li, 1989). The relative resistance of the noradrenaline response to inhibition by kainate and NMDA in the retina has also been seen in striatal neurones (Schmidt et al., 1987), hippocampus (Baudry et al., 1986; Nicoletti et al.. 1986a, b) and cerebral cortex (Godfrey et al., 1988). The reasons for the different sensitivity of the noradrenaline response are unknown. EER 50

404

N. N OSBORNE

The pharmacology of the weak inhibitory action of NMDA on agonist stimulation of InsPs in the retina is difficuIt to understand. This is because the NMDA effect is nullified by APV but has no action on kainate’s action. This is similar to what has been found to occur in the cerebral cortex (Godfrey et al., 1988). Perhaps the only conclusion to be drawn from the presented data is that the inhibitory action of NMDA and kainate may be linked to the activation of more than one subtype of excitatory amino acid receptor. The inhibitory action of NMDA and kainate may take place by analogy with the mechanism of agonistinduced inhibition of adenylate cyclase activity, i.e. through a novel inhibitory G-protein which blocks the activation of phospholipase C. Alternatively, the reduction of InsPs production may be related to the known neurotoxic action of excitatory amino acids (Schmidt et al., 1987). In conclusion, this study describes the actions of excitatory amino acids on the metabolism of InsPs in rabbit retina. It appears that a new described subtype of excitatory amino acid receptor exists, for which QA has the greatest affinity and which is coupled to inositol phospholipid turnover. Similar novel QA receptors have been described in other CNS tissues (e.g. Recasenset al., 1988; Godfrey et al., 1988). In the retina these receptors are associated with neurones, where it can be shown that when activated, an internal pool of calcium is mobilized. Secondly, it appearsthat activation of other excitatory amino acids reduced agonist-induced (carbachol in preference to noradrenaline) hydrolysis of InsPs. The mechanism by which this occurs is not known.

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